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Labinfop. 4 MLVA: a useful epidemiological tool ?
p. 8 Implementation of RT-PCR at the FLVVT within the context of the TSE Roadmap
p. 12 GC-MS/MS as a new option for confi rmatory approach of PCDD/Fs and DL-PCBs analysis in food and feed
p. 16 Tank milk, an extra tool for detection and eradication of IBR in cattle farms
p. 20 Plastic materials intended to come into direct contact with food : What is the impact on laboratories of the recently adopted Regulation (EU) No 10/2011?
p. 25 New techniques and methods developed by the Belgian NRL-GMO to identify unauthorized GMOs in the UGMMONITOR project.
p. 28 Screening of pesticides residues by the time of fl ight analyzer (ToF) : Myth or reality ?
p. 35 Introduction to the FP7 - QBOL research project
p. 43 Workshops & Symposia
LNRN A T I O N A L ER E F E R E N T I ELABORATORIA
L A B O R ATO I R E SN A T I O N A U XD E R E F E R E N C ENRLN A T I O N A L
R E F E R E N C ELABORATORIESNRL
Newsletter for the approved food safety laboratories
SEMI-ANNUEL NEWSLETTER - N°9 JANUARI 2013
FASFCAC-Kruidtuin - Food Safety Center, Kruidtuinlaan 55, 1000 Brussels
Resp
onsi
ble
edito
r : G
il H
ouin
s
2
LNRN A T I O N A L ER E F E R E N T I ELABORATORIA
L A B O R ATO I R E SN A T I O N A U XD E R E F E R E N C ENRLN A T I O N A L
R E F E R E N C ELABORATORIESNRL
LabInfoNewsletter for the approved food safety laboratories
Editors’ groupDirk Courtheyn, Mieke De Mits, Conny De Schepper, Alain Dubois, Marc Evrard, Alain Laure, Bert Vandenborre, Mieke Van de Wiele, Eva Wevers and Marie-Christine Wilem
Authors of this issueGeert De Poorter, Geertrui Rasschaert, Koen de Reu, Marc Heyndrickx, Jeroen Vancutsem, Ruth Vanhoof, Mandy Lekens, Gauthier Eppe, Jean-François Focant, Jean-Baptiste Hanon, Brigitte Caij, Els Van Hoeck, Tina N’Goy, Caroline Evrard, Fabien Bolle, Marie-Alice Fraiture, Philippe Herman, Gilbert Berben, Frédéric Debode, Eric Janssen, Isabel Taverniers, Marc De Loose, Nancy Roosens, Laure Joly, Vincent Hanot and Martine Maes
TranslationTranslation Service of the AgencyEditors’ group
Photographs and illustrationsSupplied by the laboratories
LayoutGert Van Kerckhove
Editor’s addressLabInfop.a. D. CourtheynFASFCAC-Kruidtuin – Food Safety Center4de verdieping, bureel K04/120218Kruidtuinlaan 551000 BrusselTel.: [email protected]
32
Dear reader,
2012, which was a harsh year in terms of budget and economy, is behind us. Faithful to tradition, most of us passed the turn of the year in festive mood, looking to the future.
We do not know what 2013 holds in store. Economically, we cannot yet see the light at the end of the tunnel. But that should not mean that we throw in the towel. We must take full advantage of our strengths and seize every opportunity to highlight them. In the laboratory branch, we do without any doubt hold a strong position when it comes to flex-ibility, knowhow and, above all, a wide range of analyses that allow us to address all kinds of emerging threats to food safety.
The new Royal Decree on the approval of laboratories provides us with a modern legal basis that offers good prospects for the future and should allow independent laboratories to carry out a substantial part of the annual control programme of the Agency. In value, this activity represents over 3 million euros yearly. This market along with the self-checking systems market are the preferential markets on which private laboratories should focus.
2013 will also be the year in which the Agency will put an end to the systematic testing of bovines for BSE. After more than twelve years and over 130 million euros spent on analy-ses, the curtain has finally dropped. The TSE road was long and difficult but TSE control produced results that all TSE laboratories may rightly be proud of. It is obvious that the cooperation between the public and the private sector in this field is truly an example of a successful private public partnership.
New NRLs have also been designated for various chemical fields, in accordance with Regulation (EC) No 882/2004. The Government Contracts Act was applied for that purpose and, in all honesty, I have to say that the administrative implementation of this initiative was quite burdensome. Apparently, objectification and transparency cannot easily be matched with swiftness and pragmatism.
I toast to effective cooperation, I hope you will enjoy reading the issues of our Labinfo magazine and above all, I wish you and your loved ones good health.
Geert De PoorterDirector general Laboratories
Editorial
4
MLVA: a useful epidemiological tool ?Geertrui Rasschaert, Koen De Reu and Marc Heyndrickx
ILVO-T&V, Instituut voor Landbouw en Visserij Onderzoek – Eenheid Technologie en Voeding (Institute for Agricul-tural and Fisheries Research, Technology & Food Science Unit), Brusselsesteenweg 370, B-9090 Melle
Since a few years, MLVA has been known as a new tool for epidemiological research. This article describes the principle of MLVA, examines the pros and cons of this technique and gives some examples. MLVA stands for ‘multi-locus variable number of tandem repeats assay’. The technique is based upon the natural variation of the number of tandem repeated DNA sequences found at several places, i.e. loci, within the genome of a large number of bacteria. Tandem repeats are head-to-tail repetitions of a DNA sequence motif. MLVA means that the number of repeats for a certain number of loci is defi ned. This results in a numerical code, such as 5-3-7-4, as represented in Diagram 1. The fi rst fi gure stands for the number of repeats of the fi rst DNA sequence motif of the fi rst locus examined, the second fi gure for the number of repeats of the second DNA sequence motif of the second locus examined, and so on … This variation in repeats is caused by the fact that the Taq polymerase makes inexact copies of certain segments during ‘slipped strand mispairing’. It is as if the Taq polymerase is stuck in certain areas of the genome, resulting in the duplication or the elimination of certain sequences. Such mistakes happen very fast and that is why MLVA reveals rather fast developments of a bacterial population.
A.
B.
locus1 locus2 locus3 locus4
Strain A: 5-3-7-4
Strain B: 3-4-9-2
Figure 1:(A) An example of the number of tandem repeats of four loci of two bacterial strains,(B) The gel obtained when using MLVA for typing these two strains.
54
In practice, a multiplex PCR during which the DNA of five to six loci is amplified, is performed first. Then, the length of the different fragments in the PCR product is measured very accurately by means of capillary electro-phoresis. When the length of such a repetitive element is known, the number of repeats can be calculated auto-matically by means of specialized software such as BioNumerics.
The main advantage of this technique lies in its high discriminatory power. It is often as discriminatory as pulsed field-gel electrophoresis (PFGE), generally considered as the gold standard when it comes to making a distinction between isolates. Moreover, this technique is faster and less labour intensive than PFGE. The data obtained can, like with usual typing methods, be visualized by means of dendrograms, but also by means of ‘minimum spanning trees’ (MST) which are used to visualize evolutionary relationships. The disadvantage of this technique is that every species, serotype, …. requires a new optimization, which is rather time consuming. This involves choosing the right primers, optimizing the multiplex PCR, adjusting capillary elec-trophoresis. However, international protocols are already in place for certain bacteria, such as E. coli O157, Salmonella Typhimurium, Salmonella Enteritidis and the results obtained can be compared to online databases. At the ILVO (Institute for Agricultural and Fisheries Research), Technology and Food Science Unit, MLVA has been developed and successfully applied for both MRSA ST398 and Salmonella Enteritidis. For some years, MRSA ST398, the so-called livestock-associated MRSA, has been isolated in pigs. One of the purposes of the doctoral study of M. Verhegghe was to examine the MRSA ST398 variety within a holding in order to check whether a pig is colonized with the same MRSA ST398 strain for his entire life. For this purpose, sows and their piglets were regularly sampled in the farrowing pen between the date of littering and the date of slaughter and the MRSA ST398 isolates were characterized by means of MLVA. It appeared that a large variety of MRSA ST398 types occurred within every holding, that the piglets were rarely colonized by the same MRSA ST398 type as their mother and that they could be carriers of several types in the course of their lifetime. The dendrogram (Diagram 2) shows the MLVA types of a sow (sow 3) and of three of her piglets (pig 23, pig 24 and pig 26). On day 3, day 6 and day 20 following farrowing the sow was colonized by another MLVA type and her piglets were at none of these times colonized by a MLVA type that came from their mother.
6
Figure 2: Dendrogram of the MRSA ST398 MLVA types of one sow (sow 3) sampled at regulars intervals in the farrowing pen and of three of her piglets (pig 23, pig 24, pig 26) sampled from day 3 after littering to the date of slaughter, in which d represents the number of days following littering.
In the doctoral study of I. Dewaele on Salmonella Enteritidis in persistently Salmonella positive laying hen holdings, an MLVA was optimized in order to identify the contamination sources and routes in such holdings. Five problem holdings were followed during three successive laying cycles. Contrarily to the example above, it appeared that the number of different MLVA types was rather limited in most cases and that other types were found in the egg room than in the laying pens. A visual representation of these findings is given in Diagram 3 by means of a minimum spanning tree. In this diagram, each circle stands for one MLVA type; the larger the circle, the higher the number of isolates belonging to that MLVA type. The largest circle represents the ancestral type from which the other types have been derived. The length of the “branches” stands for the evolutionary distance to the other MLVA types. In this example, a different colour has been chosen for each different place of sampling.
VNTR_vals
100
50
VNTR_vals
ClfA
ClfB
sdrC
sdrE
SIRU
33.0 55.0 36.0 6.0 3.0
34.0 58.0 36.0 6.0 3.0
32.0 54.0 34.0 6.0 3.0
33.0 57.0 37.0 1.0 3.0
33.0 57.0 37.0 6.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 36.0 7.0 3.0
33.0 57.0 36.0 7.0 3.0
33.0 57.0 36.0 4.0 3.0
33.0 58.0 36.0 7.0 3.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 58.0 38.0 8.0 4.0
34.0 58.0 38.0 8.0 3.0
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pig 23, d27
pig 24, d27
pig 23, d3
sow 3, d3
sow 3, d6
pig 24, d35
pig 26, d35
pig 24, d48
pig 26, d48
pig 24, d62
pig 23, d77
pig 23, d108
pig 23, d159
pig 26, d159
pig 23, d6
pig 26, d27
sow 3, d20
pig 24, d77
pig 24, d3
pig 24, d6
pig 26, d6
pig 23, d20
pig 24, d20
pig 26, d20
pig 23, d35
pig 23, d48
pig 23, d62
pig 26, d77
pig 24, d108
pig 26, d108
pig 24, d159
pig 26, d62
pig 26, d3
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VNTR_vals
100
50
VNTR_vals
ClfA
ClfB
sdrC
sdrE
SIRU
33.0 55.0 36.0 6.0 3.0
34.0 58.0 36.0 6.0 3.0
32.0 54.0 34.0 6.0 3.0
33.0 57.0 37.0 1.0 3.0
33.0 57.0 37.0 6.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 37.0 7.0 3.0
33.0 57.0 36.0 7.0 3.0
33.0 57.0 36.0 7.0 3.0
33.0 57.0 36.0 4.0 3.0
33.0 58.0 36.0 7.0 3.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 59.0 38.0 8.0 4.0
34.0 58.0 38.0 8.0 4.0
34.0 58.0 38.0 8.0 3.0
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pig 23, d27
pig 24, d27
pig 23, d3
sow 3, d3
sow 3, d6
pig 24, d35
pig 26, d35
pig 24, d48
pig 26, d48
pig 24, d62
pig 23, d77
pig 23, d108
pig 23, d159
pig 26, d159
pig 23, d6
pig 26, d27
sow 3, d20
pig 24, d77
pig 24, d3
pig 24, d6
pig 26, d6
pig 23, d20
pig 24, d20
pig 26, d20
pig 23, d35
pig 23, d48
pig 23, d62
pig 26, d77
pig 24, d108
pig 26, d108
pig 24, d159
pig 26, d62
pig 26, d3
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Figure 2: Dendrogram of the MRSA ST398 MLVA types of one sow (sow 3) sampled at regulars intervals in the farrowing pen and of three of her piglets (pig 23, pig 24, pig 26) sampled from day 3 after littering to the date of slaughter, in which d represents the number of days following littering.
In the doctoral study of I. Dewaele on Salmonella Enteritidis in persistently Salmonella positive laying hen hold-ings, an MLVA was optimized in order to identify the contamination sources and routes in such holdings. Five problem holdings were followed during three successive laying cycles. Contrarily to the example above, it ap-peared that the number of different MLVA types was rather limited in most cases and that other types were found in the egg room than in the laying pens. A visual representation of these findings is given in Diagram 3 by means of a minimum spanning tree. In this diagram, each circle stands for one MLVA type; the larger the circle, the higher the number of isolates belonging to that MLVA type. The largest circle represents the ancestral type from which the other types have been derived. The length of the “branches” stands for the evolutionary distance to the other MLVA types. In this example, a different colour has been chosen for each different place of sampling.
76
Figure 3: Minimum spanning tree of MLVA of a Salmonella Enteritidis persistent laying hen holding. One circle represents one MLVA type; the size of the circle is proportional to the number of isolates. A diff erent colour is used for every spot of sampling (blue : outside environment; green : egg room; red : laying pen 1; yellow : laying pen 2; violet : laying pen 3).
One may say as a conclusion that the optimization of an MLVA is time consuming but once optimization is done, MLVA is a fast tool for characterizing large numbers of isolates. An additional advantage is that MST may be used to visualize results in a more biological manner.
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8
Implementation of RT-PCR at the FLVVT within the context of the TSE RoadmapJ. Vancutsem, R. Vanhoof and M. Lekens (FLVVT, Tervuren)
The second TSE Roadmap (TSE = transmissible Spongiform Encephalopathy), published July 16th 2010, provided an outline of possible future changes to EU measures over the period 2010-2015. The majority of the short and medium term measures envisaged in the first TSE Roadmap have been achieved and the positive trend already observed in 2005 has continued since then. One of the strategic goals of the second roadmap is to further review the feed ban of 2001, because processed animal proteins (PAP) are an important source of high quality proteins. At present, the use of PAP is prohibited in feed for farmed animals, with the exception of fish meal, as laid down in Regulation 999/2001. With regard to a possible gradual lifting of the ban, the establishment of a tolerance level for PAP in feed might be considered, if a sufficiently reliable quantitative test is available, as well as the lifting of feed ban provisions for non-ruminants. As a first step towards a review of the feed ban, a proposal allowing the use of non-ruminant derived PAP in fish feed was supported by the EU Member States at the vote held during the meeting of the “Standing Committee on the Food Chain and Animal Health” on July 18th 2012 (minutes available on http://ec.europa.eu/food/commit-tees/regulatory/scfcah/biosafety/sum_18072012_en.pdf ). It should be noted that, for the time being, only fish meal is considered. Yet, allowing PAP for non-ruminants is explicitly mentioned in the Roadmap. However, such a measure would only be acceptable if validated analytical techniques to determine the species origin of PAP are available (ruminants, pigs, poultry). In addition, correct channeling of PAP from different species will require invest-ments from the industry.
Analytical techniques
As microscopy does not allow to determine the animal species of most of the animal elements present, such as muscle fibers and bone fragments, PCR may be used for identification. It was decided in 2010 that these PCR tests would be performed at the FLVVT. This analytical technique being new, the FLVVT attended an intensive training at the EURL-AP in December 2010. In 2011 the CODA (VAR) put two extra rooms at the disposal for this analysis and a RT-PCR (real-time polymerase chain reaction) apparatus (Lightcycler 480 II) and purification robot (MagMAX) were purchased.
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Figures 1 and 2 : RT-PCR apparatus (Lightcycler 480 II, left) and a purification robot (MagMAX, right)
At first, the focus will be on the detection of ruminant DNA. At the moment, the methods for detecting DNA of pigs and poultry are still being developed. In December 2011-February 2012, the EURL-AP set up a validation study and in March – May 2012, it launched an implementation test on the detection of ruminant DNA. The FLVVT took part in this study. The test was performed on 10 samples : 6 DNA extracts and 4 feedstuffs. At the same time, the FLVVT also analysed the first samples submitted by clients.
PCR analysis
The major precaution to be taken with regard to PCR analyses is to avoid contamination. During the amplification phase, DNA material is amplified millions of times and when that DNA is released, samples, lab rooms, … may be unintentionally contaminated with target sequences. That is why all steps of the analyses are performed in sepa-rate locked rooms: grinding the samples, weighing the samples, DNA extraction and purification, preparing plates and PCR. In addition, the workbench and the material were thoroughly cleaned using detergents in order to break down DNA.When the sample has been ground and weighed, the DNA is extracted and purified by means of a specific kit that uses paramagnetic particles that bind DNA. By putting a microcentrifuge tube on a magnetic rack, the DNA is purified in different steps using different reagents and is finally eluted. This process may also be automated using a robot. The purified DNA is then ready to be put on a PCR plate.
Implementatie van RT-PCR in het FLVVT in het kader van het TSE stappenplan J. Vancutsem, R. Vanhoof en M. Lekens (FLVVT, Tervuren) In het tweede stappenplan voor TSE (overdraagbare spongioforme encefalopathie) dat op 16 juli 2010 gepubliceerd werd, werd het kader geschetst van mogelijke wijzigingen van TSE-maatregelen in de EU voor de periode 2010-2015. De meeste geplande maatregelen van het eerste TSE-stappenplan voor de korte en middellange termijn zijn intussen verwezenlijkt en de positieve trend die in 2005 werd waargenomen, heeft zich verder doorgezet. Eén van de actiedoelstellingen in het tweede stappenplan is een verdere versoepeling van het voederverbod uit 2001 omdat verwerkte dierlijke eiwitten (VDE) een belangrijke bron van hoogwaardige eiwitten zijn. Op dit ogenblik is het gebruik van VDE, met uitzondering van vismeel, verboden voor landbouwhuisdieren volgens Verordening 999/2001. Voor de versoepeling wordt gedacht aan het instellen van een tolerantieniveau voor VDE in diervoeders mits de ontwikkeling van voldoende betrouwbare kwantitatieve analyses en aan het opheffen van het voederverbod voor niet-herkauwers. In een eerste stap voor het versoepelen van het voederverbod werd een voorstel tot het toelaten van VDE van niet-herkauwers in visvoer ondersteund door de Europese lidstaten tijdens de stemming op 18 juli 2012 in het “Standing Committee on the Food Chain and Animal Health” (verslag beschikbaar op http://ec.europa.eu/food/committees/regulatory/scfcah/biosafety/sum_18072012_en.pdf). Merk hierbij op dat het voorlopig enkel om vismeel gaat, maar dat het toelaten van VDE voor niet-herkauwers wel expliciet omschreven is in het stappenplan. Deze versoepeling is slechts mogelijk wanneer er gevalideerde analysemethoden beschikbaar zijn om vast te stellen van welke diersoort de VDE afkomstig zijn (herkauwers, varkens, pluimvee). Ook zal de industrie investeringen moeten doen voor de gescheiden verwerking van de VDE van verschillende oorsprong. Analysemethoden Omdat het met microscopie niet mogelijk is om van de meeste aanwezige dierlijke bestanddelen zoals spiervezels en botfragmenten de diersoort vast te stellen, kan voor de identificatie overgegaan worden op PCR. In 2010 werd de beslissing genomen om deze PCR-analyses te laten doorgaan in het FLVVT. Omdat deze analysetechniek nieuw was, nam het FLVVT in december 2010 deel aan een intensieve training georganiseerd door het EURL-AP. In 2011 werden voor deze analyse door het CODA twee extra analyseruimtes ter beschikking gesteld en werden een RT-PCR (real-time polymerase chain reaction)-toestel (Lightcycler 480 II) en een opzuiveringsrobot (MagMAX) aangekocht.
Afbeelding 1 en 2: RT-PCR-toestel (Lightcycler 480 II, links) en een opzuiveringsrobot (MagMAX, rechts)
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Figure 3 : Schematic representation of sample preparation for PCR
The primers, the probe and the mastermix with the nucleotides and polymerase are added to amplify the DNA. When the sample extracts have been added, the plate is put into the PCR apparatus and the actual 50 cycle reac-tion is started. The software calculates the C
p (Crossing point) value as the maximum of the second derivative of the amplifi ca-
tion curve, i.e. the point at which the curve increases the most. The Cp value obtained is then compared to the
cut-off value, i.e. the number of cycles expressed as a Cp value above which a signal is considered as a false-
positive sample. The cut-off value is specifi c for the used platform and cannot simply be transferred to another apparatus within the laboratory.
In order to verify whether the series of analysis has been performed correctly, additional samples are analysed in the same run for the purpose of quality control. In the fi rst place this is meant as a positive and negative extrac-tion control, the purpose of which is to check the quality (contamination) of extraction reagents. In addition, a positive DNA check and a NTC (no template control) are also analysed in order to check the quality of PCR rea-gents. Finally, the possible eff ect of the presence of inhibiting components on the PCR is examined by analyzing a tenfold dilution of each sample. If no inhibition occurs, the signal will, on principle, appear 3,3 (log
2(10)) cycles
later.
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Figure 4: RT-PCR curves: flat line for negative samples, upward line for positive samples
In the future, more samples will be analysed using PCR when the use of PAP will again be authorized and it is probable that public opinion will not, or practically not, oppose to the reintroduction of PAP in the food chain. The necessary analytical techniques, such as those for detecting PAP of pigs and poultry by means of PCR, will be further developed for that pur-pose.
Literature:
• The TSE Roadmap 2: A strategic paper on Transmissible Spongiform Encephalopathies for 2010-2015 (16/07/2010)• Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the
prevention, control and eradication of certain transmissible spongiform encephalopathies, Official Journal of the Euro-pean Union, L 147, p1–40
• Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance), Official Journal of the European Union, L 54, p1–130
• Fumière O., Marien A., Berben G. (2012) EURL-AP PCR Implementation Test 2012• Fumière O., Marien A., Berben G. (2012) Validation study of a real-time PCR method developed by TNO Triskelion bv for
the detection of ruminant DNA in feedingstuffs (Draft)• Veys P. & Baeten V. (2007) CRL-AP Interlaboratory Study 2007 (Final report)• Summary report of the standing committee on the food chain and animal health held in Brussels on 18 July 2012• ISO 84276 (draft): Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived
products - General requirements and definitions• Scientific Opinion on the revision of the quantitative risk assessment (QRA) of the BSE risk posed by processed animal
proteins (PAPs). EFSA Journal 2011;9(1):1947
which a signal is considered as a false-positive sample. The cut-off value is specific for the used platform and cannot simply be transferred to another apparatus within the laboratory. In order to verify whether the series of analysis has been performed correctly, additional samples are analysed in the same run for the purpose of quality control. In the first place this is meant as a positive and negative extraction control, the purpose of which is to check the quality (contamination) of extraction reagents. In addition, a positive DNA check and a NTC (no template control) are also analysed in order to check the quality of PCR reagents. Finally, the possible effect of the presence of inhibiting components on the PCR is examined by analyzing a tenfold dilution of each sample. If no inhibition occurs, the signal will, on principle, appear 3,3 (log2(10)) cycles later.
Figure 4: RT-PCR curves: flat line for negative samples, upward line for positive samples In the future, more samples will be analysed using PCR when the use of PAP will again be authorized and it is probable that public opinion will not, or practically not, oppose to the reintroduction of PAP in the food chain. The necessary analytical techniques, such as those for detecting PAP of pigs and poultry by means of PCR, will be further developed for that purpose. Literature:
The TSE Roadmap 2: A strategic paper on Transmissible Spongiform Encephalopathies for 2010-2015 (16/07/2010)
Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001
laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies, Official Journal of the European Union, L 147, p1–40
Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of
sampling and analysis for the official control of feed (Text with EEA relevance), Official Journal of the European Union, L 54, p1–130
Fumière O., Marien A., Berben G. (2012) EURL-AP PCR Implementation Test 2012
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GC-MS/MS as a new option for confirmatory approach of PCDD/Fs and DL-PCBs analysis in food and feed
Gauthier Eppe* and Jean-François Focant
CART University of Liège, Allée de la Chimie 3, B-6c Sart-Tilman, B-4000 Liège, BELGIUM
Introduction
Risks for human health from PCBs and dioxins are mainly related to consumption of food from animal origin. During the last decade, repeated cases of contamination of feedingstuffs highlighted the importance of feed as the potential contamination media. Reducing the dioxin uptake by human is thus highly dependent on actions taken to minimize the contamination of all feed materials, including raw materials, but also recycled products and ingredients (e.g. citrus pulp pellets, recycled fats, mineral clays, choline chloride component, hydrochloric acid related to gelatin production, guar gum thickener, biodiesel-related fatty acids, etc).Despite those actions, isolated cases of contamination might still arise. The implementation of continuous mo-nitoring strategies, the enforcement of the maximum-action-target level strategy (now simplified in maximum-action level only), as well as the availability of a Rapid Alert System for Food and Feed (RASFF), nowadays allows actions to be taken more rapidly and in a coordinated manner in order to reduce the potential human exposure to a minimum in case a contamination event is reported.
The need for efficient monitoring of dioxins and PCBs
Both biological and physico-chemical methods can be used for screening of dioxins and PCBs in foods(1). On a total toxic equivalent (TEQ) basis, both types of methods have to express false compliant rates below 5%, a within-laboratory reproducibility (RSD
R) below 25%, and obviously have a rate of false non-compliant, sufficiently low to
make the use of a screening tool advantageous. Additionally, all non-compliant results have to be confirmed by the gas chromatography-high resolution mass spectrometry (GC-HRMS) method, and up to 10% of compliant samples also have to be confirmed. Bioassays and GC-MS methods are however subjected to different levels of specific requirements and offer different information (Commission Regulation (EU) No 278/2012(2); Commission Regulation (EC) No 252/2012(3)). For GC-MS screening methods the level of expertise is higher and is similar to that of the GC-HRMS methodology used for confirmatory purposes, the only differences being the type of mass analyzer to be used and the LOQs to be reached. Therefore, although originally foreseen as an economically viable alternative to GC-HRMS, the GC-MS screening approach is rarely implemented in practice. The major reason is that, since the analytical requirements are the same, and because the sample preparation requirements are the same (preparing a sample for GC-LRMS or GC-HRMS requires the same efforts), the only difference is in fact the MS investment. The price for HRMS instruments is higher than for other MS analyzers used in screening, but the real financial impact on a sample by sample basis based on a return of investment calculated on the lifetime of the analyzer is extremely low. Many analytical laboratories thus prefer to invest in the confirmatory physico-chemical tool rather than in its screening equivalent.
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However, different studies on the applicability of new generation of triple quadrupole GC-MS/MS for determi-nation of PCDD/Fs and dioxin-like PCBs in feed and food samples were recently published(4,5,6,7). The outcome of the comparison of GC-MS/MS and GC-HRMS results indicated, that GC-MS/MS has now the potential to be used as an alternative confirmatory method. To discuss this new option, a core-working group was formed within the network of European Union Reference Laboratory (EU-RL) and National Reference Laboratories (NRLs) of EU member states for Dioxins and PCBs in Feed and Food. The objective was to assess the potentialities of new MS techniques/instruments for determination of dioxins and PCBs in feed and food.
Evaluation of GC-MS/MS systems
It was clear over 30 years ago that GC-MS was the instrumental method of choice for persistent organic pollutants (POPs) measurements and especially for PCDD/Fs. Mass spectrometry provides not only a very specific quantifica-tion but also ensures the unambiguous identification of target compounds. However, not all GC-MS instrumen-tation can measure PCDD/Fs. The complexity of the measurement of these compounds is also related to the low levels at which they occur in environmental matrices, but particularly in food and feed. The required sensitivity is achieved by a combination of high-resolution and high-mass accuracy using double focusing magnetic sector instruments, commonly called high resolution mass spectrometers (HRMS) performing in the SIM (selected ion monitoring) mode. However, the new generation of triple quadrupole instruments has shown a significant impro-vement of the sensitivity that may partially fill the sensitivity gap with GC-HRMS sector instruments. Kotz and co-workers have recently reported instrumental limit of quantification (LOQ) of 50 fg (absolute amount on-column) for TCCD6. In addition, Fürst and co-workers achieved the same level of performances. The lowest calibration point for PCDD and PCDF congeners was 100 fg injected on-column with an excellent linearity from 0.1 to 10 pg injected on column7.The working group within the EU-RL and NRLs network evaluated the potentialities of the new candidate confir-matory method by assessing: - The working range, especially focusing on the lower end of the working range- Linearity, precision, trueness and robustness of the systems- The applicability in routine analysis in food and feed analysis at the level of interest- The comparison of GC-MS/MS and GC-HRMS results (in comparison with proficiency testing consensus
values)
The conclusions of the evaluation of different available sensitive GC-MS/MS systems showed that latest GC-MS/MS systems from different vendors are applicable for routine analysis of PCDD/Fs and dioxin-like PCBs in feed and food and have a potential as alternative confirmatory method8.
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Figure 1: full scan versus MS/MS spectra of 2,3,7,8-TCDD
Proposed amendments of current criteria
A series of new performance criteria were added in the EU legislation. For confirmatory methods, the definition in-cludes now also the possibility to use gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) as confirmatory methods. The definition of the limit of quantification has been revised to take into account the issue of the very low noise level acquired in MS/MS mode. New specific identification criteria for GC-MS/MS were also added. They are based on established criteria in Commission Decision 2002/657/EC9 and EPA methods 1613 and 1668. For instance, the application of the GC-MS/MS requires the monitoring of at least two specific precursor ions performing in MRM (multiple reaction monitoring) mode, each with one specific transition product ion for all labeled and native analytes in the scope of analysis. More information is available in Commission Regulations 252/2012 and 278/2012. These amendments set comparable strict criteria for both HRMS and MS/MS systems in order to keep the highest analytical quality and reliability of PCDD/F and PCB measurements by confirmatory methods. Is it sufficient?One should note that measuring routinely at sub parts-per-trillions PCDD/Fs and DL-PCBs is challenging and it requires obviously the upmost sensitive detection system but the extraction and clean-up processes both play an important role that definitely have influences on the quality and reliability of the analytical results.
Figure 1: full scan versus MS/MS spectra of 2,3,7,8-TCDD
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References: 1. Focant J-F., Anal Bioanal Chem (2012) 403:2797–28002. Commission Regulation (EC) No 152/2009 of 27 January 2009, amended by Commission Regulation (EU) No 278/2012 of 28 March 2012 (OJ L 91, 28.3.2012, p. 8–22)3. Commission Regulation (EU) No 252/2012 of 21 March 2012 repealing Regulation (EC) No 1883/2006 (OJ L 84, 23.3.2012, p. 1–22)4. Ingelido AM, Brambilla G, Abballe A, di Domenico A, Fulgenzi AR, Iacovella N, Iamiceli AL, Valentini SDe Filep E (2011) Rapid Commun. Mass Spectrom, 26: 236-2425. Sandy C, Fürst C, Bernsmann T, Baumesiter D (2011) Organohalogen Compd 73: 1370-13716. Kotz A, Malisch R, Wahl K, Bitomsky N, Adamovic K, Gerteisen I, Leswal S, Schachtele J, Tritschler R, Winterhalter H (2011) Organohalogen Compd 73 : 688-6917. P. Fürst, T. Bernsmann, D. Baumeister, C Sandy, Agilent Technologies publication 5990-6594EN (2010); http://www.chem.agilent.com/Library/applications/5990-6594EN.pdf8. Kotz A, Malisch R, Focant J-F, Eppe G, Cederberg T, Rantakokko P, Fürst P, Bernsmann T, Leondiadis L, Lovász C, Scortichini G, Diletti G, di Domenico A, Ingelido AM, Traag W, Smith F and Fernandes A. (2012) Organohalogen compd, 74 in press9. 2002/657/EC: Commission Decision of 12 August 2002 (OJ L 221, 17.8.2002, p. 8–36)
*Corresponding AuthorGauthier EppeE-mail: [email protected]
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Tank milk, an extra tool for detection and eradication of IBR in cattle farms
Jean-Baptiste Hanon and Brigitte CaijCODA-CERVADirection opérationnelle des maladies viralesUnité des maladies enzootiques et (ré)émergentes.
Introduction
Infectious bovine rhinotracheitis (IBR) is a respiratory disease of cattle caused by bovine herpesvirus 1 (BoHV-1). Disease outbreaks can result in severe production losses, abortion and mortality. After infection with BHV-1 the animals seroconvert and become lifelong carriers of the virus that remains latent in the peripheral sensory ganglia. After reactivation caused by stress, the virus can be re-excreted and contaminate healthy animals. That is why several European countries, including Belgium, committed themselves to implement an eradication plan consisting in the detection and gradual eradication of all seropositive animals in order to obtain entirely IBR free herds. For detection of IBR positive animals, ELISA tests can be used on serum samples as well as on individual milk samples and on tank milk samples, as authorized by European legislation (Commission Decision 2004/558/EC amended by Decision 2010/433/EU).
Most European countries that implemented an IBR control plan use milk as a matrix for IBR detection in dairy herds for both acquiring and preserving the IBR free status of a herd. This is particularly so in the neighboring countries of Belgium (Germany, France, the Netherlands). In Belgium, the Royal decree on infectious bovine rhi-notracheitis (RD of 22 November 2006 amended by the RD of 16 February 2011) provides the possibility for a dairy herd to preserve the IBR status (status I3 or I4) on the basis of 6 tank milk analyses a year (plus an annual serology on other bovine animals than dairy cows). However, no IBR ELISA kits that may be used on milk have as yet been submitted for certification by the CODA-CERVA, the national reference laboratory, and can therefore not be used for official control purposes. As a result, IBR detection in herds is now done by means of analyses on the serum obtained from blood samples taken from individual adult bovine animals.
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Serological IBR statuses and detection tests used
When a herd has been infected, vaccination is recommended to limit virus circulation and contamination of naïve animals. The vaccines used for that purpose are made from a virus strain deleted in the glycoprotein gE. Based on the difference in the gE glycoprotein between a wild virus strain and the vaccine strain, animals infected with the wild virus have anti gE antibodies (gE+) and can thus be differentiated from healthy naïve or vaccinated animals that do not have anti-gE antibodies (gE -). However, vaccinated animals possess antibodies against other glyco-proteins of BHV-1 like antibodies against the glycoprotein gB (gB+). On the contrary, healthy animals that have not been infected don’t have any IBR antibodies. Based on the differences in immunological responses of naïve, vaccinated or infected animals, ELISA tests based on the detection of antibodies against gE or gB can determine the status of an animal. Different ELISA tests are available and can be used to establish the IBR status in accord-ance with the overview table below:
Status of the Animal gE ELISA gB ELISA Indirect ELISA
IBR infected + + +
Not infected and IBR vaccinated (deleted vaccine) - + +
Not infected and not vaccinated - - -
Evaluation of the use of ELISA tests for IBR detection in milk in view of using such tests in Belgium
In 2011, CODA-CERVA performed a study which resulted in a preliminary evaluation of several ELISA tests available on the European market that could be used for IBR detection in milk. The subject of that study was to estimate the features of those tests on individual milk and on tank milk samples (sensitivity, specificity, detectability) and their compliance with the official tests performed on serum. Several partners made a contribution to this study in which were involved 92 cattle farmers from all over Belgium, the regional veterinary laboratories (ARSIA and DGZ), the milk inspection bodies (Comité du lait and MCC) and 5 ELISA kits manufacturers.
Seven ELISA kits were evaluated in this study (2 gE ELISA kits, 1 gB ELISA kit and 4 indirect ELISA kits). The conclu-sions of the study were translated into recommendations that will be useful for a subsequent official certification procedure of IBR ELISA kits for milk in view for their use within the context of the IBR control program in Belgium.
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Typical features of detection in tank milk
Tank milk consists of milk obtained from one or more milking sessions of all lactating cows of a dairy farm. Hence, the analysis of a tank milk sample for the detection of antibodies should, in principle, make it possible to detect the presence of infected adult animals in that herd, except for dry cows or cows from which the milk of which has not been collected in the tank (e.g. due to mammitis). It is therefore important to perform the milk tank analysis several times until all animals of the herd have been tested in the course of one year. The concentration of anti-bodies or immunoglobulins in milk is about 10 % of that in blood serum. Moreover, this concentration varies in the course of lactation: it is lower when milk production is at its highest level, due to a diluting effect. In addition, when IBR prevalence in a herd is low, the milk of infected animals is diluted with that of the healthy cows. It is therefore important to have sufficiently sensitive ELISA tests that allow the detection of low amounts of antibod-ies.When compared to individual serological tests, IBR detection on tank milk has the following advantages:- sampling is easy and may be done through the milk inspection services- costs are low due to the smaller number of samples to be tested - early detection of a possible contamination of an IBR free herd due to repetitive testing at regular times over the year (as opposed to the annual serological screening)
The most important disadvantage of the method is the poor stability of fresh milk samples. Appropriate logistic facilities are required for the prompt transport of the samples to the lab. Adding a preservative to the sample may possibly help to increase the sample’s lifetime. On the other hand, samples can be preserved by freezing without any problem once the milk has been skimmed.
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Conclusions
Thanks to the study performed by CODA-CERVA the different types of ELISA kits for the detection of IBR from milk samples could be characterized. The overall characteristics of the 3 categories of ELISA tests may be listed as follows:
gE ELISA gB ELISA Indirect ELISA
Individual milk Sensitivity +/- ++ +++
Specificity + to +++ +++ ++
Tank milk Sensitivity - - +++
Detectability -(>10 to 15%)
+/-(>10%)
+++(<2%)
Specificity + +++ +++
Legend: +++ excellent / ++ very good / + good / +/- mediocre / - bad
It appears that indirect ELISA kits are very efficient for detecting IBR in tank milk in non vaccinated herds and should therefore be recommended in view of the preservation of I4 IBR free statuses. The sensitivity and the de-tectability of indirect ELISA in tank milk are significantly superior to those obtained with annual serological detec-tion on a random sample of animals, as required by the Royal decree on IBR: the size of the sample for serological testing is appropriate for detecting a 15 % IBR prevalence whereas with indirect ELISA on tank milk it is possible to detect an infection level of less than 2% in cows. In addition, the repetitive testing of tank milk (every 2 months) allows earlier detection of possible re-infections of a herd when compared to a serological test that is performed only once a year.
For I3 status IBR free animals (vaccination authorized), indirect ELISA is not an appropriate method since it does not allow to distinguish vaccinated animals from infected animals. The gE ELISA kits have low sensitivity in tank milk and do not allow the detection of infected animals within a herd with low prevalence (detectability >10 to 15%). This lack of sensitivity is partly compensated for by the fact that tests on tank milk are repeated every two months. It can indeed be expected that when an IBR free herd is infected, the rate of infected cows will soon exceed 15%. Moreover, there are certain milk treating methods that make it possible to increase the immunoglob-ulin concentration of tank milk samples and improve the sensitivity and the detectability of gE ELISA kits. Thanks to these methods, higher sensitivity might be achieved than with the annual serological detection on a random sample of animals, i.e. the procedure that is currently applied in Belgium.
Under these circumstances, it is recommended to adopt as soon as possible a procedure for the official certifica-tion of indirect ELISA kits for the detection of IBR in tank milk, within the context of IBR control in Belgium. In the meantime there have already been contacts with neighboring countries that have included the use of controlled IBR ELISA kits for milk in their official control program and proposals for mutual recognition of controlled batches from other countries were formulated.
Contact: [email protected]
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Plastic materials intended to come into direct contact with food : What is the impact on laboratories of the recently adopted Regulation (EU) No 10/2011?
Els Van Hoeck, Tina N’Goy, Caroline Evrard and Fabien BolleWIV-ISP, Juliette Wytsmanstraat 14, 1050 Brussel
Packagings and materials intended to come into direct contact with food (Food contact materials, FCM) must comply with several requirements that have been laid down at the European level. Within that context, Regulation (EU) No 10/2011 on plastic materials and articles intended to come into contact with food (“PIM” in short, “Plastics implementation measure”) was published on the 14th of January 2011 [1]. This regulation replaces the following documents:• the plastic materials directive (Directive 2002/72/EC) [2] • the directive laying down the basic rules for testing migration (Directive 82/711/EEC) [3] • the directive laying down the list of simulants to be used (Directive 85/572/EEC) [4] • the directive on the use of vinyl chloride monomer [5]However, this new regulation also includes some important amendments that may have a significant impact on laboratories performing migration tests in order to check the compliance of packagings. These amendments will be discussed further in this text.
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Scope of Regulation No 10/2011
Over the past few years, an increasing number of materials intended to come into contact with food have been used. These materials not only consist of plastic materials, but of multi-layer materials that are held together by adhesives. They may also be printed or covered by a coating or used as gaskets in caps and closures that together with those caps and closures compose a set of two or more layers of diff erent types of materials. Such materials are referred to as ‘multi-layer materials and objects’, composed of diff erent materials. Each of the layers of such materials must comply with the Regulation, except when there is a functional barrier between the plastic material and the food. This regulation does not apply to ion exchange resins, rubber and silicones. A number of materials to which the regulation applies are shown below.
Directive 2002/72/EC contains diff erent lists of monomers and other starting substances as well as additives categorized according to their authorization status. These lists have been assembled into one list, the so-called ‘EU list’. Only the substances mentioned on the ‘EU list’ may be used intentionally in the manufacturing of plastic materials and objects. Are included in this list :• monomers and other starting substances, • other additives than dyes, • other aids to polymerisation than solvents, • macromolecules obtained by microbial fermentation. Adhesives, inks and varnishes do not necessarily contain the same substances as plastic materials and are there-fore still regulated by other rules of the European Union or EU Member States. Another category of substances that are not covered by the Directive are dyes, solvents and non-intentional added substances (NIAS). The use of nanoparticles is forbidden except when the risk has been assessed by the EFSA and their use has been authorized.
22
How to perform migration tests?
The migration of contaminants from packagings and other materials to foodstuff s is assessed by means of migra-tion tests. Migration can be assessed in two ways, i.e. by determining the specifi c migration or the overall migra-tion. Since it is hard to bring together the material and all foodstuff s it may come into contact with, migration testing is done by means of simulants. The way in which migration tests may be performed is shown below. Packagings may be tested using a migration cell. Plates, beakers, … are fi lled with the simulant and kitchen tools are sub-merged into the simulant.
A summary of the choice of simulants and of the amendments made by the new Regulation is given in the table below:
Type of food Simulant 2002/72/EEC EU No 10/2011
Watery (pH > 4,5) A Distilled H2O 10 % Ethanol (v/v)
Sour (pH < 4,5) B 3 % Acetic acid (m/v) 3 % Acetic acid (m/v)
Alcoholic C 15 % Ethanol (v/v) 20 % Ethanol (v/v)
Half fatty D (b) 50 % Ethanol (v/v) D1: 50 % Ethanol (v/v)
Fatty Olive oil D2: Plant oil
Dry E / MPPO (Tenax)
In addition the temperature and the duration of migration for real use circumstances, or according to the worst case scenario, are determined. These conditions have been laid down in previous directives but are now as-sembled and harmonized in the new Regulation 10/2011. A distinction is made between overall migration and specifi c migration. The new standard testing conditions for overall migration are mentioned in the table below:
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Test Expected contact with food Migration test conditions
OM1 Contact with frozen and cooled food 10 days @ 20°C
OM2Long storage at room temperature + short heat-ing
10 days @ 40°C
OM3 Short heating 2 hours @ 70°C
OM4 High temperature use 1 hour @ 100°C
OM5 High temperature use (up to 121°C)2 hours @ 100°C Or 1 hour refluxing @ 121°C
OM6Use of simulant A,B or C at a temperature of more than 40°C
4 hours @ 100°C or 4 hours refluxing
OM7 Use of fatty food at a high temperature 2 hours @ 175°C
As for specific migration, both the times and temperatures of contact have been laid down in Regulation No 10/2011. Both can be found in the table below (printed in red : changes when compared to the previous Directives).
Contact time in worst foreseeable use Contact time for migration test
t ≤ 5 min 5 min
5 min ≤ t ≤ 0,5 hour 0,5 hour
0,5 hour ≤ t ≤ 1 hour 1 hour
1 hour ≤ t ≤ 2 hours 2 hours
2 hours ≤ t ≤ 6 hours 6 hours
6 hours ≤ t ≤ 24 hours 24 hours
1 day ≤ t ≤ 3 days 3 days
3 days ≤ t ≤ 30 days 10 days
30 days ≤ t Specific conditions
Interpretation of results According to Regulation (EU) 10/2011 all results must be reported in mg/kg. There are, of course, certain exep-tions to that rule:• Materials that may be filled with a volume of 500 ml to 10 l and/or materials used for babies and young infants• > In that case the real surface/volume ratio should be taken into account. • Materials with a volume of less than 500 ml or more than 10 l and/or when calculating the surface of the material and the volume of food is not practical• > In that case the conventional surface/volume ratio of 6 dm² per kg food should be taken into account.
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Conclusion
This new Regulation, 10/2011, includes certain changes in the conditions/simulants to be used in migration test-ing for the assessment of plastic materials used for manufacturing packagings and other materials that come into direct contact with foodstuffs. A transition period had been provided for, but as of January 1st 2013, all laboratories have to use these new migration simulants and apply these new testing conditions.
References
[1] Commission Regulation No. 10/2011 on plastic materials and articles intended to come into contact with food.
[2] Council Directive 2002/72/EC relating to plastic materials and articles intended to come into contact with foodstuffs.
[3] Council Directive 82/711/EEC laying down the basic rules necessary for testing migration of the constituents of plastic materials and articles intended to come into contact with foodstuffs.
[4] Council Directive 85/572/EEC laying down the list of simulants to be used for testing migration of constitu-ents of plastic materials and articles intended to come into contact with foodstuffs.
[5] Council Directive 78/142/EEC on the approximation of the laws of the member states relating to materials and articles which contain vinyl chloride monomer and are intended to come into contact with foodstuffs.
2524
New techniques and methods developed by the Belgian NRL-GMO to identify unauthorized GMOs in the UGMMONITOR project.Fraiture Marie-Alice1,3,4, Herman Philippe1, Berben Gilbert2, Debode Frédéric2, Janssen Eric2, Taverniers Isabel3, De Loose Marc3 and Roosens Nancy1
1 WIV-ISP (Platform of Biotechnology and Molecular Biology; Biosafety and Biotechnology Unit); 2 CRA-W; 3 ILVO (Institute for Agricultural and Fisheries Research); 4 UGent (Faculty of Pharmaceutical Sciences; Laboratory of Phar-maceutical Biotechnology)
Genetically modified organisms (GMOs) are defined as organisms “in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination” (Directive 2001/18/EC). The introduction and the control of GMOs in the food and feed chains in the European market are submitted to the European legislation in order to guarantee the freedom of choice to the consumers (Reg. EC n°1829/2003 and 1830/2003). However, the enforcement of this legislation is complex for several reasons. First of all, the number and the diversity of commercialised GMOs will increase significantly in the 5 next years. Second, in addition to ge-nes conferring insect resistance or herbicide tolerance, a larger range of traits will be developed (e.g. abiotic stress tolerance, disease resistance and nutritional allegations). Third, the present commercialised GMOs are principally produced by American and European companies which have a major interest to be authorized to commercialise their products on the European market. Nevertheless, in 2015, more and more GMOs will be developed by Asian technological centres intended for the local consumption. These GM crops will be very probably not submitted to European approbation. Thus, the frequency of unauthorised GMOs (UGMs) on the European market should significantly increase by their accidental (or adventitious) presence in raw material and processed food (Stein and Rodriguez-Cerezo, 2009). In this context, efficient and innovative methods and techniques must be developed to: (i) improve UGM detection in the food/feed chain and (ii) collect data enabling their safety risk assessment and the potential risk they could pose to human health. The project entitled “UGMMONITOR” consists in the development of a platform in molecular biology and a database (biosafety information) for the detection of UGMs in food and feed. It is financed by the Federal Public Service Health, Food Chain Safety and Environment (convention RF 11/6242) (starting date: 01 May 2012) and will allow to develop high-tech methods for a better management of UGMs control. One of the main goals of this project is to provide a database of UGMs containing information useful for risk assessment and also an integrated high-tech approach to detect UGM in food/feed chain.
Hereafter, a small overview of the new methods and techniques developed by the different partners are given.
26
The Scientifi c Institute of Public Heath (WIV-ISP)
On the one hand, in order to create the “UGMMONITOR BIOSAFETY DATABASE”, a large inventory of UGMs and their characterisation will be carried out, with a particular attention on rice. To this end, information will be collec-ted in a publicly available database such as the commercialised GMOs in the next coming years, the type of food/feed that can be contaminated by UGMs, the available data to conduct a risk assessment etc. By this way, this database will represent a precious tool to provide necessary data for the enforcement of food/feed chain control in particular in case of crises.
On the other hand, according the information obtained in the database, new SYBR®Green screening methods targeting unauthorized GM-rice will be developed. In a second step, a strategy coupling DNA walking methods and nested PCR will be integrated to the SYBR®Green detection methods developed at WIV-ISP in order to prove the presence of UGMs/GMOs in a food matrix by the isolation of the transgenic-endogenic junction(s). The ob-tained amplicons will be sequenced to confi rm/infi rm the presence of transgenic crop from the analysed sample. This strategy will be adapted, fi rst, on rice as crop model, and will be evaluated for its sensitivity in regards to the problem of “low level presence”.
Instituut voor Landbouw- en Visserijonderzoek (ILVO)
A novel anchor PCR protocol with automated fl uorescent detection through capillary gel electrophoresis (CGE), developed within ILVO in the FOD-RF project GMODETEC, will be tested in this project on rice DNA. First, a selec-tion of fl anking unique sequences – that have to be known – will be made, based on a screening of the gDNA library for rice. For each selected target element, 3 diff erent “anchor primers” will be developed (primers 2 and 3 thereby nested for primer 1) and combined with diff erence restriction enzymes (and primers) in anchor PCR tests. A database of unique anchor PCR fi ngerprint patterns will be developed for a set of known GM rice events. For the optimized protocol, in a later stage the sensitivity, detection limit and specifi city will be determined.
Besides development of novel high-tech methods and technologies, existing techniques will be evaluated for their suitability for UGM detection on the one hand, and applied for GM rice as a model plant on the other hand. To these belong: generic DNA quality or integrity tests, PCR inhibition tests, PCR-based screening tests and com-bination of screening elements in a matrix model, PCR element hopping, and fi nally event-specifi c PCR analysis. A combination of these tools will be used to assess in how far the nature of an “unexplainable signal” can be explained, and possibly present UGM(s) can be detected.
2726
Centre Wallon de Recherches Agronomiques (CRA-W)
The contribution of CRA-W to the project mainly includes the assessment of new DNA analytical techniques belonging to the so-called “Next Generation Sequencing” (NGS) category in detection of a genetic modifi cation on rice as model. In recent years sequencing has indeed become more and more effi cient. It is now possible to se-quence a whole genome in a short time at an aff ordable price. The limits of the approach are more and more the handling of the so-generated big data sets. Based on the results obtained with rice as model, it will be possible to state when such techniques might be of interest within a global strategy of detection of UGMs. The technique to be applied consists in the implementation of a NGS approach but where only a fraction of the total DNA extract is sequenced after an enrichment step that selects fragments in which a piece of a known screening element is present. A transfer of some real-time PCR targets to analytical pyrosequencing where targets are sequenced in only 2 hours at a price similar to that of real-time PCR will also be attempted.
Next to that, involvement of CRA-W also includes the development of new screening elements with a special focus on transgenic animals (GM fi sh).
En nu?
Corrigendum:
In the introduction of the article “GMOseek research project (2009 - 2011) for GMO detection” published in Labinfo No. 7 of January 2012, the phrase:
“The GMOseek project (SAFEFOODERA: “Food Safety – forming a European platform for protecting consumers against health risks”) was fi nanced by the European Commission under the ERA-NET platform for protecting consumers against health risks and was running from 1/06/2009 till 31/05/2011.”
should be read as:
“The GMOseek project (SAFEFOODERA: “Food Safety – forming a European platform for protecting consumers against health risks”) was fi nanced by the Food Standards Agency (FSA, UK) and the Bundesamt für Verbraucherschutz und Lebensmiddelsicherheit (BVL, Germany) under the ERA-NET platform for protecting consumers against health risks and was running from 1/06/2009 till 31/05/2011.”
28
Screening of pesticides residues by the time of flight analyzer (ToF) : Myth or reality ?
Laure Joly & Vincent HanotWIV-ISP, Juliette Wytsmanstraat 14, 1050 Brussel
The passengers “pesticides” are requested...... at the airport desk
Over 800 pesticides are currently listed by the European Union. At present, the triple quadrupole Multiple Reac-tion Monitoring (MRM) coupled to a liquid or gas chromatography is the most commonly used analyzer for de-tecting several hundreds of molecules simultaneously. With this instrument, every potential pesticide is observed by two MRM transitions. However, the amount of pesticides to be analyzed at the same time is constantly increas-ing and unfortunately the amount of MRM transitions is not unlimited. Nevertheless, alternative solutions for triple quadrupole analyses are now under development. Among these, the most endorsed solution is undoubtedly the Time of Flight analyzer (ToF) used for “screening” prior to pesticides quantification. Moreover, it seems that this type of mass spectrometer perfectly meets the new SANCO 12495/2011 criteria [1]. Finally, with regard to the latest developments made by the engineers, it seems that it is now high time for our pesticides to ...
... go to the gate in order to embark
The time of flight analyzer (ToF) makes it possible to know the mass-to-charge ratio (m/z) of an ion based on the time (t) it needs to pass through the flight tube and reach the detector. Before entering this tube, ions are acceler-ated by a potential difference (U) of known strength. The potential energy charged in this manner corresponds to
Ep = zeU, e being the elementary charge.
Once the ions are in the tube (of length L), they evolve in an ultra vacuum condition, their potential energy is entirely converted into kinetic energy :
Ec = 1/2mv2, v being the velocity of the ion.
Considering Ep = Ec and v = L/t, it appears that :
m/z = t2 . Cste
In real terms this means that an ion with an m/z ratio equal to 100 needs twice the time to pass through the flight tube of an ion with an m/z ratio equal to 25. Now that we have acquired the theory, it is time...
2928
... to breach the sound barrier
Successive technological improvements have radically changed the ToF method.First of all, a reflectron (mirror or electrostatic reflector) allowed to compensate the differences in potential energy that may occur between ions of the same m/z ratio by a difference in path length within this reflectron, so as to allow them to arrive at the detector simultaneously. Secondly, the installation of the pusher. The electrospray source which continuously generates ions makes it haz-ardous to determine an initial reference time (t = 0) that is typical of every ion and marks the introduction of it in the tube. In order to solve this tricky problem, a “pusher” has now been added. Using an orthogonal ion accelera-tion, this device allows the ions to be packed together when entering the ToF. Moreover, this ingenious technique also allows neutrons to be kept off. Finally, an extremely low pressure (~5.10-11 bar) has to be maintained in the ToF in order to prevent all fragmentation that may occur when accelerated ions and the environment collide.
Figure 1 : Ion path in a flight tube, the reflectron compensates the differences in energy which has been acquired before entering the flight tube : the blue and red ions having the same mass arrive at the same time on the detector.
… de geluidsmuur te doorbreken
De opeenvolgende technologische vorderingen hebben de werking van de ToF radicaal veranderd. Eerst en vooral kon door het plaatsen van een reflectron (spiegel of elektrostatische reflector) het verschil in potentiële energie tussen ionen met dezelfde ratio m/z worden gecompenseerd door een verschillende trajectlengte binnen dezelfde reflectron, zodat ze samen op de detector toekomen. In tweede instantie het opstellen van de pusher. Aangezien de electrospray bron op ononderbroken wijze ionen opwekt, wordt het gevaarlijk om een referentietijd (t = 0) te bepalen eigen aan elk ion en ter aanduiding van de invoering ervan in de buis. Om dit lastig probleem op te lossen, wordt vandaag de dag een "pusher" aangewend. Door toepassing van een orthogonale acceleratie van ionen kan dit onderdeeltje de ionen aan de ingang van de ToF bijeenbundelen. Bovendien kan dit ingenieus procedé eveneens de aanwezigheid van ongeladen deeltjes vermijden. Tenslotte ziet men erop toe dat een zeer lage druk (~5.10-11 bar) in de ToF wordt aangehouden en dit om elke fragmentering te vermijden die kan ontstaan doordat versnelde ionen en de omgeving met elkaar botsen.
Pusher
Detector
Reflectron
Source
Pusher
Detector
Reflectron
Source
Figuur 1: Traject van ionen in een flight tube, de reflectron compenseert het verkregen energieverschil voordat ze de flight tube binnengaan: de blauwe en rode ionen met dezelfde massa komen op hetzelfde moment in de detector toe. Vóór de take-off…
30
Co-pilot’s journal
Resolution is the capacity of the analyzer to separate two related m/z components and is specified by R=m/δm. Resolution measure-ment for an isolated mass ion (m) consists of measuring its mass defect (δm) at half peak height.
The accuracy of the m/z measure-ment of the analyzed molecule represents the possibility to meas-ure a mass as closely as possible to the theoretical mass. This measure-ment has to be true and accurate.
In mass spectrometry, the meas-urement error (defining the ac-curacy) is expressed - either in dalton (absolute value),error (Da) = M
exp - M
th
or in parts-per-million (ppm) (rela-tive value) = | M
exp – M
th| / M
th * 106
Before the plane takes off, it is time to...…contact the control tower
The identification of pesticides is partly based, in the case of a ToF, on the molecular mass of the parent ion and not on the MRM tran-sitions parent fragment as would have been the case with a triple quadrupole. So it is crucial to measure the masses accurately when using a ToF analyzer. In order to ensure that the measured mass is as close as possible to the theoretical value, sensitivity, accuracy and resolution are optimized as follows : • At first, the working parameters of the source, the transfer para-
meters and the ToF parameters are optimized by a component with a mass similar to that of the pesticides to be analyzed.
• Secondly, an external calibration has to be realized to obtain a mass measurement that is close to the exact mass at all points of the spectrum. Such calibrations are executed daily or even weekly according to the target accuracy level.
• Finally, a continuous internal calibration will be executed : a reference component or “lock mass” and analytes are introduced in the source and this during the whole analysis process. This last phase makes it possible to break variations that might occur in time (e.g. due to temperature changes). Flight conditions seem to be optimal and we can...
…draw up our flight plan
First of all, considering the fact that there has been no mass selection in advance by the ToF analyser, all ions generated in the source are simultaneously detected : most of the detected ions are matrix ions. The obtained chromatogram for a mandarin orange matrix containing no pesticide at all is shown in figure 2. The same ions are generated in the source with a triple quadrupole, but the analyzer serves as a filter and these ions are not detected.
3130
Figure 2 : Chromatogram of a mandarin orange matrix containing no pesticide at all
Finally pesticides are identifi ed by high resolution and high mass accuracy of the ToF analyzer. The monoisotopic mass corresponding to the fi rst peak of the isotopic profi le is the mass we were looking for. This peak only takes into account the masses of the most stable isotopes (fi gure 3). In comparison, the medium ion masses are the masses we are looking for with a triple quadrupole instrument.
Figure 3 : in black the theoretical medium mass of cyprodinil and in violet its theoretical monoisotopic mass
0 2 4 6 8 10 12 14 Time (min)
220 225 230
Average mass226.2969
Monoisotopicmass
226.1344
m/z
32
After these considerations, it is time to...... take a test fl ight
When using a ToF instrument for analyses, a software program compares theoretical monoisotopic masses (Mth
) of all pesticides with the diff erent masses of the mass spectrum (M
exp). A 5 to 10 ppm tolerance is generally accepted.
Starting from a total chromatogram of a mandarin orange sample (fi gure 4a) the presence of cyprodinil is con-fi rmed with a chromatographic retention time of 8.38 min by extracting the chromatogram which corresponds with its theoretical monoisotopic mass (226.1344) and a 10 ppm tolerance (fi gure 4b).
Figure 4 : (a) chromatogram of a mandarin orange sample, (b) chromatogram of the same sample fi ltered for the mass 226.1344 (theoretical mass of cyprodinil) and a 10 ppm tolerance.
The mass spectrum linked to the retention time of 8.38 min is shown in fi gure 5a. Processing software programs subduing background noise are very useful to identify a pesticide among a very large amount of masses (fi gure 5b).
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
0
100
2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
0
1001: TOF MS ES+
BPI1.02e5
1: TOF MS ES+ 226.134 10PPM
1.42e38.38
a)
b)
(min)Time
(min)Time
3332
m/z200 400 600 800 1000
%
0
100SG20110126-9 2184 (8.421) Cm (2153:2191)
b)4.78e4
300.2084
m/z200 400 600 800 1000
%
0
100100100SG20110126-9 2184 (8.421) Cm (2152:2191-(2128:2151+2191:2217))
1.19e4a)217.1115217.1115217.1115 226.1350226.1350226.1350
SG20110126-9 2184 (8.421) Cm (2153:2191)SG20110126-9 2184 (8.421) Cm (2152:2191-(2128:2151+2191:2217))
Figure 5 : mass spectra linked to a retention time of 8.38 min without (a) and with (b) subtraction of the back-ground noise.
After this successful maiden fl ight, it is time to...… go for a long-distance fl ight
Several hundreds of pesticides are examined when screening : the combination of the known retention time/theoretical monoisotopic mass of every pesticide will be assessed. Previous phases are automatically or semi-automatically repeated several hundreds of times. However, diff erent source voltages (capillary, conical) and ToF transmission voltages cannot be individualized. As it is, contrarily to what happens when using a triple quadrupole, mean generic requirements are applied to all pesticides using a ToF. Every non optimal voltage for a certain pesticide consequently reduces its chance to reach the detector undamaged. An inadequate voltage is enough for the pesticide to be badly ionized or fragmented or even for not being transmitted. A slight calibration shift is also enough for the pesticide to be tilted out of the 10 ppm tolerance zone and not to be recovered by the software program. Such shifts occur when the intensity of the signal is too weak - the form of the peak is not well defi ned and the mass accuracy diminishes - but also when the signal is too intense - satura-tion of the detector causes a peak distortion and a mass shift occurs. A calibration loss in the course of time or a momentarily blinding of the detector caused by an extremely intense matrix peak can also result in mass accuracy problems.There is a large variety of matrices in which pesticides are detected and each of them has a specifi c impact on ionization, transmission and detection of pesticides : a drastic loss of sensitivity can be observed when using a specifi c matrix for a certain pesticide without suggesting such problems in previous analyses in other matrices.Scan velocity also has an impact on sensitivity : the lower the velocity, the higher the sensitivity of the instrument, but on the contrary the amount of points per peak decreases. This aff ects the deconvolution software and conse-quently mass accuracy.Manufacturers make every eff ort to improve the sensitivity, the resolution as well as the accuracy of the new gen-eration ToF instruments. But the critical eye of the analyst will always be necessary in order to avoid false positive and false negative results when using a ToF analyzer. False positives are not a problem as such because confi rma-tion can annul the result, on the other hand false negatives do not have a second chance. This is the end of the journey, so it is time...
34
... for a soft landing
Finally the ToF analyzer is an extremely high performing instrument based on a simple principle : measuring the trajectory time of an ion so as to know its mass. High resolution, high accuracy and high sensitivity of these instru-ments allow us to measure the monoisotopic mass of the pesticide so that it can be identified.On the other hand, results always have to be scrutinized by a critical mind as regards any risk involving pesticides in extraction and purification phases, in chromatography and detection, as well as during the processing of results by computer software. Fortunately, the most important sources of errors (false positives and false negatives) are blocked because of the various warnings in the SANCO criteria. The analyst has to prove that he can detect the detection limit (SDL, screening detection limit) of the pesticide at least in 95% of the samples. Today, at European level, several labora-tories are using the ToF technology for routine screening analyses.
Logbook
[1] SANCO 12495/2011 Method validation and quality control procedures for pesticide residues analysis in food and feed
3534
Introduction to the FP7 - QBOL research projectMartine Maes
ILVO, Instituut voor Landbouw en Visserij Onderzoek, Eenheid Plant,Burg. Van Gansberghelaan 96 bus 2, 9820 Merelbeke
QBOL (Quarantine organisms Barcode Of Life) is a three year project (2009 - 2012) funded under the 7th Frame-work Program of the European Union. QBOL seeks to generate DNA barcode information for vouchered speci-mens of quarantine pathogens and pests. The DNA sequence will form the basis of a publically available, search-able, web accessible database. The free search tool will enable rapid species level identification of quarantine pathogens and pests using a DNA barcode. Full protocols will enable anyone familiar with simple molecular techniques such as PCR to identify quarantine pests quickly and easily.
Focus on: Bacteria barcoding
The research within QBOL is structured around the different groups of quarantine organisms: bacteria, fungi, nematodes, invertebrates, phytoplasmas and viruses. The individual species to be worked on have been identified from the EU directive (2000/29/EC of 8 May 2000) and the EPPO lists of pests recommended for regulation (A1, A2 and alert lists). Dr. Martine Maes from the Institute for Agricultural and Fisheries Research (ILVO) leads the ‘Bacteria Barcoding’ group.Within this group the barcoding tasks are divided between different QBOL partners: 1. For Xanthomonas: Institute for Agricultural and Fisheries Research (ILVO), Belgium2. For Clavibacter: Laboratory of Microbiology at Gent University (UGent), Belgium3. For Ralstonia solanacearum and Xylella fastidiosa: Agroscope Changins-Wädenswil Research Station (ACW),
Switzerland.To day, about 950 bacterial strains have been studied and over 1750 sequences have been produced. An impor-tant task was to collect all relevant bacterial strains that covered the diversity of the Q-bacteria under study and their taxonomic relatives and look-alikes, epiphytes and saprophytes that can reside on the same host plants. The QBOL working collection contains Q-bacteria isolated in the different geographic regions all over the world where the diseases exist and from the different host plants. They have been retrieved from internationally recog-nized bacteria collections (the majority of the strains were obtained from the Belgian BCCM/LM-UGent collection), working collections and isolation campaigns.
36
Distribution of harmful bacteria
The most important global distribution channel for bacteria is through trade and exchange of plants and seeds. For instance, some processes such as the production of propagation mate-rial, often take place in areas of the world where these bacterial diseases are indigenous. This material is then transferred for further growth in the EU. Moreover, bacterial infection of plant material is often symptomless and escapes visual inspection upon entry in the EU. Besides plant material, insects traded for use as pollinators or biological control agents can also represent a pathway of entry for harmful bacteria.
Damage and losses
The impact of the Q- and Q-alert bacteria in the EU or EPPO regions is rather speculative. Q-control and related measures were up to now relatively successful in preventing the real spread and establishment of the pathogen. Data on damage and losses based on EPPO data sheets are given below for some important Q-bacteria and refer to those areas where these bacteria are indigenous or uncontrolled.
Inleiding tot het FP7 – QBOL onderzoeksproject
Martine Maes ILVO, Instituut voor Landbouw en Visserij Onderzoek, Eenheid Plant, Burg. Van Gansberghelaan 96 bus 2, 9820 Merelbeke
QBOL (Quarantine organisms Barcode Of Life) is een project van 3 jaar (2009 - 2012) dat wordt gefinancierd binnen het 7de kaderprogramma van de Europese Unie. Het doel van QBOL is DNA-barcode informatie aan te maken voor getuigenspecimens van quarantainepathogenen en -parasieten. De DNA-sequentie wordt de basis van een publiek toegankelijke, doorzoekbare online databank. Met dat gratis instrument zal men bij middel van de DNA-barcode snel quarantainepathogenen en –parasieten kunnen identificeren op soort niveau. Er zullen volledige protocollen worden opgesteld waarmee eenieder die vertrouwd is met eenvoudige moleculaire technieken zoals PCR snel en makkelijk quarantaineorganismen zal kunnen identificeren.
Focus op: Barcodes voor bacteriën
Het onderzoek met betrekking tot QBOL is opgebouwd rond de verschillende groepen quarantaineorganismen: bacteriën, schimmels, aaltjes, ongewervelde dieren, fytoplasma’s en virussen. De afzonderlijke soorten waarnaar onderzoek wordt gedaan werden bepaald op basis van de EU-richtlijn (2000/29/EG van 8 mei 2000) en de EPPO lijst van organismen waarvoor reglementering als Q organisme aanbevolen wordt (A1, A2 en alarmlijsten).
Dr. Martine Maes van het Instituut voor Landbouw- en Visserijonderzoek (ILVO) staat aan het hoofd van de 'Bacteria Barcoding' groep.
Binnen die groep zijn de taken in verband met barcoding verdeeld over verschillende QBOL partners:
1. Voor Xanthomonas: Instituut voor Landbouw & Visserij Onderzoek (ILVO), België
2. Voor Clavibacter: Laboratorium voor Microbiologie van de Universiteit Gent (UGent), België
3. Voor Ralstonia solanacearum en Xylella fastidiosa: Agroscope Changins-Wädenswil Research Station (ACW), Zwitserland.
Er werden totnogtoe zo’n 950 bacteriestammen bestudeerd en meer dan 1750 sequenties aangemaakt. Een belangrijk werk daarbij was het verzamelen van alle relevante bacteriestammen die de diversiteit weergaven van de bestudeerde Q-bacteriën en hun taxonomisch verwanten en dubbelgangers, epifyten en saprofyten die zich op dezelfde waardplanten kunnen bevinden.
De werkcollectie voor QBOL bestaat uit Q-bacteriën die werden geïsoleerd in de diverse geografische regio’s overal ter wereld waar de ziekten voorkomen en uit verschillende waardplanten. Zij werden bijeengebracht uit internationaal erkende bacterieverzamelingen (de meeste stammen werden verkregen uit de Belgische BCCM/LM-UGent collectie), werkcollecties en isolatiecampagnes.
Verspreiding van schadelijke bacteriën
Wereldwijd is de belangrijkste weg waarlangs bacteriën worden verspreid die van de handel in en het handelsverkeer van planten en zaaigoed. Sommige processen, zoals de productie van teeltmateriaal, vinden bijvoorbeeld plaats in streken waar die bacterieziekten inheems zijn. Het materiaal wordt dan voor opkweek overgebracht naar de EU. Bovendien gebeurt een
• Clavibacter michiganensis subspecies michiganensis: tomato is the main host of economic importance while some suscep-tible solanaceous weeds may be potential reservoirs of the pathogen. This bacterium was first described in 1910 in North America, where it presumably originated. As tomato seed is the main long-distance vector of the pathogen, seed trade facilitates the worldwide spread of the disease. Serious losses to both glasshouse and field tomato crops, either by death of young plants or disfigured fruits, have been reported. In North Carolina (USA), a 70% reduction in yield has been recorded in some years. Recent experiments carried out in France have shown a yield loss of 20-30%. Locally, transfer of contamina-ted equipment may allow transmission of the disease in and between glasshouses, fields or farms. Since the first report of the disease in the USA in 1910, C. michiganensis subsp. michi-
ganensis has spread throughout the world.
bacteriële infectie van teeltmateriaal vaak zonder symptomen waardoor ze niet wordt opgemerkt tijdens de visuele inspectie bij binnenkomst in de EU. Naast teeltmateriaal kunnen ook insecten die verhandeld worden als bestuivers of als biologische bestrijding, een weg zijn waarlangs schadelijke bacteriën binnenkomen.
Schade en verlies
De omvang van de gevolgen van Q- en Q-alarm-bacteriën in de EU of de gebieden van de EPPO kan alleen maar worden geraamd. Totnogtoe waren de bestrijding van Q organismen en de daarmee gepaard gaande maatregelen vrij succesvol in het vermijden van de grootschalige verspreiding en vestiging van de ziekteverwekker. Hierna wordt informatie weergegeven, gebaseerd op gegevens van EPPO, over schade en verliezen die worden veroorzaakt door enkele belangrijke Q-bacteriën, met vermelding van de plaatsen waar die bacteriën inheems zijn of niet worden bestreden.
Clavibacter michiganensis subspecies michiganensis: tomaat is de economisch belangrijkste waardplant terwijl een aantal gevoelige nachtschadigen mogelijke reservoirs van de ziekteverwekker kunnen zijn. Deze bacterie werd het eerst beschreven in 1910 in Noord-Amerika, waar ze vermoedelijk haar oorsprong vindt. Omdat tomatenzaad de belangrijkste lange-afstandsvector van de ziekteverwekker is, werkt de handel in zaaizaad de wereldwijde verspreiding van de ziekte in de hand. Er werden grote verliezen gemeld voor zowel kas- als vollegrondsteelten van tomaten als gevolg van ofwel het afsterven van jonge plantjes of misvormde vruchten. In Noord Carolina (USA) werd in sommige jaren een opbrengstdaling van 70% vastgesteld.
Bij recente proeven in Frankrijk werd een opbrengstdaling van 20-30% opgetekend. Op lokaal vlak kan via het gebruik van besmet materieel de ziekte worden overgebracht op andere planten in dezelfde serre of naar andere serres, percelen of bedrijven. Sinds de ziekte in 1910 voor het eerst werd gemeld in de USA heeft C. michiganensis subsp. michiganensis zich over de hele wereld verspreid.
Ralstonia solanacearum heeft een brede waaier van waardplanten maar de verschillende pathogene variëteiten (rassen) binnen de soort hebben een kleinere reeks van waardplanten. Binnen het EPPO-gebied heeft ras 3 maar weinig waardplantsoorten maar daartoe behoren wel belangrijke gewassen zoals aardappelen en tomaten en het onkruid Solanum dulcamara dat een grote rol speelt als reservoir van de bacterie langsheen waterlopen en in irrigatiesystemen. Daarnaast kunnen ook andere waardplanten worden aangetast in de warmere delen van het EPPO-gebied waar de bacterie reeds voorkomt. De grootste economische schade werd gemeld in aardappelen, tabak en tomaten in het zuidoosten van de USA en in Brazilië, Colombia, Zuid-Afrika, Indonesië en India. Soms werd een verlies van de hele tomatenoogst gemeld. Het is ook zo dat meer dan 200, vooral tropische en subtropsiche, plantensoorten gevoelig zijn voor een of meer rassen van R. solanacearum. In het stroomgebied van de Amazone in Peru is bijvoorbeeld de helft van de bananenplantages aangetast.
Xanthomonas oryzae omvat twee niet-Europese ziekteverwekkers bij rijst, de pathovars oryzae en oryzicola. Die pathovars tasten een aantal wilde of minder vaak gekweekte Poaceae aan maar ook onkruidsoorten die als locale vectoren kunnen optreden terwijl de verspreiding over grote afstanden gebeurt via besmet rijstzaad. Bacterieverwelkingsziekte wordt veroorzaakt door X. oryzae pv. oryzae en is de ernstigste ziekte die in Zuid-Oost-Azië voorkomt in rijst. Op de Filippijnen lopen de verliezen in gevoelige teelten op tot zo’n 22,5% in het regenseizoen en 7,2% in het droogseizoen. Het is bekend dat stikstofbemesting de
gevoeligheid voor de ziekte vergroot. X. oryzae pv. oryzicola veroorzaakt
3736
• Ralstonia solanacearum has an extremely wide host range, but the recognized different pathogenic varieties (races) within the species show limited host ranges. Within the EPPO region, race 3 has a limited host range but includes important crops such as potato and tomato, as well as the weed Solanum
dulcamara, which plays a major role as a reservoir for the bac-terium along water courses and irrigation ponds. Furthermore, other hosts are likely to be affected in the warmer parts of the EPPO region, where the bacterium already occurs. The greatest economic damage has been reported on potatoes, tobacco and tomatoes in southeastern USA, Brazil, Colombia, South Africa, Indonesia and India. Total loss of tomato crops has been reported. Furthermore, over 200 plant species, especially tro-pical and subtropical crops, are susceptible to one or more of the races of R. solanacearum. For instance, in the Amazon basin in Peru, about half of the banana plantations are affected.
• Xanthomonas oryzae includes two non-European rice patho-gens, the pathovars oryzae and oryzicola. These pathovars also attack a number of wild or less frequently cultivated Poaceae, but also weeds which may act as local carriers, while long-distance dispersal occurs through infected rice seeds. Bacterial leaf blight is caused by X. oryzae pv. oryzae and is the most serious disease of rice in Southeast Asia. In the Philip-pines, losses are on the order of 22.5% in wet to 7.2% in dry seasons in susceptible crops. Nitrogen fertilization is known to increase the susceptibility. X. oryzae pv. oryzicola, the cause of bacterial leaf streak, is of importance in some areas during very wet seasons and under high rates of nitrogen. Losses of 5-30% have been reported from India, but in general bacterial leaf streak is a less important disease than bacterial leaf blight. X. oryzae pv. oryzae is a dangerous pathogen. It is absent from the European rice-growing areas but can probably survive in Mediterranean countries, thus representing a serious risk for the EPPO region.
bacteriële infectie van teeltmateriaal vaak zonder symptomen waardoor ze niet wordt opgemerkt tijdens de visuele inspectie bij binnenkomst in de EU. Naast teeltmateriaal kunnen ook insecten die verhandeld worden als bestuivers of als biologische bestrijding, een weg zijn waarlangs schadelijke bacteriën binnenkomen.
Schade en verlies
De omvang van de gevolgen van Q- en Q-alarm-bacteriën in de EU of de gebieden van de EPPO kan alleen maar worden geraamd. Totnogtoe waren de bestrijding van Q organismen en de daarmee gepaard gaande maatregelen vrij succesvol in het vermijden van de grootschalige verspreiding en vestiging van de ziekteverwekker. Hierna wordt informatie weergegeven, gebaseerd op gegevens van EPPO, over schade en verliezen die worden veroorzaakt door enkele belangrijke Q-bacteriën, met vermelding van de plaatsen waar die bacteriën inheems zijn of niet worden bestreden.
Clavibacter michiganensis subspecies michiganensis: tomaat is de economisch belangrijkste waardplant terwijl een aantal gevoelige nachtschadigen mogelijke reservoirs van de ziekteverwekker kunnen zijn. Deze bacterie werd het eerst beschreven in 1910 in Noord-Amerika, waar ze vermoedelijk haar oorsprong vindt. Omdat tomatenzaad de belangrijkste lange-afstandsvector van de ziekteverwekker is, werkt de handel in zaaizaad de wereldwijde verspreiding van de ziekte in de hand. Er werden grote verliezen gemeld voor zowel kas- als vollegrondsteelten van tomaten als gevolg van ofwel het afsterven van jonge plantjes of misvormde vruchten. In Noord Carolina (USA) werd in sommige jaren een opbrengstdaling van 70% vastgesteld.
Bij recente proeven in Frankrijk werd een opbrengstdaling van 20-30% opgetekend. Op lokaal vlak kan via het gebruik van besmet materieel de ziekte worden overgebracht op andere planten in dezelfde serre of naar andere serres, percelen of bedrijven. Sinds de ziekte in 1910 voor het eerst werd gemeld in de USA heeft C. michiganensis subsp. michiganensis zich over de hele wereld verspreid.
Ralstonia solanacearum heeft een brede waaier van waardplanten maar de verschillende pathogene variëteiten (rassen) binnen de soort hebben een kleinere reeks van waardplanten. Binnen het EPPO-gebied heeft ras 3 maar weinig waardplantsoorten maar daartoe behoren wel belangrijke gewassen zoals aardappelen en tomaten en het onkruid Solanum dulcamara dat een grote rol speelt als reservoir van de bacterie langsheen waterlopen en in irrigatiesystemen. Daarnaast kunnen ook andere waardplanten worden aangetast in de warmere delen van het EPPO-gebied waar de bacterie reeds voorkomt. De grootste economische schade werd gemeld in aardappelen, tabak en tomaten in het zuidoosten van de USA en in Brazilië, Colombia, Zuid-Afrika, Indonesië en India. Soms werd een verlies van de hele tomatenoogst gemeld. Het is ook zo dat meer dan 200, vooral tropische en subtropsiche, plantensoorten gevoelig zijn voor een of meer rassen van R. solanacearum. In het stroomgebied van de Amazone in Peru is bijvoorbeeld de helft van de bananenplantages aangetast.
Xanthomonas oryzae omvat twee niet-Europese ziekteverwekkers bij rijst, de pathovars oryzae en oryzicola. Die pathovars tasten een aantal wilde of minder vaak gekweekte Poaceae aan maar ook onkruidsoorten die als locale vectoren kunnen optreden terwijl de verspreiding over grote afstanden gebeurt via besmet rijstzaad. Bacterieverwelkingsziekte wordt veroorzaakt door X. oryzae pv. oryzae en is de ernstigste ziekte die in Zuid-Oost-Azië voorkomt in rijst. Op de Filippijnen lopen de verliezen in gevoelige teelten op tot zo’n 22,5% in het regenseizoen en 7,2% in het droogseizoen. Het is bekend dat stikstofbemesting de
gevoeligheid voor de ziekte vergroot. X. oryzae pv. oryzicola veroorzaakt
38
• Cultivated strawberry is the main host of Xanthomonas fragar-
iae, the cause of angular leaf spot. It was fi rst described in 1962 in North America and probably spread from there with plan-ting material to other continents. X. fragariae causes a reduc-tion in yield, but generally the disease is not destructive. Howe-ver, heavy losses may occur in wet weather conditions and also with frequent overhead sprinkler irrigation. The disease is now already present in several strawberry producing countries within the EU and it has the potential to become established in most strawberry-growing countries. In an attempt to control its spread, X. fragariae has been listed as a quarantine disease in propagation material and plants for planting.
Barcoding of bacteria
In the Barcode of Life project, there is no program for prokaryotes. The Canadian Centre for DNA Barcoding (CCDB) mentions the specifi c events and features in the bacterial genome that can com-plicate development of barcodes for bacteria. There is no ‘universal’ bacterial or prokaryotic barcode gene appointed. Although the largest biochemical and genetic diversity on Earth exists within the prokaryotic domains Archaea and Bacteria, the natural diversifi ca-tion and hence taxonomic speciation diff ers importantly from the mechanisms that are known from Eukaryotes.
Indeed, the rate of accumulation of new mutations and the phe-nomenon of lateral gene transfer results in a relatively fast evolv-ing genome, also coupled to virulence and specialization for host plants. As a result it is diffi cult and probably impossible to fi nd a universal barcode gene for bacteria and the strategy of barcod-ing the very important biodiversity of this group of organisms is underdeveloped. One of the major diffi culties is the delineation of species. This is surely also the case for the genera Xanthomonas, Clavibacter, Pseudomonas and Ralstonia, which contain important plant pathogens.
The majority of the Q-bacteria are to be identifi ed on the pathovar or subspecies level. It is not feasible to use one single gene that is representative and reliable for identifi cation of all bacterial plant pathogens on the quarantine list. Every development needs exten-sive validation.
bladstrepenziekte en is in een aantal gebieden belangrijk in zeer natte seizoenen en bij hoge stikstofgehalten. In India werden verliezen van 5-30% gemeld maar in het algemeen is bladstrepenziekte een minder belangrijke ziekte dan de bacterieverwelkingsziekte. X. oryzae pv. oryzae is een gevaarlijke ziekteverwekker die niet voorkomt in Europese rijstteelten maar waarschijnlijk wel kan overleven in de landen rond de Middellandse Zee en dan ook een ernstig risico vormt voor het EPPO-gebied.
Gekweekte aardbeien zijn de belangrijkste waardplant van Xanthomonas fragariae, de oorzaak van de bladvlekkenziekte die gekenmerkt wordt door hoekige vlekken op de bladeren. De bacterie werd voor het eerst beschreven in 1962, in Noord-Amerika en werd waarschijnlijk van daaruit naar andere landen overgebracht via plantgoed. X. fragariae leidt tot opbrengstverlies maar vernietigt de planten meestal niet hoewel bij nat weer en frequent beregenen zware verliezen kunnen optreden. De ziekte is nu al aanwezig in een aantal aardbeiproducerende landen in de EU en kan een vaste stek verwerven in de meeste landen waar
aardbeien worden geteeld. In een poging om de verspreiding ervan tegen te gaan werd X. fragariae opgenomen in een lijst van quarantaineziekten in teeltmateriaal en plantgoed.
Barcodes bepalen voor bacteriën
Prokaryoten zijn niet opgenomen in het Barcode of Life project. Het Canadian Centre for DNA Barcoding (CCDB) vermeldt de specifieke feiten en kenmerken van het bacteriegenoom dat de ontwikkeling van barcodes voor bacteriën kan bemoeilijken. Er bestaat geen
‘universeel’ barcodegen voor bacteriën of prokaryoten. Hoewel de grootste biochemische en genetische diversiteit op aarde te vinden is in de prokaryotische domeinen van de Archaea en de bacteriën verschilt de natuurlijke diversificatie en dus ook de taxonomische vorming van nieuwe soorten in aanzienlijke mate van de mechanismen die bekend zijn bij de eurokaryoten.
De mate van accumulatie van nieuwe mutaties en het verschijnsel van de laterale genoverdracht resulteert immers in een vrij snel evoluerend genoom, eveneens gepaard gaand met virulentie en specialisering in bepaalde waardplanten. Het is daarom moeilijk en wellicht onmogelijk om een universeel barcodegen te vinden voor bacteriën en de strategie om de zeer aanzienlijke biodiversiteit van die groep van organismen in barcodes te vertalen is onvoldoende ontwikkeld. Een van de grootste moeilijkheden betreft het karakteriseren van de soorten. Dat is allicht ook het geval voor de soorten Xanthomonas, Clavibacter, Pseudomonas en Ralstonia waarin een aantal belangrijke plantenziektenverwekkers voorkomen. De meeste Q-bacteriën moeten worden geïdentificeerd op pathovar- of subsoortniveau. Het is niet mogelijk om één enkel gen te gebruiken dat representatief en betrouwbaar is voor de identificatie van alle plantpathogene bacteriën die vermeld zijn op de quarantainelijst. Voor elke ontwikkeling is een uitgebreide validatie vereist.
Symptomen van Xanthomonas axonopodis pv. dieffenbachiae
bladstrepenziekte en is in een aantal gebieden belangrijk in zeer natte seizoenen en bij hoge stikstofgehalten. In India werden verliezen van 5-30% gemeld maar in het algemeen is bladstrepenziekte een minder belangrijke ziekte dan de bacterieverwelkingsziekte. X. oryzae pv. oryzae is een gevaarlijke ziekteverwekker die niet voorkomt in Europese rijstteelten maar waarschijnlijk wel kan overleven in de landen rond de Middellandse Zee en dan ook een ernstig risico vormt voor het EPPO-gebied.
Gekweekte aardbeien zijn de belangrijkste waardplant van Xanthomonas fragariae, de oorzaak van de bladvlekkenziekte die gekenmerkt wordt door hoekige vlekken op de bladeren. De bacterie werd voor het eerst beschreven in 1962, in Noord-Amerika en werd waarschijnlijk van daaruit naar andere landen overgebracht via plantgoed. X. fragariae leidt tot opbrengstverlies maar vernietigt de planten meestal niet hoewel bij nat weer en frequent beregenen zware verliezen kunnen optreden. De ziekte is nu al aanwezig in een aantal aardbeiproducerende landen in de EU en kan een vaste stek verwerven in de meeste landen waar
aardbeien worden geteeld. In een poging om de verspreiding ervan tegen te gaan werd X. fragariae opgenomen in een lijst van quarantaineziekten in teeltmateriaal en plantgoed.
Barcodes bepalen voor bacteriën
Prokaryoten zijn niet opgenomen in het Barcode of Life project. Het Canadian Centre for DNA Barcoding (CCDB) vermeldt de specifieke feiten en kenmerken van het bacteriegenoom dat de ontwikkeling van barcodes voor bacteriën kan bemoeilijken. Er bestaat geen
‘universeel’ barcodegen voor bacteriën of prokaryoten. Hoewel de grootste biochemische en genetische diversiteit op aarde te vinden is in de prokaryotische domeinen van de Archaea en de bacteriën verschilt de natuurlijke diversificatie en dus ook de taxonomische vorming van nieuwe soorten in aanzienlijke mate van de mechanismen die bekend zijn bij de eurokaryoten.
De mate van accumulatie van nieuwe mutaties en het verschijnsel van de laterale genoverdracht resulteert immers in een vrij snel evoluerend genoom, eveneens gepaard gaand met virulentie en specialisering in bepaalde waardplanten. Het is daarom moeilijk en wellicht onmogelijk om een universeel barcodegen te vinden voor bacteriën en de strategie om de zeer aanzienlijke biodiversiteit van die groep van organismen in barcodes te vertalen is onvoldoende ontwikkeld. Een van de grootste moeilijkheden betreft het karakteriseren van de soorten. Dat is allicht ook het geval voor de soorten Xanthomonas, Clavibacter, Pseudomonas en Ralstonia waarin een aantal belangrijke plantenziektenverwekkers voorkomen. De meeste Q-bacteriën moeten worden geïdentificeerd op pathovar- of subsoortniveau. Het is niet mogelijk om één enkel gen te gebruiken dat representatief en betrouwbaar is voor de identificatie van alle plantpathogene bacteriën die vermeld zijn op de quarantainelijst. Voor elke ontwikkeling is een uitgebreide validatie vereist.
Symptomen van Xanthomonas axonopodis pv. dieffenbachiae
3938
bladstrepenziekte en is in een aantal gebieden belangrijk in zeer natte seizoenen en bij hoge stikstofgehalten. In India werden verliezen van 5-30% gemeld maar in het algemeen is bladstrepenziekte een minder belangrijke ziekte dan de bacterieverwelkingsziekte. X. oryzae pv. oryzae is een gevaarlijke ziekteverwekker die niet voorkomt in Europese rijstteelten maar waarschijnlijk wel kan overleven in de landen rond de Middellandse Zee en dan ook een ernstig risico vormt voor het EPPO-gebied.
Gekweekte aardbeien zijn de belangrijkste waardplant van Xanthomonas fragariae, de oorzaak van de bladvlekkenziekte die gekenmerkt wordt door hoekige vlekken op de bladeren. De bacterie werd voor het eerst beschreven in 1962, in Noord-Amerika en werd waarschijnlijk van daaruit naar andere landen overgebracht via plantgoed. X. fragariae leidt tot opbrengstverlies maar vernietigt de planten meestal niet hoewel bij nat weer en frequent beregenen zware verliezen kunnen optreden. De ziekte is nu al aanwezig in een aantal aardbeiproducerende landen in de EU en kan een vaste stek verwerven in de meeste landen waar
aardbeien worden geteeld. In een poging om de verspreiding ervan tegen te gaan werd X. fragariae opgenomen in een lijst van quarantaineziekten in teeltmateriaal en plantgoed.
Barcodes bepalen voor bacteriën
Prokaryoten zijn niet opgenomen in het Barcode of Life project. Het Canadian Centre for DNA Barcoding (CCDB) vermeldt de specifieke feiten en kenmerken van het bacteriegenoom dat de ontwikkeling van barcodes voor bacteriën kan bemoeilijken. Er bestaat geen
‘universeel’ barcodegen voor bacteriën of prokaryoten. Hoewel de grootste biochemische en genetische diversiteit op aarde te vinden is in de prokaryotische domeinen van de Archaea en de bacteriën verschilt de natuurlijke diversificatie en dus ook de taxonomische vorming van nieuwe soorten in aanzienlijke mate van de mechanismen die bekend zijn bij de eurokaryoten.
De mate van accumulatie van nieuwe mutaties en het verschijnsel van de laterale genoverdracht resulteert immers in een vrij snel evoluerend genoom, eveneens gepaard gaand met virulentie en specialisering in bepaalde waardplanten. Het is daarom moeilijk en wellicht onmogelijk om een universeel barcodegen te vinden voor bacteriën en de strategie om de zeer aanzienlijke biodiversiteit van die groep van organismen in barcodes te vertalen is onvoldoende ontwikkeld. Een van de grootste moeilijkheden betreft het karakteriseren van de soorten. Dat is allicht ook het geval voor de soorten Xanthomonas, Clavibacter, Pseudomonas en Ralstonia waarin een aantal belangrijke plantenziektenverwekkers voorkomen. De meeste Q-bacteriën moeten worden geïdentificeerd op pathovar- of subsoortniveau. Het is niet mogelijk om één enkel gen te gebruiken dat representatief en betrouwbaar is voor de identificatie van alle plantpathogene bacteriën die vermeld zijn op de quarantainelijst. Voor elke ontwikkeling is een uitgebreide validatie vereist.
Symptomen van Xanthomonas axonopodis pv. dieffenbachiae
Symptoms of Xanthomonas axonopodis pv. dieff enbachiae
40
Barcoding strategy
For the different target groups prioritized in this study, the barcode identification strategy follows a decision scheme. • 16S rRNA gene sequencing is proposed as the first exploratory test in case there is no preliminary idea on the
type (genus) of bacterium that has been isolated from a plant commodity. • In the next decision step, we use one core gene locus to discriminate bacteria within the genus. The result can
eventually be confirmed with a second barcode.• To further discriminate at the pathovar or phylotype level, extra barcodes located in other core or accessory
gene regions are being identified.
This forms the basis for identification of quarantine bacteria and hence the whole concept of phytosanitary risk assessment.
Strategie in verband met barcoding
De barcode-identificatiestrategie voor de verschillende in deze studie geprioriteerde doelgroepen verloopt volgens een beslissingsschema. Als eerste verkennende proef wordt de sequenering
van het 16S rRNA gen voorgesteld als men vooraf geen idee heeft van het type (genus) bacterie dat uit een plantaardig product werd geïsoleerd.
In de volgende stap gebruiken wij één universele genlocus om bacteriën binnen het genus van elkaar te onderscheiden. Dat resultaat kan worden bevestigd bij
middel van een tweede barcode. Om een nog verder doorgedreven onderscheid te maken op het niveau van
de pathovar of het fylotype worden extra barcodes in andere kern- of bijgebieden van het gen geïdentificeerd.
Dat is de basis voor de identificatie van quarantainebacteriën en bijgevolg van het hele concept van de fytosanitaire risicobeoordeling.
Strategie in verband met barcoding
De barcode-identificatiestrategie voor de verschillende in deze studie geprioriteerde doelgroepen verloopt volgens een beslissingsschema. Als eerste verkennende proef wordt de sequenering
van het 16S rRNA gen voorgesteld als men vooraf geen idee heeft van het type (genus) bacterie dat uit een plantaardig product werd geïsoleerd.
In de volgende stap gebruiken wij één universele genlocus om bacteriën binnen het genus van elkaar te onderscheiden. Dat resultaat kan worden bevestigd bij
middel van een tweede barcode. Om een nog verder doorgedreven onderscheid te maken op het niveau van
de pathovar of het fylotype worden extra barcodes in andere kern- of bijgebieden van het gen geïdentificeerd.
Dat is de basis voor de identificatie van quarantainebacteriën en bijgevolg van het hele concept van de fytosanitaire risicobeoordeling.
Strategie in verband met barcoding
De barcode-identificatiestrategie voor de verschillende in deze studie geprioriteerde doelgroepen verloopt volgens een beslissingsschema. Als eerste verkennende proef wordt de sequenering
van het 16S rRNA gen voorgesteld als men vooraf geen idee heeft van het type (genus) bacterie dat uit een plantaardig product werd geïsoleerd.
In de volgende stap gebruiken wij één universele genlocus om bacteriën binnen het genus van elkaar te onderscheiden. Dat resultaat kan worden bevestigd bij
middel van een tweede barcode. Om een nog verder doorgedreven onderscheid te maken op het niveau van
de pathovar of het fylotype worden extra barcodes in andere kern- of bijgebieden van het gen geïdentificeerd.
Dat is de basis voor de identificatie van quarantainebacteriën en bijgevolg van het hele concept van de fytosanitaire risicobeoordeling.
4140
Prioritized list of Q-bacteria for barcoding
The combined EPPO/EU (EU-Directive 2000/29/EC) list actually contains 27 Q and Q-alert bacteria (phytoplasmas and ‘candidatus’ bacteria not included). Several listed Q-bacteria are now known to be heterogenic and many are also named as pathovars, which is a special purpose classifi cation on the basis of a pathological feature that does not have a taxonomic relevance.
Within QBOL, a fi rst group of Q-bacteria are targeted; a set of Xanthomonas species and pathovars, 3 Clavibacter michiganensis subspecies, Ralstonia solan-
acearum and Xylella fastidiosa. This selection has been made taking into account the Q-relevance and the expected variability and complexity within the taxa.
Ralstonia solanacearum
Lijst van prioritaire Q-bacteriën voor barcoding
Op de gecombineerde lijst van de EPPO en de EU (EU-Richtlijn 2000/29/EG) staan thans 27 Q- en Q-alert-bacteriën (fytoplasma’s en ‘candidatus’ bacteriën niet inbegrepen). Men weet intussen dat een aantal in de lijst vermelde Q-bacteriën heterogenetisch zijn en dat vele ook als pathovars worden vermeld. Die indeling beoogt een bijzonder doel en steunt op een pathologisch kenmerk dat taxonomisch irrelevant is.
Binnen QBOL werd een eerste doelgroep van Q-bacteriën bepaald; het gaat om een aantal Xanthomonas soorten en pathovars, 3 Clavibacter michiganensis subsoorten, Ralstonia solanacearum en Xylella fastidiosa. Bij die keuze werd rekening gehouden met de Q-relevantie en de verwachte rendabiliteit alsook met de complexiteit binnen de taxa.
Ralstonia solanacearum
De lijst van prioritaire bacteriën voor barcoding conform de wetgeving betreffende planten van de EU en de EPPO.
Bijlage 2000/29/EC EPPO
Xylella fastidiosa I A A1
Clavibacter michiganensis subsp. sepedonicus I A A2
Pseudomonas solanacearum = Ralstonia solanacearum I A A2
Xanthomonas stammen die pathogen zijn voor citrusvruchten
II A A1
Xanthomonas oryzae pv. oryzae & oryzicola II A A1
Clavibacter michiganensis subsp. insidiosus II A A2
Clavibacter michiganensis subsp. michiganensis II A A2
Xanthomonas campestris (axonopodis) pv. phaseoli II A A2
Xanthomonas vesicatoria &
X. campestris (axonopodis) pv. vesicatoria
II A A2
Xanthomonas axonopodis pv. dieffenbachiae A2
Xanthomonas axonopodis pv. allii A1
Xanthomonas fragariae II A A2
Xanthomonas translucens pv. translucens A2
- Xylella fastidiosa is een belangrijke ziekteverwekker bij planten; veroorzaakt dwerggroei bij perzik, brandvlekkenziekte bij oleander, Pierce’s disease en bonte chlorose bij citrusvruchten (CVC).
- Clavibacter michiganensis subsp. sepedonicus veroorzaakt een aardappelziekte die bekend staat als ringrot vanwege de manier waarop ze het vaatweefsel in aardappelknollen infecteert.
- Ralstonia solanacearum veroorzaakt bacteriële verwelking bij een groot aantal mogelijke waardplanten zoals aardappel, tomaat, peper, aubergine en Pelargonium.
Lijst van prioritaire Q-bacteriën voor barcoding
Op de gecombineerde lijst van de EPPO en de EU (EU-Richtlijn 2000/29/EG) staan thans 27 Q- en Q-alert-bacteriën (fytoplasma’s en ‘candidatus’ bacteriën niet inbegrepen). Men weet intussen dat een aantal in de lijst vermelde Q-bacteriën heterogenetisch zijn en dat vele ook als pathovars worden vermeld. Die indeling beoogt een bijzonder doel en steunt op een pathologisch kenmerk dat taxonomisch irrelevant is.
Binnen QBOL werd een eerste doelgroep van Q-bacteriën bepaald; het gaat om een aantal Xanthomonas soorten en pathovars, 3 Clavibacter michiganensis subsoorten, Ralstonia solanacearum en Xylella fastidiosa. Bij die keuze werd rekening gehouden met de Q-relevantie en de verwachte rendabiliteit alsook met de complexiteit binnen de taxa.
Ralstonia solanacearum
De lijst van prioritaire bacteriën voor barcoding conform de wetgeving betreffende planten van de EU en de EPPO.
Bijlage 2000/29/EC EPPO
Xylella fastidiosa I A A1
Clavibacter michiganensis subsp. sepedonicus I A A2
Pseudomonas solanacearum = Ralstonia solanacearum I A A2
Xanthomonas stammen die pathogen zijn voor citrusvruchten
II A A1
Xanthomonas oryzae pv. oryzae & oryzicola II A A1
Clavibacter michiganensis subsp. insidiosus II A A2
Clavibacter michiganensis subsp. michiganensis II A A2
Xanthomonas campestris (axonopodis) pv. phaseoli II A A2
Xanthomonas vesicatoria &
X. campestris (axonopodis) pv. vesicatoria
II A A2
Xanthomonas axonopodis pv. dieffenbachiae A2
Xanthomonas axonopodis pv. allii A1
Xanthomonas fragariae II A A2
Xanthomonas translucens pv. translucens A2
- Xylella fastidiosa is een belangrijke ziekteverwekker bij planten; veroorzaakt dwerggroei bij perzik, brandvlekkenziekte bij oleander, Pierce’s disease en bonte chlorose bij citrusvruchten (CVC).
- Clavibacter michiganensis subsp. sepedonicus veroorzaakt een aardappelziekte die bekend staat als ringrot vanwege de manier waarop ze het vaatweefsel in aardappelknollen infecteert.
- Ralstonia solanacearum veroorzaakt bacteriële verwelking bij een groot aantal mogelijke waardplanten zoals aardappel, tomaat, peper, aubergine en Pelargonium.
The prioritized bacteria for barcoding, listed according to the EU plant legislation and EPPO.
2000/29/EC Annex EPPO
Xylella fastidiosa I A A1
Clavibacter michiganensis subsp. sepedonicus I A A2
Pseudomonas solanacearum = Ralstonia solanacearum I A A2
Xanthomonas strains pathogenic to Citrus II A A1
Xanthomonas oryzae pv. oryzae & oryzicola II A A1
Clavibacter michiganensis subsp. insidiosus II A A2
Clavibacter michiganensis subsp. michiganensis II A A2
Xanthomonas campestris (axonopodis) pv. phaseoli II A A2
Xanthomonas vesicatoria &
X. campestris (axonopodis) pv. vesicatoriaII A A2
Xanthomonas axonopodis pv. dieff enbachiae A2
Xanthomonas axonopodis pv. allii A1
Xanthomonas fragariae II A A2
Xanthomonas translucens pv. translucens A2
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- Xylella fastidiosa is an important plant pathogen that causes phoney peach disease, oleander leaf scorch, and Pierce’s disease, and citrus variegated chlorosis disease (CVC).
- Clavibacter michiganensis subsp. sepedonicus causes a disease in potatoes known as ‘ring rot’ due to the way it infects vascular tissue inside potato tubers.
- Ralstonia solanacearum causes bacterial wilt in a very wide range of potential host plants, e.g. potato, tomato, pepper, eggplant and Pelargonium.
- Xanthomonas axonopodis and Xanthomonas citri have different pathovars and variants that cause citrus canker types.
- The major host of Xanthomonas oryzae is rice. The species contains two non-European pathovars: Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola.
- Clavibacter michiganensis subsp. insidiosus infects alfalfa.- Clavibacter michiganensis subsp. michiganensis causes bacterial wilt in tomatoes.- Xanthomonas axonopodis pv. phaseoli is the causal agent of common bacterial blight of beans.- The principal hosts of Xanthomonas vesicatoria & X. axonopodis pv. vesicatoria are tomatoes and Capsicum.- Xanthomonas axonopodis pv. dieffenbachiae is pathogenic on Aroids and is most aggressive on Anthurium.- Xanthomonas axonopodis pv. allii damages Allium crops.- Xanthomonas fragariae causes angular leaf spot in strawberry.- Xanthomonas translucens pv. translucens is the causal agent of bacterial leaf streak of small grain cereals.
The Q-bank database contains all relevant data that have been produced in QBOL for correct barcode-identifica-tion of the plant pathogenic quarantine organisms.
http://www.q-bank.eu/
Contact: Martine Maes ([email protected])
- Xanthomonas axonopodis en Xanthomonas citri hebben verschillende pathovars en varianten die kanker veroorzaken bij citrusplanten.
- Rijst is de belangrijkste waardplant van Xanthomonas oryzae. De soort heeft twee niet-Europese pathovars: Xanthomonas oryzae pv. oryzae en Xanthomonas oryzae pv. oryzicola.
- Clavibacter michiganensis subsp. insidiosus besmet alfalfa.
- Clavibacter michiganensis subsp. michiganensis veroorzaakt bacteriële verwelking bij tomaten.
- Xanthomonas axonopodis pv. phaseoli is de verwekker van bacteriebrand bij bonen.
- De belangrijkste waardplanten van Xanthomonas vesicatoria & X. axonopodis pv. vesicatoria zijn tomaten en Capsicum.
- Xanthomonas axonopodis pv. dieffenbachiae is pathogeen voor araceeën en is het agressiefst op Anthurium.
- Xanthomonas axonopodis pv. allii veroorzaakt schade in Allium teelten.
- Xanthomonas fragariae veroorzaakt bladvlekkenziekte bij aardbei.
- Xanthomonas translucens pv. translucens is de verwekker van bacteriële bladstrepenziekte in kleinkorrelige graansoorten.
De database Q-bank bevat alle relevante gegevens die in QBOL werden aangemaakt met het oog op een correcte barcode-identificatie van plantpathogene quarantaineorganismen.
http://www.q-bank.eu/
Contact:
Martine Maes ([email protected])
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The trainings for the approved laboratories organized by the FASFC in co-operation with the National Reference Laboratories are available on the website of the FASFC
(www.favv.be > Business Sectors > Laboratories > Trainings).
The schedule is updated regularly, it is therefore recommended to check the website from time to time.
Other interesting workshops and symposia are mentioned below.
Workshops & SymposiaDate Subject Place More information (website)
21-23.01.2013 8th Conference RME 2013: Food Feed Water Analysis: innovations and break-throughs!
Noordwijkerhout, The Netherlands
http://www.feedsafety.org/
28.01-1.02.2013 Advanced Food Analysis Wageningen, The Netherlands
http://www.vlaggraduateschool.nl/courses/food-analys.htm
20-21.02.2013 11th International Fresenius ConferenceFood Safety and Dietary Risk Assess-ment
Mainz/Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=5&kurs=350
26.02-1.03.2013 4th MoniQA International Conference Budapest, Hungary http://budapest2013.moniqa.org
4.04.2013 ‘Waarborgen van voedselveiligheid: hoe gerust kun je zijn?’
Wageningen, The Netherlands
http://www.fimm.nl/symposium-microbio-logie/symposium-levensmiddelenmicrobi-ologie/
18-19.04.2013 New Trends on Methods for Pesticides and Drug Residues
Paris, France AOACI Europe section with ASFILAB www.aoaceurope.com
22-23.04.2013 8th International Fresenius Conference“Contaminants and Residues in Food”
Mainz/Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=5&kurs=354
7-10.05.2013 EuroFoodChem XVII Istanbul, Turkey http://www.arber.com.tr/eurofoodchemxvii.org/index.php/homewith state-of-the-art knowledge and appli-cations in food chemistry and complemen-tary disciplines
22-24.05.2013 35th Mycotoxin Workshop Ghent, Belgium http://en.mytox.be/conferences/
23-24.05.2013 Workshop EU Ref Lab for Parasites Rome, Italy http://www.iss.it/crlp/index.php
3-7.06.2013 ISO/IDF Analytical Week Rotterdam, Nederland
24-25.06.2013 15th International Fresenius AGRO ConferenceBehaviour of Pesticides in Air, Soil and Water
Mainz/Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=5&kurs=346
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25-30.08.2013 The 33rd International Symposium on Halogenated Persistent Organic Pollutants (POP’s) - DIOXIN 2012
Daegu, Korea http://www.dioxin2013.org/
25.08-28.08.2013 127th AOAC Annual Meeting & Exposition
Chicago, USA http://www.aoac.org
25-29.08.2013 24th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP)
Perth Exhibition Centre, Perth, Western Australia
http://www.waavp.org/
12-13.09.2013 18th Conference on Food Microbiology Brussels, Belgium www.bsfm.be
17.09.2013 Trends in Food Analysis VII Gent, Belgium
18.10.2013 5th symposium BWDS: Spatial Approach of Wildlife Diseases
Tervuren, Belgium http://www.bwds.be
24.10.2013 ‘Methoden in de levensmiddelen- microbiologie’
The Netherlands http://www.fimm.nl/symposium- microbiologie/microbiologische-methoden- onderzoek/
28.10-1.11.2013 IDF World Dairy Summit Yokohama, Japan http://www.fil-idf.org/Public/SiteEventType.php?ID=23123
5-8.11.2013 6th International Symposium on RECENT ADVANCES IN FOOD ANALYSISRAFA 2013
Prague, Czech Republic
http://www.rafa2013.eu/RAFA_2013_flyer_ 1.pdf
28-31.01.2014 HTC-13: International Symposium on Hyphenated Techniques in Chroma-tography and Hyphenated Chromato-graphic Analyzers
Brugge, Belgium http://www.kvcv.be/kalender/
26-30.10.2014 IDF World Dairy Summit Tel Aviv, Israel http://www.idfwds2014.com/
4544
25-30.08.2013 The 33rd International Symposium on Halogenated Persistent Organic Pollutants (POP’s) - DIOXIN 2012
Daegu, Korea http://www.dioxin2013.org/
25.08-28.08.2013 127th AOAC Annual Meeting & Exposition
Chicago, USA http://www.aoac.org
25-29.08.2013 24th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP)
Perth Exhibition Centre, Perth, Western Australia
http://www.waavp.org/
12-13.09.2013 18th Conference on Food Microbiology Brussels, Belgium www.bsfm.be
17.09.2013 Trends in Food Analysis VII Gent, Belgium
18.10.2013 5th symposium BWDS: Spatial Approach of Wildlife Diseases
Tervuren, Belgium http://www.bwds.be
24.10.2013 ‘Methoden in de levensmiddelen- microbiologie’
The Netherlands http://www.fimm.nl/symposium- microbiologie/microbiologische-methoden- onderzoek/
28.10-1.11.2013 IDF World Dairy Summit Yokohama, Japan http://www.fil-idf.org/Public/SiteEventType.php?ID=23123
5-8.11.2013 6th International Symposium on RECENT ADVANCES IN FOOD ANALYSISRAFA 2013
Prague, Czech Republic
http://www.rafa2013.eu/RAFA_2013_flyer_ 1.pdf
28-31.01.2014 HTC-13: International Symposium on Hyphenated Techniques in Chroma-tography and Hyphenated Chromato-graphic Analyzers
Brugge, Belgium http://www.kvcv.be/kalender/
26-30.10.2014 IDF World Dairy Summit Tel Aviv, Israel http://www.idfwds2014.com/
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