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Validation studies of Real Time Validation studies of Real Time PCR protocols for PCR protocols for
Salmonella and Salmonella and ListeriaListeriaSalmonella and Salmonella and ListeriaListeriamonocytogenesmonocytogenes
Monica Gianfranceschi, David Rodriguiez-Lazaro,
Elisabetta Delibato, Antonietta Gattuso, Damiano Comin
and Dario De Medici,
Components of a microbiological criterion (Codex)
• Micro-organism of concern
• Analytical method
• Sampling plan
• Microbiological limits
• The food-stuff
Task 7.3 and Task
7.5
Task 7.4 and Task
2
• The food-stuff
• The point of the food chain
where the limit applies
• Actions to be taken when
unsatisfactory
• Results
Task 7.4 and Task
7.5
3
Background
Testing against criteria
Food Business Operators (Reg 2073/2005)For validation and verification of procedures
based on HACCP and GHP
For batch acceptability testing during the storage life
4
For batch acceptability testing during the storage life
(PO)
Competent Authorities (Reg 882/2004 onofficial controls)To verify that food is in compliance with the Community
criteria during the entire shelf life (FSO)
Classical Cultural Methods
• No expensive infrastructure • Cheap in consumables• Cheap equipments
5
• Laborious to perform• Large volumes of materials• Time consuming
• rapid methods replaced “alternative methods”.
• Rapid =
– a shorter time to detection
– better flow through
– automation
Alternative method or rapid methods
6
– automation
Based on “Golden standard”:
a “rapid method” can be defined as any method or system that
reduces the time taken to obtain a microbiological test result
(Feng, 1996; Fung, 1994)
Rapid (Alternative) methods
Advantages for Food Business Operators:• Rapid corrective measures and actions • Possible to verify the efficency of the GHP and HACCP
Results available more rapidly than by
using classical cultural methods
7
Advantages for Competent Authorities:• More rapid management of emergency situation and crisis• Evaluate microbiological hazard prevalence/concentration
in a specific commodity (large monitoring), in order to evaluate the risk for the population
The use of alternative analytical methods is
acceptable when the methods are:
• validated against the reference method
Alternative method in Regulation EC 2073/2005 on microbiological criteria
8
• if a proprietary method, certified by a third
party in accordance with the protocol set out
in EN/ISO standard 16140 or other
internationally accepted protocols, is used.
Proprietary Methods
Validation bodies
• AFNOR (Association Français de
Normalisation, France)
• NordVal (part of the Nordic Committee
on Food Analysis, Norway)
• MicroVal (European Validation and
Certification Organisation, Europe)
9
Certification Organisation, Europe)
• AOAC (Association of Official Analytical
Chemist, USA)
Validation certificates of AFNOR, NordVal and MicroVal are based on ISO 16140
• AOAC ~ ISO 16140
ISO for molecular methods
Standard reference Title Status
EN ISO 20837:2006Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-
borne pathogens - Requirements for sample preparation for qualitative detection published
EN ISO 20838:2006Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-
borne pathogens - Requirements for amplification and detection for qualitative methods published
EN ISO 22118:2011Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection and
quantification of food-borne pathogens - Performance characteristics published
EN ISO 22119:2011Microbiology of food and animal feeding stuffs - Real-time polymerase chain reaction (PCR) for the detection
of food-borne pathogens - General requirements and definitions published
EN ISO 22174:2005Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-
publishedEN ISO 22174:2005Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-
borne pathogens - General requirements and definitions published
CEN ISO/TS 13136:2012
Microbiology of food and animal feed - Real-time polymerase chain reaction (PCR)-based method for the
detection of food-borne pathogens - Horizontal method for the detection of Shiga toxin-producing
Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups
published
ISO/TS 15216-1Microbiology of food and animal feeding stuffs- Horizontal method for detection of hepatitis A virus and
norovirus in food using real-time RT-PCR- Part 1: Method for quantitative determinationpublished
ISO/TS 15216 -2Microbiology of food and animal feeding stuffs- Horizontal method for detection of hepatitis A virus and
norovirus in food using real-time RT-PCR- Part 2: Method for qualitative determinationpublished
ISO/PDTS WI 00275221
“Microbiology of food, feeding stuffs and environmental samples — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — Detection of botulinum type A, B, E and F neurotoxin producing
clostridia”
draft
“General requirements relating to molecular methods for detection and quantification of
microorganisms” - Draft standard of PCR detection of enteropathogenic Yersinia spp.in discussion
Microbiology of food and animal feeding stuffs - Detection of Vibrio parahaemolyticus in seafoods: Part 1 -
Quantitative determination of total, thermostable direct haemolysin (TDH) and thermostable direct-related
haemolysin (TRH) positive Vibrio parahaemolyticus using nucleic acid hybridisation
in discussion
Revision of EN ISO 16140
PG 1: Terminology
PG 2: Proprietary methods
PG 3: Intermediate validation
PG 4: Method verification
11
PG 5: In house method validation
PG 6: Technical requirements for the
establishment/revision of standardised
methods
Revision of EN ISO 16140
PG 1: Terminology
PG 2: Proprietary methods
PG 3: Intermediate validation
PG 4: Method verification
12
PG 5: In house method validation
PG 6: Technical requirements for the
establishment/revision of standardised
methods
Intermediate validation (non proprietary methods)
First Draft text EN ISO 16140-1 - Terminology of Method Validation”
Intermediate validation
A validation study including an inter-laboratory study with a limited
number of participating laboratories
NOTE
13
NOTE
This type of validation study is restricted to non-proprietary methods. It is
applicable when the method of analysis deals with a complex
methodology or with an analyte for which only a restricted number of
laboratories have the competence and the infrastructure to participate in
a validation study.
Since the validation process has high costs, the
laboratories involved should be preferably reduced
VALIDATION STRATEGYPRE-VALIDATION EVALUATION
Objective: Define the best conditions for alternative tests compatible with ISO standards for detection of Listeria monocytogenes and Salmonella spp.
Prerequisites: Prerequisites:
• Rapid protocol (next-day results)
• As easy and simple as possible (easy to be implemented in a routine lab)
• Compatible with ISO standard (i.e. the ISO standard must be continued
once the results are obtained by the alternative methods)
• Not too expensive (similar price as standard)
Approach:
• Use the same enrichment broths (Half-Fraser for L. monocytogenes,
BPW for Salmonella spp.)
• Evaluate several strategies for reduction of time for final
Validation Strategy:Pre-Validation Evaluation for Listeria and Salmonella
results:
� Reduce the time of incubation
� Use of easy and simple bacterial DNA extraction protocols
� Use real-time PCR assays for detection
Parameters to evaluate:
� Size of sample (25 g or 50 g)
� Dilution of the sample (1:3, 1:5 or 1:10)
Validation Strategy Pre-ValidationEvaluation for Listeria and Salmonella
� Dilution of the sample (1:3, 1:5 or 1:10)
� Incubation time (i.e. 5, 8-10 or 18-24 hours of (pre)-enrichment)
� Bacterial DNA extraction protocol (boiling, resin protocol, or commercial
column-based kit)
Listeria monoctyogenes1-10 ufc
10-100 ufc
(100-1000 ufc)
25 g
50 g
ISO
Dilutions: 1:3
1:5
Listeria
monoctyogenes
VALIDATION STRATEGYEVALUATION APPROACH
50 g1:5
1:10
Incubation:
8 h
18 h
24 hDNA extraction:
Boiling
Chelex
Column
monoctyogenes
DilutionIncubation
timesISO
Baseline
QIAGEN
Baseline
CHELEX
Baseline
BOILING
1 cfu /
sample1:3
8 h 3/3 1/3 0/3 0/3
18 h 3/3 1/3 0/3 0/3
24 h 3/3 3/3 1/3 0/3
VALIDATION STRATEGYPRE-VALIDATION EVALUATION RESULTS
1:5
8 h 3/3 0/3 0/3 0/3
18 h 3/3 2/3 0/3 0/3
24 h 3/3 2/3 1/3 0/3
1:10
8 h 3/3 0/3 0/3 0/3
18 h 3/3 0/3 1/3 0/3
24 h 3/3 3/3 0/3 0/3✔
1:10 Half Fraser
(ISO)
Incubation 24 h
Validation StrategyFinal protocol: Listeria
monocytogenes in Cheese
Listeria monoctyogenes
DNA extraction: QIAGEN column
Real-time PCRRodríguez Lázaro et al (2004) Appl. Environ. Microbiol.
ISS
Strains
1-10
10-
100
3.4 cfu/g
0.34 cfu/g
5 hours1°
detection
18 hours
3 a.m. 2°
detection
3 hours 3°
detectionAfter 24 hours
ISO method Real-Time PCR
CFU
L. monocytogenes/samplesDilution Rate*
pre-
enrichment
(HFB)
hours
Numbers of
replicates
Positive
samples
Positive
samples
hly system
(FAM)
Ct±SD
IAC system
(HEX)
Ct±SD
1-10 CFU
(0.34 CFU/g)
25g + 50mL
5h 5 2 0 - 32.92±1.25
8h 5 5 0 - 33.00±0.48
24h 5 5 5 35.91±0.67 32.61±1.46
25g + 100mL
5h 5 2 0 - 32.55±0.79
8h 5 5 0 - 32.12±0.90
24h 5 5 5 29.59±0.66 32.89±1.10
25g + 225mL
5h 5 2 0 - 32.86±1.10
8h 5 5 0 - 33.51±0.5825g + 225mL 8h 5 5 0 - 33.51±0.58
24h 5 5 5 29.16±0.91 32,17±0.64
10-100 CFU
(3.4 CFU/g)
25g + 50mL
5h 5 2 0 - 33.28±0.84
8h 5 5 0 - 33.63±0.71
24h 5 5 5 31.26±0.31 32,70±1.83
25g + 100mL
5h 5 2 0 - 33.30±0.42
8h 5 5 0 - 33.32±0.70
24h 5 5 5 26.61±0.37 32,46±1.43
25g + 225mL
5h 5 2 0 - 33.10±0.63
8h 5 5 0 - 33.62±1.03
24h 5 5 5 25.38±0.16 32.86±1.40
Reduction of volume for expensive media order to reduce cost for analysis
No differences were found when we use 100 or 225 ml of Half Frazer with
25 g of sample after 24 hours of pre-enrichment in Real Time PCR
Real Time PCR positive after 24 hours pre-emrichment in HF
After 5 and 8 hours of pre-enrichment in HF all the samples
were negative
23
Salmonella spp.
1-10 ufc
10-100 ufc
100-1000 ufc
25 g
50 g
VALIDATION STRATEGYEVALUATION APPROACH
Incubation:
6 h
10 h
DNA extraction:
Boiling
Chelex
Column
10 h
24 h
DilutionIncubation
timesISO
Baseline
QIAGEN
Baseline
CHELEX
Baseline
BOILING
1 cfu /
sample1:10
6 h 3/3 1/3 1/3 0/3
10 h 3/3 3/3 3/3 3/3
24 h 3/3 3/3 3/3 3/3 ✔
VALIDATION STRATEGYEVALUATION APPROACH
DilutionIncubation
timesISO
Baseline
QIAGEN
Baseline
CHELEX
Baseline
BOILING
1 cfu /
sample1:10
6 h 3/3 1/3 1/3 0/3
10 h 3/3 3/3 3/3 3/3
24 h 3/3 3/3 3/3 3/3
Salmonella spp.(it is possible also use 50 g)
Dilution 1:10 Buffered Peptone Water
(ISO)
VALIDATED STRATEGY FOR SALMONELLA
Incubation 24 h (it is possible at 10 h, but better
Ct values after 24 h)
DNA extraction: Chelex
Real-time PCRJosefsen MH, et. al (2007). AEM 73:3040-3048.
Malorny B, et al (2004) AEM. 70:7046-7052.
Preparation of spiked samples for Ring Trial
SalmonellaListeria monocytogenes
27
Lenticules Lyophilized strains
Protocols
28
Ring trialLaboratories Salmonella
Listeria
monocytogenesCountry
Accredited
17025
Università di Bologna X X Italy
National Veterinary Institute X X Norway Yes
Centro National de Tecnologia y
Seguridad AlimentariaX X Spain Yes
National Food Chain Safety Office X X Hungary Yes
Istituto Superiore di Sanità X X Italy Yes
University of Zagreb X X Croatia Yes
Istituto Tecnologico Agrario de Castilla
y LeonX X Spain
y LeonX X Spain
University of Copenhagen X X Denmark
Agence nationale de sécurité sanitaire
de l’alimentation, de l’environnement et
du travail
X France Yes
IZS Venezie X X Italy Yes
IZS Lazio e Toscana X X Italy Yes
IZS Lombardia e Emilia Romagna X X Italy Yes
IZS Meridione X Italy Yes
IZS Abruzzo e Molise X Italy Yes
NRL
Ring Trial Listeria monocytogenes
Worst-case scenario
Soft Cheese
� High level of natural competitive micro-flora
Participants: 12 laboratories (all labs are very expert in the detection of
Listeria monocytogenes using ISO 11290-1 or/and Real Time PCR
methods
30
� High level of natural competitive micro-flora
� High level of amplification inhibitors (high level of fat and proteins)
The samples were spiked using a lyophilized standard containing:
� an ATCC strain of Listeria monocytogenes difficult to isolate (ATCC
19111)
� a strains of Listeria innocua ( with a concentration one log less
concentrate than Listeria monocytogenes)
Platform
31
6 3 2 1
12
Ring Trial Listeria monocytogenes Results
Laborato
ry
Sample A Sample B Sample C Sample D Sample E Sample F Sample G Sample H
ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR
1 — — ���� ���� — — — ���� — ���� — ���� ���� ���� ���� ����
2 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
3 ���� ���� ���� ���� ���� ���� — — ���� ���� ���� ���� ���� ���� ���� ����
4 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
5 ���� ���� ���� — ���� — ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
6 ���� ���� ���� ���� — — ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
7 — ���� — ���� — ���� — ���� — ���� — ���� — ���� — ����
8 ���� ���� — — — — — ���� ���� ���� ���� ���� ���� ���� ���� ����
9 ���� ���� — ���� — — ���� ���� — ���� ���� ���� ���� ���� ���� ����
10 ���� ���� ���� ���� ���� ���� — ���� — ���� ���� ���� ���� ���� — ����
11 ���� ���� ���� ���� — — — — ���� ���� ���� ���� ���� ���� ���� ����
Low Concentration
32
Laborato
ry
Sample A Sample B Sample C Sample D Sample E Sample F Sample G Sample H
ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR ISO qPCR
1 — ���� — ���� ���� ���� — ���� — ���� — ���� — ���� — ����
2 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
3 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
4 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
5 ���� ���� ���� ���� ���� — ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
6 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
7 — ���� — ���� — ���� — ���� — ���� — ���� — ���� — ����
8 — ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� — — — ����
9 — ���� ���� ���� ���� ���� ���� ���� ���� ���� — ���� ���� ���� ���� ����
10 ���� ���� — ���� — ���� ���� ���� — ���� — ���� ���� ���� ���� ����
11 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ����
Medium Concentration
Contamination
level
Diagnost
ic
Specifici
ty
Diagnost
ic
Sensitivt
y
Positive
predictiv
e value
Negative
predictive
value
Accordance
(%)
Concordance
(%)
Concordanc
e odd ratio
(COR)
ISO
LOW
— 70.45 — —
72.11
(58.42 ,
86.02)
56.60
(47.63 ,
76.02)
1.98
(1.0 , 4.78)
MEDIUM
— 72.73 — —
83.79
(69.53 ,
96.09)
57.80
(46.79 ,
84.81)
3.77
(1.3 , 13.8)
Contamination levelDiagnostic
Specificity
Diagnostic
Sensitivty
Positive
predictive
value
Negative
predictive
value
Accordance
(%)
Concordance
(%)
Concordance
odd ratio
(COR)
ISO
LOW — 70.45 — —72.11
(58.42 , 86.02)56.60
(47.63 , 76.02)1.98
(1.0 , 4.78)
MEDIUM — 72.73 — —83.79
(69.53 , 96.09)57.80
(46.79 , 84.81)3.77
(1.3 , 13.8)
LOW+MEDIUM — 71.59 100.00 —78.52
(65.98 , 90.7)57.32
(47.4 , 79.9)2.72
(1.17 , 6.87)
NONE 100.0 — — 87.12 100.00 100.00 1.00
Statistical analysis and ISO 16140 evaluation parameters
obtained for the qPCR-based method from the trial data
96.09) 84.81)(1.3 , 13.8)
LOW+MEDIUM
— 71.59 100.00 —
78.52
(65.98 , 90.7)
57.32
(47.4 , 79.9)
2.72
(1.17 , 6.87)
NONE 100.0 — — 87.12 100.00 100.00 1.00
qPCR
LOW
— 87.50 — —
76.6
(64.62 ,
87.72)
76.10
(65.78 ,
87.18)
1.03
(0-90 , 1.16)
MEDIUM
— 97.73 — —
96.10
(88.3, 100.00)
95.50
(87.03 ,
100.00)
1.20
(1.04 , 1.17)
LOW+MEDIUM
— 92.61 100.00 —
86.45
(79.19 ,
93.63)
86.24
(79.74 ,
93.44)
1.02
(0.98 , 1.07)
NONE 100.0 — — 64.72 100.00 100.00 1.00
qPCR
LOW — 87.50 — —76.6
(64.62 , 87.72)76.10
(65.78 , 87.18)1.03
(0-90 , 1.16)
MEDIUM — 97.73 — —96.10
(88.3, 100.00)95.50
(87.03 , 100.00)1.20
(1.04 , 1.17)
LOW+MEDIUM — 92.61 100.00 —86.45
(79.19 , 93.63)86.24
(79.74 , 93.44)1.02
(0.98 , 1.07)
NONE 100.0 — — 64.72 100.00 100.00 1.00
Relative accuracy
(%)
Relative specificity
(%)
Relative sensitivity
(%)
False negative ratio
(%)
Falso positive ratio
(%)
82.75
(78.01 , 87.48)
96.70
(93.00 , 100.00)
97.62
(96.00 , 100.00)
1.18 0.0
Relative accuracy Relative specificity Relative sensitivity False negative ratio Falso positive ratio
L. monocytogenes detection by the qPCR method was affected in a
lower extend in that worst scenario, being therefore proved to be
the more reliable than the reference method. At top of that, the
qPCR method is cost effective (3 € vs 15 €) and time saving (27
hours vs 7 days).
CONCLUSIONS
Relative accuracy
(%)
Relative specificity
(%)
Relative sensitivity
(%)
False negative ratio
(%)
Falso positive ratio
(%)
82.75
(78.01 , 87.48)
96.70
(93.00 , 100.00)
97.62
(96.00 , 100.00)
1.18 0.0
Ring Trial Salmonella
Pork loin was aseptically removed in a slaughtering; the meat was stored
for ageing for three days at 4°C.
325 samples of 25-g were prepared used ageing pork loin.
Each sample was used for artificially contaminated..
The contamination of the pork samples were performed using commercial
lenticules.
Partecipants: 13 laboratories
35
lenticules.
The contamination was performed using three different blinded
contamination levels of lenticules:
L0 = negative control
L1 = lenticules contaminated with 10-20 CFU (0.4-0.8 CFU/g of pork
meat) of Salmonella Typhimurium NCTC 12023 (HPA, Salisbury, UK)
L2 = lenticules contaminated with 20-160 CFU (0.8- 6.4 CFU/g) of
Salmonella Typhimurium NCTC 12023 (Oxoid, Milan, Italy)
Platform
36
6 3 2 1
13
1
Contamination
level
Diagnost
ic
Specificit
y
Diagnost
ic
Sensitivt
y
Positive
predictiv
e value
Negative
predictive
value
Accordance
(%)
Concordance
(%)
Concordanc
e odd ratio
(COR)
LOW — 100.0 — — 100.0 100.0 100.0
Contamination
level
Diagnostic
Specificity
Diagnostic
Sensitivty
Positive
predictive
value
Negative
predictive
value
Accordance
(%)
Concordance
(%)
Concordanc
e odd ratio
(COR)
ISO
LOW — 100.0 — — 100.0 100.0 100.0
MEDIUM — 100.0 — — 100.0 100.0 100.0
LOW+MEDIUM — 100.0 100.0 — 100.0 100.0 100.0
NONE 100.0 — — 100.00 100.0 100.0 100.0
qPCR
LOW — — — 100.0 100.0 100.0
MEDIUM — 100.0 — — 100.0 100.0 100.0
LOW+MEDIUM — 100.0 100.0 — 100.0 100.0 100.0
NONE 100.0 — — 100.00 100.0 100.0 100.0
ISO
LOW — 100.0 — — 100.0 100.0 100.0
MEDIUM — 100.0 — — 100.0 100.0 100.0
LOW+MEDIUM — 100.0 100.0 — 100.0 100.0 100.0
NONE 100.0 — — 100.00 100.0 100.0 100.0
qPCR
LOW — — — 100.0 100.0 100.0
MEDIUM — 100.0 — — 100.0 100.0 100.0
LOW+MEDIUM — 100.0 100.0 — 100.0 100.0 100.0
NONE 100.0 — — 100.00 100.0 100.0 100.0Relative accuracy
(%)
Relative specificity
(%)
Relative sensitivity
(%)
False negative ratio
(%)
Falso positive ratio
(%)
100.0 100.0 100.0 0.0 0.0
Salmonella IAC
Results of Salmonella Ring Trial
38
A lot of IAC negative in positive samples, probably the
method does not work well in duplex using Roche (the
lab has 100% of concordance with the real samples)
6 negative IAC in negative samples (presence
of inhibitors) 1 in lab 3 and in lab 8, 2 in lab 7
and 9
University of BolognaGerardo Manfreda
Alessandra De Cesare
Frederique
National Veterinary InstituteTaran Skjeral
GroJohannessen
CNTAJavier Perez
Maria Jose Saiz
Hungarian Food Safety Office
AnsesLegall Francoise
Marianne Chemmay
Berthand Lombard
Nathalie Gnanou-Besse
IZS VenezieDamiano Comin
Maria Grimaldi
Antonia Ricci
Antonia Lettini
IZS Lombardia e Emilia RomagnaMarina Nadia Losio
Barbara Bertasi
Acknowledgements
39
Zsuzsanna Sréterné Lancz
Mészáros László
University of ZagrebLidija Kozačinski
Danijela Horvatek Tomić
ITACyLDavid Rodriguez Lazaro
Marta Hernanez-Perez
University of CopenhagenJonh Olsel
Dziuginta Jakociune
Barbara Bertasi
Enrico Pavoni
IZS Lazio e ToscanaStefano Bilei
Paola De Santis
Sarah Lovari
IZS MezzogiornoFederico Capuano
Yolande Proroga
Istituto Superiore di SanitàMonica Virginia Gianfranceschi
Elisabetta Delibato
Antonietta Gattuso
Michele Sonnessa
Validation of Sampling scheme of
Campylobacter in poultry (Antonio Valero,
Damiano Comin & Alessandra De Cesare)
PO= less than 2 % positive with a contamination > 10.000 cfu of
Campylobacter/g of skin
Validation protocol:Validation protocol:
30 different lots
3 samples for lots
All the samples was sampled after chilling (according with EU
regulation 2073)
2 slaughterhouses:
1. 90 carcasses from 30 batches of a medium slaughterhouse
2. 90 carcasses from 30 batches of a large slaughterhouse.
Validation of Sampling scheme of
Campylobacter in poultry (Antonio Valero,
Damiano Comin & Alessandra De Cesare)
The analysis were performed using 35 grams of skin (10 g for quantitative
detection, and 25 for qualitative)
EFSA suggested to use the skin of the neck and half breast (we use the
skin of the entire breast, because half breast was insufficient.
The validation were performed in three slaughterhouses:
A. 90 carcasses from 30 batches of heavy broiler of a large
slaughterhouse.
B. 90 carcasses from 30 batches of light broiler of a large
slaughterhouse.
C. 90 carcasses from 30 batches of a medium slaughterhouse
In A e B were analyzed 36 batches (20% more)
Validation of Sampling scheme of
Campylobacter in poultry (moving window
approach?)
Validation of Sampling scheme of
Campylobacter in poultry (Damiano
Comin IZS Venezie)
A. 58 samples out 108 samples were positive (53.7) 23 lots out 36 were positives (63.9%); all the positive samples werecontaminated with less of <104 ufc/g of Campylobacter
B. 75 samples out 108 were positives (69.44), 28 lots out 36 wereB. 75 samples out 108 were positives (69.44), 28 lots out 36 werepositive 8 (77.8%); 3 samples out 108 samples (2.7 %) or 3 lotsout 36 lots (9.3%) were contaminated with more than<104 ufc/g (one lot with 18.000, one lot with 100.000 and one lotwith 100.000 and 10.000)
C. 51 samples out 90 were positive (56.6%), 16 lot out 30 werepositives (53.3%); one out 90 samples (1.1%) or one lot out 30 lots (3.3%) was contaminated with more than of 104 ufc/g ofCampylobacter (15.000 CFU/g)