Rapid Test for the Detection of Pathogens in Dairy Products · Rapid Test for the Detection of Pathogens in Dairy Products . 2 ... Pre-Conditions for Using Rapid Tests . 14 ... dairy

June 13, 2013

Bioq. Mariano Veltri

Product Manager for BioMonitoring

Rapid Test for the Detection of Pathogens in Dairy Products

2

Content

From classical culture to rapid testing

Overview Merck Millipore rapid detection solutions

General principle of real-time PCR

Overview Merck Millipore real-time PCR solutions

Pathogenic E.coli testing from ground beef using RapidCultTM

Other Pathogens in Food:

- Salmonella

- Listeria

- Bacillus Cereus and Cronobacter Sakazakii

Food Pathogens

Cultivation Rapid Enrichment Microorganisms Identification

RTU RTU

DCM

Bactident

Lateral Flow

RT PCR

Salmonella

Cronobacter

Listeria

E. coli O157

Bac. cereus

Staph. aureus

Campylobacter

Enterococci

Enterobacteriaceae

Vibrio

Pseudo. aerug

Immunology

Singlepath

Real Time PCR

Foodproof MMB

DCM DCM

RTU

24-48 h 20-90 min 24-48 h 24-48 h

4

Food & Beverage / Enrichment media Food & - Key products for QC Lab

1.07228. Buffered Peptone Water ( Salmonella)

1.10266. Lauryl Sulphate Broth ( Coliforms in Water)

1.10398. FRASER Broth ( Listeria)

1.10549. LEB (antibiotics incl.); ( Listeria)

1.14582. mEC Broth /mTSB ( Enterohemorrhagic E.coli)

1.07661. Lactose Broth ( Salmonella)

5

Food & Beverage / Plating Agar Key products – for QC Lab

1.05463. Plate Count Agar ( Total Count)

1.05463 Chromocult Coliform Agar ( Coliforms & E.coli)

1.01406. VRB Agar ( Coliforms & E.coli)

1.16000. YGC Agar ( Yeasts)

6

ISO 11133

ISO 11133-1

Microbiology of food and animal

feeding stuffs —

Guidelines on preparation and

production of culture media

- Part 1

ISO 11133-2

Microbiology of food and animal

feeding stuffs –

Practical guidelines on performance

testing of culture media - Part 2

This new standard describes for the first time in history

of Microbiology the definition of quality control for

commercial culture media.

Quantitative testing and reporting of results (CoA)

with recovery rates is the new preferred testing

method

7

Quality Control of Plating Agar

> 70% for non selective media

> 50% for selective media

Productivity

Selectivity

Most important change !

ISO 11133 – Part 2 Practical Guideline on Performance testing of culture media - Certificate of Analysis

Quality Control of Selective Liquid Media

10*6 to 10*8 c.f.u per ml

below 10*4 c.f.u per ml

Target bacteria

Unwanted

Extremely important to

avoid false-negative results

Methods for use in performance testing of culture media

RVS / TTn / Selenite Cystine broth

Methods for use in performance testing of culture media

Quality control Minimum level must

be 10*5 c.f.u

Test strains Inoculum Growth after

24 hours

Escherichia coli

ATCC 25922

Approx. 99 % < 10 %

Salmonella typhimurium

ATCC 14028

Approx. 1 % > 90 %

Quality Control of Selective Liquid Media

1 Page 10

more than 70 % Recovery Rate for each test strain used

Specification of Tryptic Soy Agar Merck COA in

Compliance with ISO 11133


12

Classical Culture + ID

Culture + ELISA

Culture +PCR/

REAL TIME PCR

BIOCHIP

3 4 5 1 2 DAYS

log10 cfu

1

2

3

4

5

6

7

6 7

REAL TIME

TEST

SHORTENED

CULTURE

RAPID TESTING

13

• All rapid testing methods are fully

dependent on the quality of the

corresponding non/selective enrichment

media

• If an enrichment medium does not allow

growth of a minimum level of bacteria

necessary for detection, a false-negative

results will occur !

• Minimum levels PCR: 103 -104 CFU / ml

• Minimum levels Immunoassays: 105 CFU / ml

Pre-Conditions for Using Rapid Tests

http://images.google.com/imgres?imgurl=http://www.emdchemicals.com/analytics/Micro_Manual/TEDISdata/prods/4979-1_10398_0500-1.jpg&imgrefurl=http://www.emdchemicals.com/analytics/Micro_Manual/TEDISdata/prods/1_10398_0500.html&h=300&w=199&sz=46&tbnid=qjj4nX7N0hYJ:&tbnh=110&tbnw=73&start=2&prev=/images?q=fraser+broth&hl=de&lr=&sa=N

http://tugll.tugraz.at/51446/files/-1/1575/mikrotiterplatte.png

14

Content







- Salmonella

- Listeria


Overview rapid detection solutions

15

Superior Granulated Dehydrated

Culture Media

Innovative Immunoassays – Lateral FlowTests

- Singlepath® and Duopath®

Broad range of preparation & detection kits

for real-time PCR

Safe Minimizes air-borne toxins, allergens and workplace contaminations

Accurate Prevents component separation and clumping

Fast Dissolves rapidly in water

Easy Ensure easier handling by superior flow properties & non-sticking media

Reliable Homogenous distribution of ingredients assures high reproducibility

16

Superior Granulated Culture Media

No dust

vs Powder Granulated media

No clumping

17

3 BLENDING 2 ADD SAMPLE 1 ADD MEDIUM 4 INCUBATE 5 PIPETTE

SAMPLE

6 PLACE

SAMPLE

NEGATIVE

POSITIVE

Read

after

20 -25

min

General Application of Lateral Flow Tests

Innovative Immunoassays – Lateral Flow Tests

18

Immunochromatographic principle:

Antibody-linked colloidal gold particles react

with its complimentary antigenic determinant

to provide a visual reaction read-out

Fast

Definite results within 20–30 minutes

Easy to use

Clear yes/no results after simple sample

application

Safe

Positive control and specially adapted

enrichment media for precise, reliable

results

Economic

Rapid results save operating costs

19

Principle of Lateral Flow Test

bacterium gold particle

antibody

membrane

20

1. Control reaction in-built in LFT.

No need for separate reaction to demonstrate proper function,

as necessary for ELISA.

Can save costs esp. when low numbers of samples to be tested

2. LFT’s are 1-step tests.

After application of test sample to sample port, NO further pipetting

steps necessary.

No separate color reaction for LFT’s necessary to make reactions

visible.

3. LFT are provide results faster than ELISA

Only incubate test on lab bench for 20 – 30 min and read result.

Advantages of Lat. Flow tests vs. ELISA

21

Merck‘s Lateral Flow Test Portfolio

Listeria: Salmonella: E. coli O157&VTEC: Campylobacter: Bacillus cereus: Legionella:

LEB Tetrathionate mEC + n Bolton Broth M.Y.P. Agar CYE Agar Base

Fraser Rappaport.-Vas. mTSB + n CCD Agar CGY Broth BCYE Suppl.

UVM Selenit Cystin CT-SMAC BHI GVPC Suppl.

PALCAM Salmosyst SMAC CYE-Plates

OXFORD M Broth BHI GVPC-Plates

Chromocult® XLT4 , XLD, CAYE Anaerocult® C

Listeria Agar Rambach®

22

Content






- Other Pathogens in Food:

- Salmonella

- Listeria


Presentation title in footer | 00 Month 0000 23

What is real-time PCR?

PCR = Polymerase Chain Reaction

Specific amplification of DNA fragments from

bacteria, viruses, human, animal or plant cells

&

the simultaneous automated detection of this

amplified DNA on the computer monitor by

fluorescence measurement = real-time

24

PCR: Polymerase Chain Reaction

Invented by Karl Mullis, 1979

Nobel Price in Chemistry,1993

Specific amplification of DNA fragments

from bacteria, viruses, human, animal

or plant cells

&

simultaneous automated detection

of these amplified DNA by fluorescence

measurement

Real-Time PCR

©www.forensic-science.org

Basic principle T T T T T A A A C C C C C

A A A A T T T C C C C C C T

A

A

G G G G G G

G G G G G

95°C

55°C

72°C

Taq

Taq PCR

T T T T T A A A C C C C C


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G G G G G G

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25

26 26

PCR – Principle of Amplification

Detection formats

27

Hybridisation Probes

Probes remain intact

TaqMan Probes (5‘Nuclease)

Probes get destroyed

28

Real-time PCR: Online data measurement

Agarose gel electrophoresis

Staining

Photography

Classical PCR Real-Time PCR

Ct-value

Threshold Cycle (Ct) value

Advantage

High specifity

Low risk of “false positive“ results

29

PCR workflow

Start 20 min 40 min 130 min

90-120 min

PCR Set-up PCR run Sample

preparation

Enrichment,

Culture or

Single Colony

Detection

& Analysis

30

Real Time PCR – Sample preparation

From manual to full automated handling

<30 min

ShortPrep SamplePrep RoboPrep

<60 min

31

Content






Other Pathogens in Food: - -

- Salmonella -

- Listeria


Corbett/QIAGEN

Rotor-Gene 3000

Broad range of real-time thermal cycler

Agilent/Stratagene Mx3000p

BioRad IQ5

Roche LightCycler 1.5 Roche LightCycler 2.0

ABI 7700

Cepheid SmartCycler

Eppendorf realplex

ABI 7000

Roche LC480

ABI 7500

BioRad myiQ

ABI 7900

ABI StepOne System

32

33

Test for several bacteria in 1 PCR run

Salmonella

L. mono., L. Genus

E. coli O157

Campylobacter Quant.

GMO screening

RR Soya Quant.

Identical Thermoycler conditions

PCR Setup at room temperature

96 well plate

34 34

Benefits of Real-Time PCR: Saving of labour / time

Applying real-time PCR, all samples are read by just one mouse-click

Clear results, no hidden colonies in the background

Objective results; no personal influence of lab manager who reads the plates

http://www.impetus.uni-koeln.de/content/a5/images/highres/image007.jpg

35

Presence/absence tests of pathogenic bacteria

Quantitative investigation of pathogenic bacteria

Large scale identification of spoilage organisms

Detection and quantification of gene modified organisms

Real-time PCR product portfolio – Foodproof & MMB detection kits

36

Foodproof Detection Kits: For Roche LightCycler 1.x, 2.0

Salmonella

Listeria monocytogenes

Listeria Genus

E. coli 0157

Campylobacter

Enterobacteriaceae plus E. sakazakii

E. sakazakii

E. coli and Shigella

Beer Screening bacteria

Saccharomyces cerevisiae var. diastaticus

GMO Maize Quantification

GMO Soya Quantification

GMO Screening

Hybridization Probes Kits

37

Salmonella spp.


Listeria Genus

E. coli O157

Campylobacter quantitatively

Enterobacteriaceae + E. sakazakii

Brucella

Dekkera (= Brettanomyces: Wine spoilage)

Alicyclobacillus

GMO Screening (4 events multiplexed!!!)

GMO Soya Quantification

GMO Maize Quantification

Foodproof 5‘Nuclease Kits: For TaqMan Instruments & LC 480

38

Foodproof Sample Preparation Modules I

Rapid & Convenient Kits

foodproof ShortPrep I Kit (for Salmonella kits)

foodproof ShortPrep II Kit (for E. coli O157, Campylobacter & Listeria)

foodproof ShortPrep III Kit (for beer screening kit)

Kits for difficult matrices

foodproof Sample Preparation Kit I (Gram-neg. bacteria in raw & processed foods)

foodproof Sample Preparation Kit II (Gram-pos. bacteria in raw & processed foods)

foodproof GMO Sample Preparation Kit

Rapid Kits (with bulk reagents) = Value for Money

foodproof StarPrep One Kit (for all Enterobacteriaceae)

foodproof StarPrep Two Kit (for Gram-positive bacteria) Available Q1 / 2013

foodproof StarPrep Four Kit (for yeasts)

foodproof Magnetic Separation Kit I (for Gram-negative bacteria)

39 39

Foodproof products from Merck for various instruments

• Brand label: foodproof®

• Lightcycler (Roche): foodproof® Salmonella Detection Kit

- Hybridization Probes (LC 1.x, 2.0) -

• TaqMan-Instruments: foodproof® Salmonella Detection Kit

- 5‘Nuclease -

NO kit based on SYBR Green from Merck !!!!

40

All foodproof real-time PCR kits contain:

Internal Amplification Control (IAC) present in every capillary / well and every test

PCR run positive control (PC) as a separate reaction. Target DNA from kit added.

PCR Negative control (NC) as a separate reaction. Ultra-pure H2O from kit added.

Taq / UNG enzyme Mix present in every capillary / well and every test

Highest Quality Assurance with foodproof real-time PCR kits

No false-negative results

from food matrix

No false-negative

results from defective kit

No false-positive results

due to contaminated kit

No false-positive results

due to carry-over

contamination

41

Prevention of Carry-over Contamination

Mechanism of Uracil N Glycosylase

a au uaa a u a aau aaau aa u a aa uu aua

t t a at t t a t t ta t t t a t t a t t t aa tat

Uracil-N-Glycosylase

a au uaa a u a aau aaau aa u a aa uu aua

PCR mit Thymin

a a t taa a t a aa t aaa t aa t a aa t t ata

a a t taa a t a aa t aaa t aa t a aa t t atat t a at t t a t t t a t t t a t t at t t aa tat

a a t taa a t a aa t aaa t aa t a aa t t atat t a at t t a t t t a t t t a t t a t t t aa tat

t t a at t t a t t ta t t t a t t a t t t aa tat

DNA-Matrix DNA-Matrix

a a t taa a t a aa t aaa t aa t a aa t t ata a a t taa a t a aa t aaa t aa t a aa t t ata

u ua auu u a u uua uuua uu a u uu aa uau

u ua auu u a u uua uuua uu a u uu aa uau

t t a a t t t a t t t a t t t a t t a t t t aa tat

PCR with uracil PCR with thymine

Native DNA Amplified DNA

42

Prevention of Carry-over Contamination

Necessary Kit component UNG

Sample Enrichment

DNA Extraction Purification

PCR

Detection no false positive results possible

due to lab contamination

UNG degrades ONLY „old“ amplified GAUC-DNA

UNG cannot degrade native GATC-DNA

UNG is inactivated before „new“ PCR starts

(during initial denaturation of DNA)

UNG - Glycosylase step

Contamination by “old“

amplified GAUC-DNA

possible

43

Content






Other Pathogens in Food: - -

- Salmonella -

- Listeria


EHEC‘s: Emerging Pathogens

44

45

EHEC‘s: Emerging Pathogens Highly pathogenic enterohaemorrhagic E. coli (EHEC) strains like

verotoxin (= shiga-toxin)– producing (VTEC/STEC) strains gain importance

Serovars: O157:H7, O26, O103, O111, O145 and other strains are of

interest

Production of verotoxins is the most common criteria for detection:

Verotoxin 1 (VT1, SLT1, Stx1) and 2 (VT2, SLT2, Stx2)

EHEC strains produce either VT1 or VT2 only or even both

VT2 is causative agent for severe diseases, like hemolytic uremic

syndrome (HUS)

Infection with E. coli O157 are known as “Hamburger Disease”

Fast

Reduced time to result – from more than 18 down to 8-12 hours

Flexible

Incubation for 12 or less up to 22 hours to fit with optimal workflow

Efficient

Pooled samples: 25 g and 375 g are validated

High yield

Increased growth of all E. coli due to special composition

Reliable

Validation with Singlepath® E.coli 0157 & foodproof ® E. coli O157 Detection Kit

consistent with USDA-FSIS guidance

High-quality

Comprehensive inclusivity study: superior growth of all E .coli strains tested

Granulated medium meet ISO 11133 guidelines

46

Features of Rapidcult™ E. coli

47

Confirmation by culture and biochemical tests required

Day

1

Day

2

Day

3

25 g/ml TEST SAMPLE

225 ml m-EC or m-TSB

37 °C FOR 24 h

CT-SMAC Agar 37°C / 24 h Fluorocult E.coli 0157 Agar

Sorbitol (+)

are non

pathogenic

E.coli

ISO Standard for testing of E.coli 0157 in Food

Caution: Some E.c. O157 are

Sorbitol (+) -> false-neg. result

One broth detects all EHEC strains Enrichment*

25g/375g in pre-warm Rapidcult 8-22h/42°C

Classical culture media Lateral Flow Test* Real-time PCR* Post-enrichment

160 µl in Sample Port

Result: 8h 20 min

After immunoconcentration streak out on

CT-SMAC Agar

Incubation: 24h/35-37°C

Conformation e.g. Fluorocult E.coli 0157

48

DNA Extraction

StarPrep One Kit

PCR

foodproof E.coli 0157 Detection Kit

Result: 48 h Result: 10 h

CAYE Broth & supplement

Screening

Duopath ® Verotoxin

Result: 14 h

* USDA FIS validated

Rapidcult™ results with real-time PCR

Sensitive screening for most important virulence genes: eae, stx 1 and stx 2 with

new foodproof® STEC Screening Kit – prototype version

After 8h enrichment using Rapidcult E.coli

The virulence patterns of more than 50 STEC strains were identified correctly

strains included: O26 (n=11), O103 (n=3), O111 (n=3), O121 (n=1), O145

(n=3), O157 (n=14) and others (n=15).

O104 as the cause of the severe outbreak in Germany in 2011 included

8h enrichment for 25g of ground beef

shown for E. coli O157, O26, O103, O111 and O104 compared to the

Microbiology Laboratory Guidebook USDA-FSIS,(MLG 5.05B ) and ISO/PRF TS

13136

Tests for O45, O121 and O45 ongoing

49

Rapidcult™ results with real-time PCR

50

Real-time PCR detection of important virulence genes from E. coli 0157:H-

after 8 h RapidCult incubation

51

Content







- Salmonella

- Listeria


From presence/absence to quantitative risk assessment

Bacillus cereus © CDC Public Health Image Library Cronobacter Sakazakii

Salmonella Listeria

52

Salmonella in Food

53

54

Interpretating of Growth on Plates

Day

1

Day

2

Day

3

Day

4

Day

5

Day

6

For confirmation take 5 suspected colonies from each plate

and streak onto Nutrient agar 18 - 24 h / 35 - 37 °C

Biochemical / Serological Confirmation Tests

TSI / Urea / Lysin / -Gal / VP / Indol

25 g/ml test sample

in 225 ml BPW

16 - 20 h / 35 - 37 °C

1 ml BPW to

10 ml MKTTn Broth

24 h at 37°C

0.1 ml BPW to

10 ml RVS Broth

24 h at 41.5°C h

XLD Agar

24 h / 35-37°C

1 plate 14 cm /

2 plates 9 cm

Any other

Salmonella Agar

1 plate 14 cm /

2 plates 9 cm

3 days

11 media

XLD Agar

24 h / 35-37°C

1 plate 14 cm /

2 plates 9 cm

Any other

Salmonella Agar

1 plate 14 cm /

2 plates 9 cm

ISO Standard 6579 for Detection of Salmonella

55

Cultural reference method ISO 6579:2002 Annex D

Non-selective pre-enrichment

Selective plating on XLD

Biochemical Identification

Selective enrichment

Confirmation

*Modified Semi-Solid

Rappaport-Vassiliadis Medium

25g/225ml BPW 18+2h/37°C

3 drops/0.1ml on MRSV* 24h/41,5°C

Negative plates: Further 24h incubation

Detection principle: Motility of Salmonellae to migrate into the semi-solid medium

56

Day

1

Day

3

Day

4

25 g/ml TEST SAMPLE

In 225 ml Buff. Peptone Water

35 - 37°C for 16 - 20 h

Day

2

NO Salmonella

not present

0.1 ml BPW to 9.9 ml RVS Broth

42°C for 24 h

only 2 media

only 2 days

YES Salmonella

present

STREAK OUT

ONTO RAMBACH 37°C FOR 24 h

If positive, confirmation by culture and

biochemical tests required

Rapid Testing Singlepath® Salmonella - Screening

57

Detection of Salmonella by RT-PCR: Within ONLY 24 h and only BPW

Conventional

Non-selective enrichment

in BPW 16 – 20h


24h

1. Solid media

24h

2. Solid Media

24h

Isolation/Incubation

24h

Biochemical

verification 4h

Serological

verification 4h

Non-selective

enrichment in BPW

16 – 24h

Sample preparation

0.5h

Amplification/Detection

1h

PCR

Time savings: neg. result > 2 d

pos. result > 4 d

58

Real-time PCR-Analysis

Dilute possible PCR inhibitiors

Non-selective pre-enrichment

25g/225ml BPW 18+2h/37°C

Selective plating on XLD

Biochemical Identification


Confirmation

3 drops/0.1ml on MRSV* 24h/41,5°C

Negative plates: Further 24h incubation

0.5 ml BPW in 5 ml prewarmed BHI* 3h/37°C

Subcultivation 1:10

* Brain Heart Infusion

DNA extraction

StarPrep One Kit

PCR

Time: 3-5 days Time: 1.5 days

foodproof Salmonella Kit

59

Results on Milk

Positive control

Negative control

Samples 1c 18 h / 18+3 h

Amplification Plots LC480II spiked with 12,9 cfu

Listeria in Food

60

Milk and milk products: Soft cheese, raw milk, pasteurized milk, ice cream, fresh cream

Day

1

Day

3-6

25 g/ml TEST SAMPLE

IN 225 ml 1/2 STRENGTH FRASER

30°C FOR 24h

STREAK OUT

ON ALOA® + PALCAM

37°C FOR 24 - 48h

Day

3-4

ISO Standard 11290-2004 for testing of Listeria

in Food and Animal feeds

Day

4-7

0.1 ml IN 10 ml FRASER

35 OR 37°C FOR 48h

All colonies showing a blue-green color with opaque halo / grey-green color with a black zone are

presumptive L. monocytogenes colonies (typical colonies) and counted as such. Suspect colonies

must be confirmed.

STREAK OUT

ON ALOA® +

PALCAM

37°C FOR 24 - 48h

BIOCHEMICAL CONFIRMATION: GRAM (+), CATALASE (+), MOTILITY (+),

CAMP (+), HAEMOLYSIS (+), GLUCOSE (+), RHAMNOSE (+), XYLOSE (-)

62

Appearance of L. monocytogenes on Chromocult®:

• blue-green colonies = -D-glucosidase activity

• opaque halo = phosphatidylinosit-phospholipase C activity

Listeria spp. Listeria monocytogenes

Chromocult® Listeria Selective Agar

63

Chromocult® Listeria Selective Agar complies with Agar Listeria

according to Ottaviani and Agosti (ALOA®) in line with the

recommendations of ISO 11290 (2004) and FDA / BAM (2003)

1.00427 Chromocult®

Listeria Selective Agar Base


Listeria Selective Supplement Contains 4 different antibiotics


Listeria Enrichment Supplement Contains L-α- phosphatidylinositol

Chromocult® Listeria Selective Agar

64

Rapid Testing Singlepath® Listeria

Lateral Flow Test for the SPECIFIC detection of all Listeria species

For screening of environmental and food samples.

Very useful for screening in food plants acc HACCP guidelines

US-food companies screen for Listeria spp.

For the confirmation of presumptive Listeria spp. positive colonies

on Listeria selective agars like Chromocult® Listeria Agar

ALOA®) PALCAM, Oxford

Enrichment media used: Fraser, LEB, UVM

65

Rapid Testing Singlepath® L’ mono

Worldwide first Lateral Flow Test for the SPECIFIC detection of


For screening of environmental and food samples

Very useful for screening in food plants acc HACCP guidelines

European food companies screen for Listeria monocytogenes

For the confirmation of presumptive positive colonies

on Listeria selective agars like Chromocult® Listeria Agar

ALOA®) PALCAM, Oxford

Enrichment media used: Fraser, LEB, UVM

Day

1

Day

3

0,1 ml IN 10 ml LEB or

Full Fraser OR UVM

30° / 37°C FOR 21 - 24 h

ALOA®

37°C for 24 - 48h

Day

2

L. monocytogenes

present

Singlepath® L’ mono / Singlepath Listeria - Screening

L. monocytogenes

not present

160µl

25 g/ml TEST SAMPLE

IN 225 ml 1/2 STRENGTH FRASER

30°C FOR 24h

Confirmation

Listeria spp.

not present

suspend 1 - 3 presumptive colonies in 250 µl BHI or

CASO or Fraser or PALCAM Broth

L. monocytogenes

confirmed

PALCAM Agar OXFORD Agar ALOA® Agar

L. monocytogenes

not confirmed

Singlepath® L’ mono - Confirmation

150 µl

1 h at 37°C

• Confirmation within 1,5 h

• Fastest confirmation assay available on the market

Detection of Listeria spp. & L. monocytogenes by real-time PCR:

Within ONLY 24 h

Conventional


½ Fraser 24 h

Selective

enrichment

Full Fraser 24 – 48 h

1. Solid media

24 h

Isolation/Incubation

24 h

Biochemical

verification 4 h

Serological

verification 4 h


½ Fraser 24 h

Sample preparation 0.5 h

Amplification/Detection 1 h

PCR

by foodproof Listeria Genus

or

by foodproof Listeria monocytogenes

Cronobacter spp.

69

Rapid Detection of Cronobacter spp.

Enterobacter sakazakii is renamed since 2008

New name is: Cronobacter spp. nov.

71

Considered as a clear species since 1980. Renamed in

2008 due to pheno-and genotypic diversity.

C. spp. is an opportunistic pathogen.

High risk for newborns, especially low-birth weight infants.

Causes severe neonatal sepsis, meningitis

Powdered infant formula (PIF) is major vehicle and cause

of Cronobacter spp. illness.

Very resistant to osmotic & dry stress. Can survive >2

years in PIF

Cronobacter spp. (formerly E. sakazakii)

72

Cronobacter spp.

EC Regulation 2073

Absence of Enterobacter sakazakii in 10 g sample Milk

powder

Mandatory for all infant formula producer

Dr. A. Bubert, Merck KGaA 73

1. pre-enrichment in BPW

2. enrichment in EE broth,

3. plating + isolation on violet red bile glucose agar (VRBGA)

4. biochemical identification of typical colonies

is time consuming and requires up to 7 days

Cronobacter spp.: Current FDA method

FDA method only yielded 26% positive results

in a comparative study with old ISO draft (40%)

Day

1

Day

3

Test sample

in BPW (1:10)

37°C for 16 - 20 h

10 ml Modified LST Broth – Vancomycin Medium

45°C for 22 - 26 h

Day

2

Streak onto

Enterobacter sakazakii Isolation Agar

44°C for 22 - 26 h

Biochemical Confirmation

ISO TS 22964 for isolation of E. sakazakii

Day

4

0,1 ml

Bottleneck: Not all E. sak.

can grow under

these conditions

75

foodproof Enterobact. plus E. sakazakii Kit

Features

Unique real-time PCR kit for detection of all Enterobacteria

AND Cronobacter spp. simultaneously with ONLY 1 reaction

Highest reliability for Cronobacter Screening in powdered

Infant Formula and other babyfoods

Very useful for Hygiene Monitoring of Enterobacteriaceae

Kit is designed according to latest ISO standards for real-

time PCR to prevent false-positive and false-negative results

Kit does NOT require mLST enrichment step

76

Conventional (ISO 22964:2006)

Enrichment

22 - 26 h

Isolation

22 - 26 h

Biochemical

Identification

44 - 48 h

Pre-Enrichment

16 – 20 h

PCR

Subcultivation

in BPW 3 h

DNA Isolation

0.5 h

Amplification/Detection

1 h

Pre-Enrichment

in BPW for 18 h

Cronobacter spp. – PCR vs. Conventional Detection

Time to result : 1 day only

77

Detection of Enterobacteriaceae /

Cronobacter spp. by RT-PCR

PCR 1

Enterobacteriaceae/Cronobacter

Enrichment

20-24 h

DNA-Extraction

Internal Pos.

Control

Enterobacteriaceae Cronobacter

Positive Result

PCR 2

Salmonella

Negative Result

Enterobacteriaceae neg.

Cronobacter neg. E. coli neg.

Salmonella neg. Coliforms neg.

Positive

78

Reagent D for dead-live discrimination

Reagent D efficiently eliminates amplification of DNA from

dead bacteria.

Mechanism: Reagent D invades ONLY into dead bacteria

and binds to DNA. DNA is not amplifiable any more.

Reagent D is a light sensitive substance. Exposure to visible light leads to

covalent binding of this substance to DNA and prevents the DNA from being

amplified via PCR.

Works until 106 dead bacteria / gram food

Currently validated for Enterobacteriaeceae only

Only Merck offers Reagent D for dead-live discrimination

79

No false-positive results even at high Entero-

bacteriaceae concentrations using Reagent D

with Reagent D

Without Reagent D

80

Advantages of foodproof® Enterobacteriaceae

plus E. sakazakii Detection Kit

• Unique 5 – in - 1 Test:

- Negative result of Enterobacteriaceae reaction means:

No Enterobacteriaceae

No Cronobacter spp. (formerly E. sakazakii)

No Salmonella

No E. coli

No Coliforms

• Unique kit which can discriminate between live and dead bacteria

• Unique kit which give no false-positive results for Enterobacteriaceae

These unique features save time and costs

81

• Sybr green real-time PCR System:

- Only for the detection of E. sakazakii

- Disadvantage: High rate of false-positive (due to SYBR Green) results

• foodproof Enterobacteriaceae plus E. sakazakii

- Detects ALL Cronobacter spp. strains

- Advantage: No false-positive or false-negative results

Foodproof kit is successfully validated by Nestlé, Switzerland!!

- Nestlé recommends their babyfood plants to use foodproof kit –

- ISO is working on a revision of C. sakazakii method because the existing method

works not very well. Up to now there is no improved method available.

foodproof® Enterobacteriaceae plus E. sakazakii

Detection Kit vs. Sybr green system

MMB Food Pathogen System

MMB Food Pathogen Prep Kits MMB Food Pathogen Detection Kits

MMB Bacteria Prep Kit

MMB DNA/RNA Virus Prep Kit

V = VIC for Applied Biosystems, Agilent, Eppendorf, QIAGEN

R = ROX for BioRad Systems LC = For Lightcycler Systems

MMB Staphylococcus aureus (V), (R), (LC) Kit

MMB Legionella Screening (V), (R), (LC) Kit

MMB Vibrio cholerae (V), (R), (LC) Kit

MMB Legionella pneumophilia (V), (R), (LC) Kit

MMB Clostridium perfringens (V), (R), (LC) Kit

MMB Bacillus cereus (V), (R), (LC) Kit

MMB Norovirus (V),(R),(LC) Kit

82

MMB Food Pathogen Prep Kits 2 x 50 extractions

Membrane filter technology

Content:

1 x Lysis Buffer

1 x Binding Buffer

1 x Wash Buffer

1 x Elution Buffer

2.0 ml Receiver tube for washing

1.5 ml Receiver tube for elution

Spin filter

83

1. Preparation of food matrix – for example

2. DNA Isolation with MMB Food Pathogen Prep Kit

225 ml

Buffered Peptone Water

Wash Wash

again

Elute Sample

DNA

25 g

chocolate

MMB Food Pathogen Prep Kits procedure

Incubation

16-20

hours

in

Lysis

84

85

Bacillus cereus detection General

Environmentally widespread Gram-positive, spore-forming, motile rod

Cause gastrointestinal (e.g. diarrhea) as well as non-gastrointestinal diseases

(e.g. septicemia,endocarditis, infections of the central nervous system)

Self-limiting and of short duration, relative low number of registered cases; < 1%

Contamination risk by meat and milk products, vegetables, soups, spices and

especially baby food

Ability to produce one or more toxins: Cytotoxic enterotoxins are produced by

almost 95% of isolates

Pre-enrichment : Tryptone soya broth (TSB)

PCR detection: B. cereus, B. mycoides, B. thuringiensis, B. anthracis

86

Duopath® Cereus Enterotoxins

Worldwide first Lateral Flow Test for the SPECIFIC detection of Bacillus cereus via their enterotoxins

For screening of foods (tested with cream of wheat, spinach,

coffee white, egg powder, chocolate powder)

For confirmation of enterotoxin-producing presumptive

positive B. cereus colonies on M.Y.P. Agar

CGY Broth (+ 1% Glucose) for the enhancement of toxin production

~ 90% of B. cereus are NHE toxin positive;

~ 50% of B. cereus are HBL toxin positive

Advantage:

Broad coverage of nearly all B. cereus (except emetic strains) due to

detection of 2 enterotoxins

87

Singlepath® Emetic Toxin MRK

Worldwide FIRST Rapid Test for detection of emetic Bacillus cereus

Singlepath does not detect Emetic toxin but a protein which is co-produced with

Emetic Toxin in B. cereus = Surrogate Marker.

For screening of foods (tested with soup, rice)

For confirmation of emetic B. cereus on agar plates

Test successfully evaluated by Kagawa Food Univ. Japan

NOW available !!!! Singlepath Emetic is already standard method

for testing UHT milk in Malaysia)

88

Day

1

Day

2

20 ml CGY + 1% Glucose

Sensitive Screening

(>1 CFU / g)

Rapid Screening

(>100 CFU / g)

in 20 ml CGY + 1% Glucose

Test Protocols – Screening of B. cereus in foods

Duopath® Cereus Enterotoxins AND / OR

Singlepath® Emetic Toxin MRK

B. cereus

present

B. cereus

not present

NO YES 150 µl

18 – 24 h at 30°C

10 g/ml sample into

90 ml CGY Broth + 1% Glucose or

BHI

200 µl

200 µl

18 – 24 h at 30°C

6 h at 30 °C

150 µl

Emetic B. cereus

not present

Emetic B. cereus

present

89

Day

1

suspend

1 - 3 presumptive colonies into 1

ml CGY Broth + 1% Glucose,

M.Y.P. Agar

B. cereus

present

B. cereus

not present

4 h at 30°C

150 µl

NO YES

B. cereus by Duopath® Cereus Enterotoxins

Emetic B. cereus by Singlepath Emetic TOX MRK

Confirmation:

Emetic B. cereus

not present

Emetic B. cereus

present

90

Clostridium perfringens detection

General

Anaerobic, Gram-positive, spore-forming rod

Widely distributed in the environment and frequently in the intestines of humans and

domestic and feral animals

Spores persist in soil, sediments, and areas subject to human or animal fecal pollution -

heat resistant and not killed by ordinary cooking

Poisoning associated with temperature abuse of prepared foods

Growth in foods with high starch or protein content, such as cooked beans, meat

products, thick soups, and gravy

8–22 h after consumption of foods containing large numbers of bacteria capable of

producing food poisoning toxins

Pre-enrichment: Thioglycolate broth

PCR detection: Positive reference C. perfringens ATCC 13124

91

Norovirus detection

General

Norovirus particles with diameter of ~ 35 nm, highly stable in environment

Capside & a single strand RNA, 7300 – 7700 nucleotides

Cause of 90 % of non bacterial gastroenteritis

Outbreaks in community facilities, e.g. hospitals, rest or nursery homes

Symptoms: vomiting, diarrhoea and nausea

Minimal infection dose is 10-100 virus particles

Food incl. shellfish (oysters, clams, mussels), salads, peeled fruits, cold meats,

sandwiches, dairy products, egg dishes, ice and fecally contaminated water or of surfaces

of foods and food preparation

Pre-enrichment: None, direct detection

RT-PCR detection: Genotypes of genogroup I & genogroup II, dominant in outbreaks

92

Staphylococcus aureus detection General

Facultative anaerobic Gram-positive coccus, which is non-motile, catalase and coagulase

positive.

Some strains produce staphylococcal enterotoxins (SEs), the causative agents of food

poisoning.

Common symptoms: nausea, vomiting, retching, abdominal cramping, and diarrhe.a

Exist in air, dust, sewage, water, milk, and food, as well as on food equipment,

environmental surfaces, humans, and animals.

Able to grow in a wide range of temperatures (7–48.5°C, with an optimum of 30–37°C), pH

(4.2 to 9.3, with an optimum of 7–7.5) and sodium chloride concentrations (up to 15% NaCl)

Foods include meat and meat products, poultry and egg products, salads, bakery products

(e.g. cream-filled pastries, cream pies, and chocolate eclairs), sandwich fillings and milk and

dairy products

Pre-enrichement : Tryptone soya broth (TSB) / PCR detection : MRSA Typ I, II, III, IV, IVa,

IVb,IVc and other S. aureus species, not detection of other Staphylococci

93

Vibrio cholerae detection

General

Gram-negative, asporogenous, motile rods, straight or comma-shaped

Strictly aqueous organism, brackish and marine waters are natural environments

Transmission is fecal-oral, indirectly via polluted water supplies or irrigation water or

contaminated shellfish that is raw or undercooked

Infection of the small intestines caused by cholera enterotoxin (CT) producing Vibrio

cholerae of serogroups O1 and O139

Mild symptoms, 10% of patients may experience classical cholera symptoms with profuse

watery diarrhea (“rice-water stool”) and often vomiting

Pre-enrichment: Alkaline peptone water with 2% sodium chloride (APW)

PCR detection: Positive reference V. cholerae Mop 413 & 415 - Mop – a putative

protease modulating virulence, no detection of other Vibrio species

Questions?

94

Muchas Gracias

[email protected]

[email protected]

Documents

Rapid Test for the Detection of Pathogens in Dairy Products · Rapid Test for the Detection of Pathogens in Dairy Products . 2 ... Pre-Conditions for Using Rapid Tests . 14 ... dairy