28.05.2008 Page 1….

"Schneller und spezifischer Nachweis von

pathogenen Keimen in Milchprodukten“

Axel Dellenbusch, Merck KGaA, Germany

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Inhalt

• Time-to-Result verschiedener Testmethoden

• Grundprinzipien: Lateral Flow Tests &Real time-PCR

• Innovative Schnelltests von Merck

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Classical Culture + ID

Culture + Immunotest

Culture +PCR/REAL TIME PCR

BIOCHIP

3 4 51 2DAYS

log10 cfu

12

3

4

56

7

6 7

REAL TIME TEST

Time-to-result verschiedener Methoden

SHORTENED CULTURE

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Principle of Lateral Flow TestsRapid Testing

negative

positive

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3 BLENDING 2 ADD SAMPLE1 ADD MEDIUM 4 INCUBATE 5 PIPETTE SAMPLE

6 PLACE SAMPLE NEGATIVE POSITIVE

Readafter20 -25

min

General Procedure of Lateral Flow Tests

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1. Control reaction built in LFT.No need for separate reaction to demonstrate proper function, as necessary for ELISA.

2. LFT’s are 1-step tests.After applying test sample to sample port, NO further pipetting steps necessary.No separate colour reaction for LFT’s necessary to make reactions visible.

3. LFT provide results faster than ELISAIncubate test on lab bench for 20 – 30 min and read result.

Saves costs esp. when only a few samples are to be tested

Advantages of Lateral Flow tests

compared to ELISA

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2n5‘- -3‘

dsDNA

Denaturation 94°C

Primer Annealing 60°C

DNA Elongation 72°C

Target Sequence

Target Sequence

Target Sequence

Target Sequence

Target Sequence

Target Sequence

Target Sequence

Target Sequence

P

P

5‘-

5‘-

5‘-

-5‘

-5‘

-5‘

-5‘

-3‘

-3‘

3‘-

3‘-

3‘-

3‘-

-3‘

-5‘

5‘--5‘

5‘-

PCR: Basic Principle

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Agarose gel electrophoresisStainingPhotography

PCRReal-Time PCR

Real Time-PCR vs. PCR: What´s the difference?

Threshold Cycle (CT-value)

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Quantitative Polymerase Chain Reaction:Intercalation of Fluorophor

SYBR Green; Intercalation Fluorophor

Advantage

Cheapest quantitative PCR format

Usable for detection of a whole group of microorganisms

Disadvantage

High risk of false positive result because of unspecific amplification signals

ISO does not allow SYBR Green in food sector

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Fluorescence Resonance Energy Transfer (FRET)

Quantitative Polymerase Chain Reaction:FRET on Roche Light Cycler using Hybridisation probes

AdvantageHigh specifity

Lowest risk of „false positive“ results

Usable for detection of specified microorganisms

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Quantitative Polymerase Chain Reaction:TaqMan® Probe Detection

Nucleotide (Probe) coupled with

fluorescence dye and quencher

Polymerase with exonucleaseactivity

TaqMan® Probe Detection

Reporter dye is cleaved Increasing level of reporter dye

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Technology Platforms I LightCycler® 1.5 or 2.0 Instrument (Roche)

© Roche Applied Science © Roche Applied Science

Carousel-based System Capillaries

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Technology Platform II:TaqMan®-based Systems

Applied Biosystems DuPont Qualicon, BAX Q7

StratageneEppendorf

Roche

Bio-Rad

© each supplieretc….

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Real time-PCR from MERCK

Start 20 min 40 min 100 min90 min

PCR-set up PCR runSample

preparationEnrichment, Culture or

Single ColonyDetection

and Analysis

In only 100 minutes from start to result

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Micobiology Testing with PCR – Why?

• Convenience• Time Savings • Accuracy• Ease-of-use• No further confirmation(s) necessary

• Cost savings overall (storage, insurance, recalls etc.)

Main reasons

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Sample Preparation Modules

foodproof ShortPrep I Kit (for Salmonella spp.: useful for 98% of all food)foodproof ShortPrep II Kit (for E. coli O157, Campylobacter and Listeria spp.)foodproof ShortPrep III Kit (for beer screening)

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

StarPrep One Kit (for Enterobacteriaceae /E.sakazakii)Reagent D (for Enterobacteriaceae /E.sakazakii)

Suspension Buffer

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Real-Time PCR Kits for Food Safety Testing:

Products based on Roche Light Cycler

foodproof GMO Maize Quantification Kitfoodproof GMO Soya Quantification Kitfoodproof GMO Screening Kitfoodproof Salmonella Detection Kitfoodproof Listeria monocytogenes Detection Kitfoodproof Listeria Genus Detection Kitfoodproof E. coli 0157 Detection Kitfoodproof Campylobacter Detection Kitfoodproof Beer Screening Detection Kitfoodproof Enterobacteriaceae plus E. sakazakii

1. Wave: compatible with Light Cycler 1.X and 2.0

The big 4!

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Salmonella spp.

Listeria monocytogenes

Listeria Genus

E. coli O157

Campylobacter

Enterobacteriaceae + E. sakazakii

2. Wave: compatible with many other instruments(e.g. ABI, Stratagene, Eppendorf, Rotorgene)

Foodproof RT-PCR Kits for Food Safety Testing:Products based on other Instruments

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ISO-Standards for PCR and real time-PCR

• ISO-Standards define specific requirements for PCR techniques:

General requirements for the detection of foodborne pathogens (ISO/DIS 22119:2007)

Sample preparation for qualitative PCR methods (ISO 20837:2006)

• Additional documents on regulations for single pathogens:

• Salmonella: (EU: Draft, Germany: DIN 10135)

• L. monocytogenes: (EU: Draft, Germany: Draft)

• STEC: (EU: Draft, Germany: Draft)

• Foodborne viruses: (EU: Draft, Germany: Draft)• Clostridium botulinum: (EU: Draft, Germany: Draft)

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APPLICATION: Rapid Detection of Listeria monocytogenes

in food with Singlepath® L’mono

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• Genus Listeria comprises 6 different species

• Only L. monocytogenes can be pathogenic for humans and animals

Disease: Systemic Infections: Meningitis, Encephalitis, Septicemia

Local Infections: Gastroenteritis

Frequency: Only 7 / 1 Mio. individuals suffer from Listeriosis

But: Mortality rate is extremely high (up to 30%)

Resistance: Multiply at 2°C, survive many preservation methods(10% osmolarity, pH 5.0, 55°C)

Established Pathogen: Listeria monocytogenes

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Day1

Day3-6

25 g/ml TEST SAMPLEin 225 ml 1/2 STRENGTH FRASER

30°C FOR 24h

STREAK OUTON ALOA® + PALCAM

37°C FOR 24 - 48h

Day3-4

ISO Standard 11290-2004 for testing of Listeria in Food and Animal feeds

Day4-7

0.1 ml in 10 ml FRASER35 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 OUTON ALOA® +

PALCAM37°C FOR 24 - 48h

BIOCHEMICAL CONFIRMATION: GRAM (+), CATALASE (+), MOTILITY (+), CAMP (+), β HAEMOLYSIS (+), GLUCOSE (+), RHAMNOSE (+), XYLOSE (-)

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Singlepath® L’ monoRapid Testing

• Worldwide first Lateral Flow Test for the SPECIFIC detection of Listeria monocytogenes

• For screening of environmental and food samples

• For the confirmation of presumptive positive colonieson Listeria selective agars like Chromocult® Listeria Agar(ALOA®), PALCAM Agar, Oxford Agar

• Multiple food enrichment media: Fraser, LEB, UVM

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Day1

Day3

0,1 ml in 10 ml LEB orFull Fraser OR UVM

30° / 37°C FOR 21 - 24 h

ALOA®

37°C for 24 - 48h

Day2

L.monocytogenespresent

Singlepath® L’ mono - ScreeningRapid Testing

L.monocytogenesnot present

150µl

25 g/ml TEST SAMPLE in 225 ml 1/2 STRENGTH FRASER

30°C FOR 24h

Confirmation

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Traditional ListeriaIdentification/confirmation tests

Features of traditional biochemicalidentification tests such as API:

1. Multiple handling steps2. Additional incubation time

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suspend 1 - 3 presumptivecolonies in 250 µl BHI or

CASO or Fraser or PALCAM Broth

L.monocytogenesconfirmed

PALCAM Agar OXFORD AgarALOA® Agar

L.monocytogenesnot confirmed

Singlepath® L’ mono - ConfirmationRapid Testing

150 µl

1 h at 37°C

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Evaluation of Singlepath® L‘mono(University of Giessen, Germany)

Singlepath L‘ mono as screening test:• 40 food samples (smoked salmon, pâté, minced meat, cheese) artificially spiked with 7-

230 CFU / 10 g

• 38 / 40 samples positive after 24 h in Full Fraser; 2 samples positive after additional 24 h

• 10 / 17 native contaminated smoked salmon samples positive Singlepath® results by testing Full Fraser after 24 h incubation (100% agreement with culture method)

100% sensitivity/specificity with food samples

Singlepath L‘ mono as confirmatory test:

• 100% sensitivity/specificity with pure cultures: 37x L. monocytogenes21x Other Listeria spp.15x Other bacteria

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Evaluation of Singlepath® L`mono(MUVA, Kempten, Germany)

Singlepath L `mono for screening of L. monocytogenes in ice cream:

• 30 vanilla ice cream samples each 25 gr. spiked with 2 diff. L. mono. strains and 2 diff. spiking levels (10-100 CFU /25 gr & 100-1000 CFU /25 gr)

• After inoculation, samples were refrozen and stored for 24 h at -20°C

• For pos. controls, samples were spiked with 107 CFU/25 gr and tested as above.

• Spiked samples were homogenized in ½ Fraser and incubated for 24 - 48h.

• Application of 180 µl enriched sample on Singlepath L’ mono

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Evaluation of Singlepath® L`mono(MUVA, Kempten, Germany)

RESULTS:• 180 µl sample volume should be used when 1 broth is used only. In this

case, the flow of the sample over the membrane is not completed.

• After 24 h enrichment at 30°C in ½ Fraser, results are consistent with ISO-11290-1.

• For detection of very low CFU (<10 CFU/25 gr food), 48h is recommended

CONCLUSION:• Singlepath L’ mono reliably detected even low numbers of L.

monocytogenes within 24 -48h using only 1 enrichment step.

• Even if 48h incubation is necessary, the method is 3 days (for negative samples to 8 days (for positive samples) faster than the ISO method.

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Application:Rapid Detection of Bacillus cereus in food

with Duopath® Cereus Enterotoxins

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Bacillus cereus – Foodborne illness

2 Types of Infections:

1. Diarrheal syndrome = Toxicoinfection

• Enterotoxin(s) produced during vegetative growth of B. cereus in small intestine

• Associated with ingestion of B. cereus producing heat-labile toxins (occurs within 8-12 hours)

2. Emetic syndrome (vomiting) = Intoxication

• Cereulide Toxin preformed in food• Usually associated with the ingestion of a heat-stable toxin

from contaminated rice (occurs within 1-6 hours)

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Bacillus cereus: Sources of infections

Meat products, soups, milk & milk products, vegetables,

puddings & sauces

– for diarrhoeal syndrome

Rice, pasta, pastry, starchy products

– preferentially emetic syndrome

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Enterotoxins produced by B. cereus

Five enterotoxins:

• Hemolysin BL (Hbl)

• Nonhemolytic enterotoxin (Nhe) Major toxins

• Cytotoxin K (CytK)

• Enterotoxin T (BceT)

• Enterotoxin FM (EntFM)

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Hemolysin BL (Hbl)

• About 50% of B. cereus produce Hbl

• 3 components (B, L1, L2) – all necessary for enterotoxin activity

• B – binds to the target cells

• L1, L2 – have lytic functions

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Nonhemolytic enterotoxin (Nhe)

• >90 % of all B. cereus produce Nhe

• Consists of 3 components

• Most biological activity when all components are present

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• Worldwide first Lateral Flow Test for the detection of Bacillus cereus via Enterotoxins HBL and NHE

ELISA TECRA: detects only NHE Toxin

RPLA Oxoid: detects only HBL Toxin

• For food screening

• For confirmation of presumptive B. cereus colonies fromselective agars (e.g. M.Y.P. Agar acc. to MOSSEL)

• To be used in combination with new Casein Peptone Glucose Yeast Extract (CGY) Broth

Duopath® Cereus Enterotoxins

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1. Rapid Screening of Bacillus cereus in foods within 24 h

Detection limit: >100 CFU / g or ml food

2. Sensitive Screening of Bacillus cereus in foods within 30 h

Detection limit: 1 CFU / g or ml food

3. Confirmation of suspect Bacillus cereus colonies from selective agars within 4 hDetection limit: 1 colony on agar plate

Enrichment of B. cereus in new CGY (+ 1% Glucose) Brothinduces Enterotoxin production significantly

Duopath® Cereus Enterotoxins Applications

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day1

day2

10 g/ml sample into90 ml CGY Broth +1% Glucose

or BHI or PBS

Screening of enterotoxinogenic Bacillus cereusin Food using Duopath® Cereus Enterotoxins

Transfer 200 µl in 20 ml CGY+1% Glucose: 18 –24 h at 37°C

Sensitive Screening(>1 CFU / g)

Rapid Screening(>100 CFU / g)

200 µl in 20 ml CGY + 1% Glucose6 h at 37 °C

150 µl onto Duopath

YESET-B. cereus

present

NO ET-B. cereus

present

Transfer 200 µl in 20 ml CGY+1% Glucose or BHI: 18 –24 h at 37°C

150 µl onto Duopath

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day1 suspend

1- 3 presumptive colonies into 1 ml CGY Broth + 1% Glucose,

4 h at 37°C,150 µl onto Duopath

Confirmation of enterotoxinogenicBacillus cereus using Duopath® Cereus

M.Y.P. Agar

YESET-B. cereus

present

NO ET-B. cereusnot present

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5 different rice qualities (risotto, native, etc.) used for evaluation

Evaluation (Univ. Munich) of Duopath® Cereus: Native rice

Native Rice sample # 25 # 28 # 36 # 59 # 73

MPN B. cer. / g 7,5 0,92 0,74 0,92 240

NHE (Rapid) n.d. n.d. n.d. n.d. n.d.

HBL (Rapid) n.d. n.d. n.d. n.d. n.d.

NHE (Sensitive) + + + + +

HBL (Sensitive) - - + - +

Conclusion:Duopath Sensitive Method correctly detected even low numbers of differentB. cereus (NHE- or NHE+HBL producers) in rice samples

n.d.= not done

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Native baby foods: 3x Milk powders for babies (1. months and 4. months),

3x Milk porridge with fruits (4. months and 8. months)

* Sample #90 contained NHE-producing B. cereus in high number; all other samples contained low numbers of NHE-producing B. cereus

Evaluation (Univ. Munich) of Duopath® Cereus: Native baby foods

Baby food No. # 90 # 91 # 92 # 101 # 107 # 111

MPN B. cer. / g 93 2,1 0,74 0,36 0,74 0,92

NHE (Rapid) +* - - - - -

HBL (Rapid) - - - - - -

NHE (Sensitive) n.d. +* +* +* +* +*

HBL (Sensitive) n.d. - - - - -

Conclusion:Duopath Sensitive Method correctly detected B. cereus in ALL baby food samples

n.d. = not done

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Internal food trials:

- Food samples tested: Semolina pudding,

Frozen spinach,

Coffee whitener,

Egg powder,

Cocoa powder

Evaluation of Duopath® Cereus: Internal trials

Results:Duopath Cereus correctly detected B. cereus in ALL tested food samples

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Merck‘s Rapid Test Product Portfolio8 Rapid tests for the most common pathogens

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

LEB Tetrathionate mEC + n Bolton Broth M.Y.P. Agar CYE Agar BaseFraser Rappaport.-Vas. mTSB + n CCDA Agar CGY Broth BCYE Suppl.UVM Selenite Cystine CT-SMAC BHI GVPC Suppl.PALCAM Salmosyst SMAC CYE-PlatesOXFORD M Broth BHI GVPC-PlatesChromocult® XLT4 , XLD, CAYE Anaerocult® CListeria Agar Rambach®

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foodproof Enterobacteriaceae plus E. sakazakii

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Enterobacter sakazakii

EC Regulation 2073

• Absence of E. sakazakii in 10 g milk powder

• Mandatory for all infant formula producers

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Day1

Day3

Test samplein BPW (1:10)

37°C for 16 - 20 h

10 ml mLST – Vancomycin Medium45°C for 22 - 26 h

Day2

Streak ontoEnterobacter sakazakii Isolation Agar

44°C for 22 - 26 h

Biochemical ConfirmationBiochemical Confirmation

ISO TS 22964

Enterobacter sakazakii Agar

Day4

0,1 ml

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• The alpha-D-Glucosidase, an enzyme specific for E. sakazakii, is used for identification

• Only colonies of E. sakazakii appear turquoise, other bacteria grow colourless

•Detection in only 1 day on agar. Potential inhibitors and high incubation temperature suppress growth of accompanying bacteria

Chromocult® Enterobacter sakazakii Agar

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Chromogenic Culture Medium for the Detection and Enumeration of Enterobacter sakazakii in Milk Powder andPowdered Infant Formula.

Chromocult® Enterobacter sakazakii Agar # 1.00873

E. sakazakii

Chromocult® Enterobacter sakazakii Agar

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E. sakazakii Isolation Agar – Comparison

Enterobacter sakazakii Agar

M. Manafi and Kerstin Lang, Hygiene Institute, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, AustriaComparison of three chromogenic Media for Detection of Enterobacter sakazakii;Poster presented at ASM Meeting 2005, Atlanta , USA

*

Colony colourBackground colourEasy readingIncubation temperatureSensitivity*Specificity*

blue-greenyellowish

+++44°C ± 1°C

100 %100 %

greenbrownish

++36°C ± 1°C

100 %95 %

MERCK Competitor 2 Competitor 1

blueviolet

++44°C ± 1°C

100 %97 %

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Detection of Enterobacteriaceae /

E. sakazakii / Salmonella by RT-PCR

PCR 1Enterobacteriaceae/E. sakazakii

Enrichment20-24 h

DNA Extraction

CH 610IPC

CH 640Enterobacteriaceae

CH 670E. sakazakii

Positive Result

PCR 2Salmonella

Negative Result

Enterobacteriaceae neg.E. sakazakii neg.Salmonella neg.

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The RT-PCR foodproof Enterobacteriaceae plus E. sakazakii Detection System

Specific and validatedprotocolEnrichment

Kit ModuleSample preparationDNA Extraction

Amplification / Detection

Kit ModuleLightCycler- PCR

StartPrep One Kit

e.g. BPW + BPW

Sample Preparation Procedure: Use StarPrep One Kit + Reagent D

100 µl enriched sampleAdd 300 µl Reagent DIncubate 5 min. in the darkExpose to light for 5 minCentrifugation for 5 minRemove supernatant, add 200 µl lysis buffer and resuspend pelletIncubate 10 min. at 95°CCentrifugation for 2 min Use 2.5 - 5 µl supernatant for PCR

Total time for sample preparation:ca. 45 minutes for 30 samples

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E. sakazakii – PCR vs. Conventional Detection

Conventional(ISO 22964:2006)

Enrichment

22 - 26 h

Isolation 22 - 26 h

Biochemical Identification

44 - 48 h

Pre-Enrichment16 – 20 h

Time to result < 1 day

PCR

Subcultivation3 h

DNA Isolation 0.5 h

Amplification/ Detection 1 h

Pre-Enrichment18 h

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Reduction of Dead Enterobacteriaceaein the Background

Milk powder and commercially available powdered infant formula may

show high background of inactive cells of Enterobacteriaceae which

can caused a positive signal – what to do about it ?

Two options are available:

Subsequent dilution and subcultivation

Use of Reagent D

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Reagent D for detection of live batceria

Reagent D efficiently eliminates amplification of dead bacteria

Mechanism: Reagent D invades ONLY into dead bacteria and intercalates in DNA. DNA is then not amplifyable any more

Works up to 106 dead bacteria / g

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Reduction of Dead Enterobacteriaceaeby Reagent D

With Reagent D

Without Reagent D

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Competitor RT- PCR System for Detection of E. sakazakii:

Disadvantage: High risk for false positive results

(Nestle is validating foodproof kit for this reason)

Research and “home brewed” PCR methodsDisadvantage: Often not carefully validated

foodproof Enterobacteriaceae plus E. sakazakii Detection Kit vs. other PCR tests

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Foodproof kits Approvals

AOAC: foodproof Salmonellafoodproof Listeria monocytogenesfoodproof E. coli O157

NordVal: foodproof Salmonellafoodproof Listeria monocytogenesfoodproof E. coli O157

MicroVal: foodproof Enterobacteriaceae plus E. sakazakii (end of 2008)

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Thank you for your attention