"Schneller und spezifischer Nachweis von ... - Interlab-ev.de

"Schneller und spezifischer Nachweis von
pathogenen Keimen in Milchprodukten“
Axel Dellenbusch, Merck KGaA, Germany
28.05.2008 Page 1
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Inhalt
• Time-to-Result verschiedener Testmethoden
• Grundprinzipien: Lateral Flow Tests &
Real time-PCR
• Innovative Schnelltests von Merck
28.05.2008 Page 2
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Time-to-result verschiedener Methoden
7
6
5
4
3
log 10 cfu
Culture + Immunotest
Culture +PCR/
REAL TIME PCR
BIOCHIP
2
1
REAL TIME
TEST
1 2
SHORTENED
CULTURE
3 4 5
Classical Culture + ID
6 7
DAYS
28.05.2008 Page 3
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Rapid Testing
Principle of Lateral Flow Tests
negative
positive
28.05.2008 Page 4
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General Procedure of Lateral Flow Tests
1 ADD MEDIUM 2 ADD SAMPLE 3 BLENDING 4 INCUBATE 5 PIPETTE SAMPLE
6 PLACE SAMPLE NEGATIVE POSITIVE
Read
after
20 -25
min
28.05.2008 Page 5
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Advantages of Lateral Flow tests
compared to ELISA
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 ELISA
Incubate test on lab bench for 20 – 30 min and read result.
Saves costs esp. when only a few samples are to be tested
28.05.2008 Page 6
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PCR: Basic Principle
3‘-
5‘-
Target Sequence
Target Sequence
-5‘
-3‘
dsDNA
3‘-
Target Sequence
-5‘
Denaturation 94°C
5‘-
Target Sequence
-3‘
3‘-
5‘-
5‘-
Target Sequence
Target Sequence
-5‘
-5‘
-3‘
Primer Annealing 60°C
3‘-
5‘-
Target P Sequence
-5‘
5‘- Target P Sequence
-3‘
DNA Elongation 72°C
28.05.2008 Page 7
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-5‘
2 n

Real Time-PCR vs. PCR:
What´s the difference
Real-Time PCR
PCR
Threshold Cycle (CT-value)
Agarose gel electrophoresis
Staining
Photography
28.05.2008 Page 8
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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
28.05.2008 ….
Page 9

Quantitative Polymerase Chain Reaction:
FRET on Roche Light Cycler using Hybridisation probes
Fluorescence Resonance Energy Transfer (FRET)
Advantage
• High specifity
• Lowest risk of „false positive“ results
• Usable for detection of specified microorganisms
28.05.2008 Page 10
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Quantitative Polymerase Chain Reaction:
TaqMan ® Probe Detection
TaqMan ® Probe Detection
Nucleotide (Probe) coupled with
fluorescence dye and quencher
Polymerase with exonuclease
activity
Reporter dye is cleaved
Increasing level of reporter dye
28.05.2008 Page 11
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Technology Platforms I
LightCycler ® 1.5 or 2.0 Instrument (Roche)
Carousel-based System
Capillaries
© Roche Applied Science © Roche Applied Science
28.05.2008 Page 12
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Technology Platform II:
TaqMan ® -based Systems
Applied Biosystems
DuPont Qualicon, BAX Q7
Bio-Rad
Stratagene
Eppendorf
© each supplier
etc….
Roche
28.05.2008 ….
Page 13

Real time-PCR from MERCK
In only 100 minutes from start to result
Start 20 min 40 min 90 min 100 min
Enrichment,
Culture or
Single Colony
Sample
preparation
PCR-set up
PCR run
Detection
and Analysis
28.05.2008 Page 14
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Micobiology Testing with PCR – Why
Main reasons
• Convenience
• Time Savings
• Accuracy
• Ease-of-use
• No further confirmation(s) necessary
• Cost savings overall
(storage, insurance, recalls etc.)
28.05.2008 Page 15
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Sample Preparation Modules
‣ foodproof ShortPrep I Kit
foodproof ShortPrep II Kit
‣ foodproof ShortPrep III Kit
(for Salmonella spp.: useful for 98% of all food)
(for E. coli O157, Campylobacter and Listeria spp.)
(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
‣ Reagent D
(for Enterobacteriaceae /E.sakazakii)
(for Enterobacteriaceae /E.sakazakii)
‣ Suspension Buffer
28.05.2008 Page 16
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Real-Time PCR Kits for Food Safety Testing:
Products based on Roche Light Cycler
‣ foodproof GMO Maize Quantification Kit
‣ foodproof GMO Soya Quantification Kit
‣ foodproof GMO Screening Kit
‣ foodproof Salmonella Detection Kit
‣ foodproof Listeria monocytogenes Detection Kit
‣ foodproof Listeria Genus Detection Kit
‣ foodproof E. coli 0157 Detection Kit
‣ foodproof Campylobacter Detection Kit
‣ foodproof Beer Screening Detection Kit
‣ foodproof Enterobacteriaceae plus E. sakazakii
The big 4!
1. Wave: compatible with Light Cycler 1.X and 2.0
28.05.2008 Page 17
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Foodproof RT-PCR Kits for Food Safety Testing:
Products based on other Instruments
‣ 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)
28.05.2008 Page 18
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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:
• STEC:
• Foodborne viruses:
• Clostridium botulinum:
(EU: Draft, Germany: Draft)
(EU: Draft, Germany: Draft)
(EU: Draft, Germany: Draft)
(EU: Draft, Germany: Draft)
28.05.2008 Page 19
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APPLICATION:
Rapid Detection of Listeria monocytogenes
in food with Singlepath ® L’mono
28.05.2008 Page 20
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Established Pathogen:
Listeria monocytogenes
• 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)
28.05.2008 Page 21
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ISO Standard 11290-2004 for testing of
Listeria in Food and Animal feeds
Day
1
STREAK OUT
ON ALOA ® +
PALCAM
37°C FOR 24 - 48h
25 g/ml TEST SAMPLE
in 225 ml 1/2 STRENGTH FRASER
30°C FOR 24h
Day
3-4
0.1 ml in 10 ml FRASER
35 OR 37°C FOR 48h
Day
3-6
Day
4-7
STREAK OUT
ON ALOA ® + PALCAM
37°C FOR 24 - 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.
BIOCHEMICAL CONFIRMATION: GRAM (+), CATALASE (+), MOTILITY (+),
CAMP (+), β HAEMOLYSIS (+), GLUCOSE (+), RHAMNOSE (+), XYLOSE (-)
28.05.2008 Page 22
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Rapid Testing
Singlepath ® L’ mono
• 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 colonies
on Listeria selective agars like Chromocult ® Listeria Agar
(ALOA ® ), PALCAM Agar, Oxford Agar
• Multiple food enrichment media: Fraser, LEB, UVM
28.05.2008 Page 23
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Rapid Testing
Singlepath ® L’ mono - Screening
Day
1
25 g/ml TEST SAMPLE
in 225 ml 1/2 STRENGTH FRASER
30°C FOR 24h
Day
2
0,1 ml in 10 ml LEB or
Full Fraser OR UVM
30 ° / 37°C FOR 21 - 24 h
150µl
Day
3
L.monocytogenes
not present
L.monocytogenes
present
Confirmation
ALOA ®
37°C for 24 - 48h
28.05.2008 Page 24
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Traditional Listeria
Identification/confirmation tests
Features of traditional biochemical
identification tests such as API:
1. Multiple handling steps
2. Additional incubation time
28.05.2008 Page 25
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Rapid Testing
Singlepath ® L’ mono - Confirmation
suspend 1 - 3 presumptive
colonies in 250 µl BHI or
CASO or Fraser or PALCAM
Broth
ALOA ® Agar
1 h at 37°C
PALCAM Agar
OXFORD Agar
150 µl
L.monocytogenes
not confirmed
L.monocytogenes
confirmed
28.05.2008 Page 26
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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. monocytogenes
21x Other Listeria spp.
15x Other bacteria
28.05.2008 Page 27
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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 10 7 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
28.05.2008 Page 28
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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 (

Application:
Rapid Detection of Bacillus cereus in food
with Duopath ® Cereus Enterotoxins
28.05.2008 Page 30
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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)
28.05.2008 Page 31
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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
28.05.2008 Page 32
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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)
28.05.2008 Page 33
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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
28.05.2008 Page 34
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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
28.05.2008 Page 35
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Duopath ® Cereus Enterotoxins
• Worldwide first Lateral Flow Test for the detection of Bacillus cereus via
Enterotoxins HBL and NHE
ELISA TECRA:
RPLA Oxoid:
detects only NHE Toxin
detects only HBL Toxin
• For food screening
• For confirmation of presumptive B. cereus colonies from
selective agars (e.g. M.Y.P. Agar acc. to MOSSEL)
• To be used in combination with new Casein Peptone Glucose Yeast Extract (CGY)
Broth
28.05.2008 Page 36
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Duopath ® Cereus Enterotoxins Applications
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 h
Detection limit: 1 colony on agar plate
Enrichment of B. cereus in new CGY (+ 1% Glucose) Broth
induces Enterotoxin production significantly
28.05.2008 Page 37
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Screening of enterotoxinogenic Bacillus cereus
in Food using Duopath ® Cereus Enterotoxins
day
1
Rapid Screening
(>100 CFU / g)
10 g/ml sample into
90 ml CGY Broth +1% Glucose
or BHI or PBS
Sensitive Screening
(>1 CFU / g)
Transfer 200 µl in 20 ml CGY+1%
Glucose: 18 –24 h at 37°C
Transfer 200 µl in 20 ml CGY+1%
Glucose or BHI: 18 –24 h at 37°C
day
2
150 µl onto Duopath
200 µl in 20 ml CGY + 1% Glucose
6 h at 37 °C
150 µl onto Duopath
NO
ET-B. cereus
present
YES
ET-B. cereus
present
28.05.2008 Page 38
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Confirmation of enterotoxinogenic
Bacillus cereus using Duopath ® Cereus
day
1 suspend
1- 3 presumptive colonies into 1
ml CGY Broth + 1% Glucose,
4 h at 37°C,
150 µl onto Duopath
M.Y.P. Agar
NO
ET-B. cereus
not present
YES
ET-B. cereus
present
28.05.2008 Page 39
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Evaluation (Univ. Munich) of Duopath ® Cereus:
Native rice
5 different rice qualities (risotto, native, etc.) used for evaluation
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) - - + - +
n.d.= not done
Conclusion:
Duopath Sensitive Method correctly detected even low numbers of different
B. cereus (NHE- or NHE+HBL producers) in rice samples
28.05.2008 Page 40
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Evaluation (Univ. Munich) of Duopath ® Cereus:
Native baby foods
Native baby foods: 3x Milk powders for babies (1. months and 4. months),
3x Milk porridge with fruits (4. months and 8. months)
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. - - - - -
n.d. = not done
* Sample #90 contained NHE-producing B. cereus in high number; all other samples contained low numbers of
NHE-producing B. cereus
Conclusion:
Duopath Sensitive Method correctly detected B. cereus in ALL baby food samples
28.05.2008 Page 41
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Evaluation of Duopath ® Cereus:
Internal trials
Internal food trials:
- Food samples tested: Semolina pudding,
Frozen spinach,
Coffee whitener,
Egg powder,
Cocoa powder
Results:
Duopath Cereus correctly detected B. cereus in ALL tested food samples
28.05.2008 Page 42
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Merck‘s Rapid Test Product Portfolio
8 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 Base
Fraser Rappaport.-Vas. mTSB + n CCDA Agar CGY Broth BCYE Suppl.
UVM Selenite Cystine CT-SMAC BHI GVPC Suppl.
PALCAM Salmosyst SMAC CYE-Plates
OXFORD M Broth BHI GVPC-Plates
Chromocult ® XLT 4 , XLD, CAYE Anaerocult ® C
Listeria Agar Rambach ®
28.05.2008 Page 43
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foodproof Enterobacteriaceae plus E. sakazakii
28.05.2008 Page 44
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Enterobacter sakazakii
EC Regulation 2073
• Absence of E. sakazakii in 10 g milk powder
• Mandatory for all infant formula producers
28.05.2008 Page 45
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Enterobacter sakazakii Agar
Day
1
ISO TS 22964
Test sample
in BPW (1:10)
37°C for 16 - 20 h
Day
2
0,1 ml
10 ml mLST – Vancomycin Medium
45°C for 22 - 26 h
Day
3
Streak onto
Enterobacter sakazakii Isolation Agar
44°C for 22 - 26 h
Day
4
Biochemical Confirmation
28.05.2008 Page 46
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Chromocult ® Enterobacter sakazakii Agar
• 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
28.05.2008 Page 47
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Chromocult ® Enterobacter sakazakii Agar
Chromogenic Culture Medium for the Detection and
Enumeration of Enterobacter sakazakii in Milk Powder and
Powdered Infant Formula.
E. sakazakii
Chromocult ® Enterobacter sakazakii Agar # 1.00873
28.05.2008 Page 48
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Enterobacter sakazakii Agar
E. sakazakii Isolation Agar – Comparison
MERCK Competitor 2 Competitor 1
Colony colour
blue-green
green
blue
Background colour
yellowish
brownish
violet
Easy reading
+++
++
++
Incubation temperature
44°C ± 1°C
36°C ± 1°C
44°C ± 1°C
Sensitivity*
100 %
100 %
100 %
Specificity*
100 %
95 %
97 %
*
M. Manafi and Kerstin Lang, Hygiene Institute, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria
Comparison of three chromogenic Media for Detection of Enterobacter sakazakii;
Poster presented at ASM Meeting 2005, Atlanta , USA
28.05.2008 Page 49
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Detection of Enterobacteriaceae /
E. sakazakii / Salmonella by RT-PCR
Enrichment
20-24 h
DNA Extraction
PCR 1
Enterobacteriaceae/E. sakazakii
PCR 2
Salmonella
CH 610
IPC
CH 640
Enterobacteriaceae
CH 670
E. sakazakii
Negative Result
Positive Result
Enterobacteriaceae neg.
E. sakazakii neg.
Salmonella neg.
28.05.2008 Page 50
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The RT-PCR foodproof Enterobacteriaceae plus
E. sakazakii Detection System
Sample Preparation Procedure: Use StarPrep One Kit + Reagent D
‣ 100 µl enriched sample Specific and validated
Enrichment
e.g. BPW + BPW
‣ Add 300 µl Reagent D protocol
‣ Incubate 5 min. in the dark
‣ Expose to light for 5 min
Kit Module
DNA ‣ Extraction
Centrifugation for 5 min
Sample preparation StartPrep One Kit
‣ Remove supernatant, add 200 µl lysis buffer and resuspend pellet
‣ Incubate 10 min. at 95°C
‣ Centrifugation for 2 min
Amplification /
Kit Module
‣ Use 2.5 - 5 µl supernatant
Detection
LightCycler- for PCR PCR
Total time for sample preparation:
ca. 45 minutes for 30 samples
28.05.2008 Page 51
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E. sakazakii – PCR vs. Conventional Detection
Conventional
(ISO 22964:2006)
Pre-Enrichment
16 – 20 h
Enrichment
22 - 26 h
Isolation
22 - 26 h
Biochemical
Identification
44 - 48 h
PCR
Pre-Enrichment
18 h
Subcultivation
3 h
DNA Isolation
0.5 h
Amplification/
Detection 1 h
Time to result < 1 day
28.05.2008 Page 52
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Reduction of Dead Enterobacteriaceae
in 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
28.05.2008 Page 53
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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 10 6 dead bacteria / g
28.05.2008 Page 54
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Reduction of Dead Enterobacteriaceae
by Reagent D
Without Reagent D
With Reagent D
28.05.2008 Page 55
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foodproof Enterobacteriaceae plus
E. sakazakii Detection Kit vs. other PCR tests
‣ 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 methods
‣ Disadvantage: Often not carefully validated
28.05.2008 Page 56
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Foodproof kits Approvals
AOAC:
NordVal:
MicroVal:
foodproof Salmonella
foodproof Listeria monocytogenes
foodproof E. coli O157
foodproof Salmonella
foodproof Listeria monocytogenes
foodproof E. coli O157
foodproof Enterobacteriaceae plus E. sakazakii
(end of 2008)
28.05.2008 Page 57
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Thank you for your attention
28.05.2008 Page 58
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