Project Title:
VALIDATION OF A LOCALLY-DEVELOPED DNA AMPLIFICATION SYSTEM (DAS) FOR THE DETECTION OF Staphylococcus aureus AND E.coli 0157:H7 IN MEAT, MILK AND THEIR PRODUCTS
Research Leader: Margarita A. MercadoUniversity ResearcherM.S. Food Science
Studies and Study Leaders• Optimization of conditions for enrichment of meat and dairy
samplesStudy Leaders: MAMercado and MTMPerez
• Standardization of DNA extraction and PCR conditions for detection of S. aureus in dairy products and E. coli O157:H7 in meat and dairy products
Study Leaders: MAMercado and MTMPerez
• In-house validation of S. aureus DAS kit in dairy products and E. coli O157:H7 DAS kit in meat and dairy products
Study Leaders: MAMercado / SMMercado / MTMPerez
• Validation of S. aureus DAS kit in HACCP implementation of meat and dairy products
Study Leaders: MAMercado / SMMercado / MRCalapardo
Implementing Agency/Station• Lead Agency
– National Institute of Molecular Biology and Biotechnology (BIOTECH) University of the Philippines Los Baños, College, Laguna
• Cooperating Agencies– Philippine Carabao Center (PCC)
UP Los Baños– Dairy Training Research Institute (DTRI)
UP Los Baños– Philippine National Collection of Microorganisms (PNCM) Laboratory,
BIOTECH, UP Los Baños– National Meat Inspection Services (NMIS)– Alaska Milk Corporation
San Pedro, Laguna
• Project Site– National Institute of Molecular Biology and Biotechnology (BIOTECH)
UP Los Baños, College, Laguna
Funding Agency
• Philippine Council for Advanced Science and Technology Research and Development, Department of Science and Technology (PCASTRD-DOST),
Bicutan, Taguig City
Duration: 13 months
• Date Started: July 1, 2007
• Expected Date of Completion: July 31, 2008
• Total Budget: PhP 465,621.00
Significance of the Study
• Rapid and definitive analytical methods targeting the detection of microorganisms are important in the HACCP program.
• Pathogenic microbes are capable of causing illness.
• Conventional methods for detection of pathogens are labor-intensive and time-consuming.
• Disease surveillance and control of pathogens in food production require detection methods that offer greater precision, rapid results and decreased cost.
• BIOTECH, UPLB developed DAS™ kits through DOST funding.
• causes food poisoning
• strains produce potent &
heat-stable enterotoxins
which are extremely
hazardous if ingested
• ubiquitous, occurring in
the mucous membrane &
skin of most warm-
blooded animals, in
wounds or lesions
Staphylococcus aureus
• In hospital-acquired infections, it remains the most
aggressive, most lethal, widespread and among the fastest
to develop resistance to antibiotics.
Large numbers of S. aureus in processed food indicate improper
hygiene, inadequate sanitation and temperature control.
• enterohemorrhagic strain
• causes life-threatening complications in children & the immunocompromised
• produces Shiga-like toxin(s) SLT-I /SLT-II or both
• responsible in foodborneillness outbreaks with a variety of foods
E. coli O157:H7
Implicated Foods
• undercooked hamburgers• raw meat products• apple cider juice • raw and pasteurized milk & water • raw vegetables
General Objective
To validate the S. aureus DASTM andE. coli O157:H7 DAS kits in meat, dairy products and in HACCP applications
Specific Objectives
To determine the enrichment conditions of meat and dairy samples prior to analysis
To determine the DNA extraction method suitable for PCR detection of S. aureus in dairy products and E. coli O157:H7 in meats and dairy products
To optimize the PCR conditions for analysis of meat and dairy products
To compare the S. aureus DAS and E. coli O157:H7 DAS kits with conventional method in terms of percent agreement through in-house validation in meat and dairy products
To validate the S. aureus DAS kit in HACCP implementation of meat and dairy products under artificial contamination and natural conditions
Part 1. S. aureus DASTM
Spiking of samples
with known levels of
S. aureus
Preparation of samples
Gathering of
samples
Cultivation in enrichment brothIncubation for 20 h at
37o C 100 rpm
Enrichment of samples
Serial dilutions of
enriched samples
Plating in BPA
Observation of
presumptive S. aureus
colonies after 48 h
Coagulase test
results after 30 h
DNAse test results
after 24 h
Conventional plating method
Preparation of cell
lysates
Addition of diluted cell
lysates to DAS tubes
Incubation of reaction
tubes in the thermal
cycler for 5 hGel
electrophoresis
Visualization of
PCR products
S. aureus DAS kit method
A. DAS Kit
B. Conventional Plating Method
Comparison of analysis time using S.aureus DASTM kit
and conventional method
Day 1 Day 2
Receive
samples
Enrichment
Pre-PCR
processing
PCRResult
Receive
samples
Enrichment
Plating
Incubation
Pick
presumptive
colonies
Coagulase
and other
ancillary
tests
Day 1 Day 2 Day 3 Day 4 Day 5
Day 6 Day 7 Day 8
Figure 1. Amplification of S. aureus in fresh milk spiked with 4 (low) and 100 (high) total cells of
analyte. Samples were enriched for 24 h using Nutrient-rich (NR) broth + 2.5% NaCl or Buffered
Peptone Water (BPW). DNA was extracted either by boiling (E1) or addition of pronase before
boiling for 10 min (E2).
1kb
Protocol improvement for fresh milk samples
Research AccomplismentsS.aureus DASTM kit
Figure 2. Amplification of S. aureus in five different brands (A,B,C,D,E) of fresh milk
inoculated with analyte at 0,7 (low) and 70 (high) cells/ml. Samples were enriched in
Nutrient-rich broth +2.5% NaCl for 24h at 37°C. Lane (M) 1kb plus ladder (1) A, 0 cell/ml
(2) B, 0 cell/ml (3) C, 7 cells/ml (4) A, 7 cells/ml (5) A, 70 cells/ml (6) B, 7 cells/ml (7) B, 70
cells/ml (8) C,70 cells/ml (9) C, 0 cell/ml (11) D, 70 cells/ml (12) D, 0 cell/ml (13) E 0
cell/ml (14) positive control genomic DNA S. aureus 1350 (15) no template (16) E,
7cells/ml (17) E, 70 cells/ml (18) positive control genomic DNA S. aureus 1350
1kb 1kb
UHT Fresh milk samples
Sample Code Cultural Method (S. aureus colonies in BPA)
S. aureus DAS kit (1.0kb amplification)
Remarks
1A - -
1B - - multiband
1C - - multiband
1D - -
2A - - multiband
2B - -
2C - -
2D - - multiband
3A + +
3B + +
3C + +
3D + +
4A + +
4B - -
4C + + faint
4D + faint 1.0 kb multiband
5A + faint 1.0 kb multiband
5B - - multiband
5C - -
5D - -
Table 1. Cultural and PCR methods of detecting S.aureus from 40 samples of unpasteurized cow’s
milk from Batangas City.
Sample Code Cultural Method
(S. aureus colonies in BPA)
S. aureus DAS kit
(1.0kb amplification)
Remarks
6A + 1.0 kb multiband
6B + 1.0 kb multiband
6C + 1.0 kb multiband
6D + 1.0 kb multiband
7A + - multiband
7B - - multiband
7C - - multiband
7D - - multiband
8A + +
8B - - multiband
8C - -
8D - -
9A - - faint multiband
9B - - faint multiband
9C - - faint multiband
9D - -
10A - -
10B + 1.0 kb multiband
10C + -
10D - -
Table 1 continuation
Percent agreement: 31 x 100 = 77.5%
40
Percent disagreement: 2 x 100 = 5%
40
Percent indeterminate: 7 x100 = 17.5% 40
Raw cow’s milk samples
White Cheese Samples Cultural Method PCR Method (1.0kb)
Uninoculated, replicate 1
Uninoculated, replicate 2
+
+
+
+
21 cells/g, replicate 1
21 cells/g, replicate 2
+
+
+
+
210 cells/g, replicate 1
210 cells/g, replicate 2
+
+
+
+
Table 2. Detection of S. aureus from white cheese unspiked and
spiked (low = 21 cells/g; high = 210 cells/g) with S. aureus
1350. Samples were enriched in nutrient broth +2.5%
NaCl at 37°C for 20h.
Figure 3. Amplification of S. aureus in five different brands
(A,B,C,D,E) of white cheese inoculated with the analyte 0, 24 (low),
240 (high) cells/g. Samples were enriched in Nutrient-rich broth
+2.5% NaCl at 37°C for 24h. Lane (M) 1kb plus ladder (1) positive
control, gDNA S. aureus 1350 (2) no template (3) A, 0 cell/g (4) A, 24
cells/g (5) A, 240 cells/g (6) B, 0 cell/g (7) B, 24 cells/g (8) B, 240 cells/g
(9) C, 0 cell/g (10) C, 24 cells/g (11) C, 240 cells/g (12) D, 0 cell/g (13)
D, 24 cells/g (14) D, 240 cells/g (15) E, naturally-contaminated,
enriched (16) E, naturally-contaminated, enriched.
1kb
White cheese samples
Figure 4. Amplification of S. aureus in uninoculated white cheese (WCu) and
inoculated white cheese (WCi). The white cheese was inoculated with 30 CFU/g
S. aureus 1350. Samples were enriched in Nutrient-rich broth supplemented with
varying salt concentrations (0,3,5,7,10,15,20%) at 37°C at 24h without shaking.Lane
(M) 1 kb+ Ladder (1) WCi, 0% NaCl (2) WCi, 3% NaCl (3) WCi, 5% NaCl (4) WCi, 7% NaCl (5) WCi,
10% NaCl (6) WCi, 15% NaCl (7) WCi, 20%NaCl (8) WCu, 0% NaCl (9) WCu, 3% NaCl (10) WCu, 5%
NaCl (11) WCu, 7% NaCl (12) WCu, 10% NaCl (13) WCu, 15% NaCl (14) WCu, 20%NaCl (15) positive
control, gDNA S. aureus1350.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
1kb
M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 M
Figure 5. Detection of S. aureus using DAS kit in ten unspiked
raw/unpasteurized cow’s milk (CW) and three unspiked raw
carabao’s milk (CB). Samples were enriched in Nutrient-rich broth
with 7% NaCl at 37°C for 24h without shaking. Lane (M) 1kb+ Ladder (1)
CW1 (2) CW2 (3) CW3 (4) CW4 (5) CW5 (6) CW6 (7) CW7 (8) CW8 (9) no template (10)
positive control, gDNAS. aureus1350 (11) CW9 (12) CW10 (13) CB1 (14) CB2 (15) CB3 (16)
positive control, gDNAS. aureus1350
1kb
Raw cow’s milk samples
Table 3. Different samples (N=171) obtained from five collaborators and analyzed for the presence of S. aureus using PCR and cultural methods.
Collabo-
ratorSamples PCR ,
1.0 kb
Cultural,
plating in BPA
S. aureus
CFU/ml
C1 6 – white cheese from carabao’s milk - - 0
1- white cheese from carabao’s milk + + 107
1- raw carabao’s milk + + 107
C2 12- uninoculated liquid blend, powdered filled
milk, lecithin
- - 0
20- uninoculated swab samples - - 0
7- inoculated (103 CFU/g S.aureus 1526) liquid
blend, powdered filled milk, lecithin
+ + 107 - 108
2- inoculated (436 CFU/g S.aureus 1526) swab
samples
+ + 107 - 108
C3 9-raw cow’s milk, white cheese, pasteurized
fresh and choco milk, yoghurt
- - 0
18- raw cow’s milk + + 107 - 109
4-swab from working space, cow’s udder + + 106 - 108
21-swab from equipment, cow’s udder, food
handlers, hose
- - 0
Continuation…Table 3
Collabo
rator
Samples PCR ,
1.0kb
Cultural,
plating in
BPA
S. aureus
CFU/ml
C4 17 - sausage, smoked mortadella, lean pork,
ground pork, pork fat, ground pork fat, pork
and beef emulsion, ground beef, natural
casings
+ + 107 - 108
4 - mortadella, hotdog, beef sausage, lean beef - - 0
22 - swab from equipment, food handlers,
food-contact surfaces
- - 0
3 - swab from manual stuffer, hands of butcher + + 107 - 108
C5 18 - inoculated (108 cells/g S. aureus 1526)
processed meat – longaniza slices, ham,
hotdog, corned beef, skinless longaniza,
cheesedog, deliburger beef, deliburger chicken
+ + 104 - 106
6 - uninoculated processed meat – corned
beef, deliburger beef, deliburger chicken
- - 0
No. of
Isolates
Origin Coagulase
Test
Mannitol
Test
DNAse
Test
Gram
reaction
10 Raw milk, pork, pork emulsion, ground
pork fat, sheep casing, manual stuffer,
swab from hand of butcher
4+ + + (+) cocci
2 Ground pork, pork sausage 4+ - + (+) cocci
9 Raw milk, swab from hand of butcher
and cow’s udder, pork fat, pork
emulsion plus fat cubes
3+ + + (+) cocci
6 Beef emulsion, pork sausage,
mortadella, swab from cow’s udder
3+ - + (+) cocci
1 Pork emulsion 2+ + + (+) cocci
1 Sheep casing 2+ - + (+) cocci
1 Swab from working space 1+ + + (+) cocci
3 Pork casing, ground beef 1+ - + (+) cocci
Staphylococcus aureus 1526 4+ + + (+) cocci
Staphylococcus epidermidis 1800 - - - (+) cocci
Table 4. Ancillary tests of 33 presumptive S. aureus from different
samples that exhibited 1.0 kb amplicon in PCR
Table 5. Inclusivity and exclusivity of S. aureus DASTM kit
No. of strains Source
Target
Amplicon
1.0 kb
S. aureus reference strains 3 PNCM +
S. aureus clinical isolates 13 CPH +
Closely-related bacteria 10
S. saprophyticus ; S. epidermidis ; Streptococcus bovis ;
Proteus vulgaris ; Enterococcus faecalis ; Corynebacterium
flavescens ; C. glutamicum ; Micrococcus luteus ;
Pediococcus acidilactici ; Enterobacter aerogenes
PNCM -
Escherichia coli 20
EIEC/EPEC/EHEC ; clinical isolates;
food isolates ; nonpathogenic strain
CPH/RITM/PNCM
BIOTECH
-
Salmonella typhii / sp. 11 CPH/PGH/PNCM -
Shigella sp. 4 CPH -
Vibrio cholerae 2 PGH -
Listeria monocytogenes 2 RITM/ Korea -
Yersinia enterocolitica 1 RITM -
Bacillus spp. 8 Ohio, USA/PNCM -
Total strains tested 74
Table 6. Correlation of positive result using S. aureus DAS kit with identification by conventional method, coagulase test and
BBL Crystal Identification kit
Number of Strains BIOTECH
DAS kit
Identification
Method
Coagulase
Test
S. aureus reference strain 1
S. aureus clinical isolates 53
S. cohnii ssp. cohnii 8
S. xylosus 6
E. coli 5
+
+
-
-
-
Conventional
Conventional
BBL ID kit
BBL ID kit
Conventional
3+
2+/ 3+ / 4+
-
-
-
Total no. of strains analyzed 73
Summary and ConclusionPart 1. S.aureus DASTM kit
• The protocol for detection of S.aureus from milk and dairy products was optimized. Nutrient rich broth +2.5% was used in the enrichment of UHT-processed fresh cow’s milk, while NR broth +7% NaCl was best for white cheese and raw or unpasteurized milk and for the rest of the samples analyzed.
• The crude DNA from the enriched samples was extracted by boiling with 0.12N NaOH for 10 min. PCR was performed using the developed S.aureus DASTM kit and the result was compared with the culture method of plating in Baird Parker Agar (BPA)
• The S.aureus DASTM kit was validated using 171 different samples (73 swab samples, 52 raw materials and 46 finished products) obtained from two meat and three dairy processing plants in the Philippines.
Summary and ConclusionPart 1. S.aureus DASTM kit
• Results showed that S.aureus was detected in 27% (46 samples) of all the samples (N=171) analyzed by both PCR and cultured methods. The incidence of the microbe was detected most often from 33 raw materials (18 raw cow’s milk, 10 raw meats, 4 natural casings, one raw carabao’s milk). Only 9 out of 73 swab samples were positive for S.aureus. The least incidence of the microbe was detected from 4 finished products (one soft white cheese, one breakfast pork sausage, and 2 smoked mortadella)
• A perfect agreement was obtained between the PCR and cultural methods in the analysis of all samples (N=171). A total of 33 presumptive S.aureus isolates were obtained and purified from all the samples analyzed and their identities were confirmed by ancillary tests such as coagulase production, gram reaction, DNAse test and mannitol utilization.
Part 2. E. coli O157:H7 DAS kit
Table 7. Bacterial strains used
STRAIN CODE BIOTECH Accession No.
SOURCE/REFERENCE
E. coli O157:H7 Ec24 10084 PNCM1
E. coli O157:H7 RITM 10307 RITM2
E. coli O157:H7 (DNA only) TWO2302 - MSU3
E. coli O157:H7 CwM2 10311 CSU4
E. coli O157:H7 CwM3 10310 CSU
E. coli O157:H7 CwM6 10309 CSU
E. coli O157:H7 CwM7 10308 CSU
E. coli O55:H7 (DNA only) - MSU
E. coli DH5a Ec8 PNCM
Shigella sp. Sh1 - CPH5
1 Philippine National Collection of Microorganisms, BIOTECH, UPLB2 Research Institute for Tropical Medicine, Alabang MM3 Michigan State University, East Lansing, Michigan USA4 Cavite State University, Indang, Cavite
Table 8. Inclusivity and exclusivity of E. coli O157:H7 DAS kit
Species/Strain No. of strains Target Amplicon0.3 kb
E. coli O157:H7 reference strains 3 +
E. coli O157:H7 local cattle isolates 4 +
E. coli EIEC 3 -
E. coli EPEC 3 -
E. coli non-pathogenic 4 -
Shigella sp. 2 -
Other enterobacteria: Salmonella, Klebsiellaoxytoca, K. pneumoniae, Enterobacter aerogenes, Proteus vulgaris
10 -
Serratia liquefaciens, S. marcescens 2 -
Erwinia carotovora 1 -
Other foodborne pathogens: Listeriamonocytogenes, Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Vibriocholerae
12 -
B. amyloliquefaciens 1 -
Thermal cycling Gel electrophoresis
Read-out
Washing of cells
DNA Extraction
Set up PCR
E. coli O157:H7 ValidationEnrichment in modified trypticase soy broth (mTSB) +0.02 mg/ml
novobiocin for 20-24 h, 37°C, without shaking
Cultural Method for detection of
E. coli O157:H7
Plating in Sorbitol Mac Conkey agar (CT-SMAC) + 5x10-5
mg/L cefixime + 2.5mg/L tellurite
Pure isolates on tryptic soy agar slants: 36 white cheese isolates; 48 (batch 1) meat isolates; 39 + 83 (batch 2) meat
isolates; 50 (batch 3) isolates
Biochemical tests: IMVIC test, Lysine decarboxylase test,
growth on Eosin Methylene Blue agar & Fluorocult agar
Enrichment in modified trypticase soy broth (mTSB) +0.02
mg/ml novobiocin for 20-24h, 37°C, without shaking
O157 serotyping by latex agglutination
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M M 15 16 17 18 19
0.3 kb
Figure 6. PCR-based detection of EHEC O157:H7 by DAS kit in
artificially- seeded cheese samples. Lane (M) 1 kb plus ladder, (1) positive control,
EHEC O157:H7 genomic DNA B-10307, (2) no template control (3) CS-1, uninoculated (4) CS-2,
seeded 1 cfu/ml (5) CS-3, seeded 200 cfu/ml (6) CS-4, uninoculated, (7) CS-5 seeded 1 cfu/ml
(8) CS-6, seeded 200 cfu/ml (9) CS-7, uninoculated (10) CS-8, seeded 1 cfu/ml (11) CS-9,
seeded 200 cfu/ml (12) CS-10, uninoculated (13) CS-11, seeded 1 cfu/ml (14) CS-12,
seeded 200 cfu/ml (M) 1 kb plus ladder, (15) gDNA B-10307 (16) no template (17) CS-13,
uninoculated (18) CS-14, seeded 1 cfu/ml (19) CS-15, seeded 200 cfu/ml
White cheese samples
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M M 15 16 17 18 19
0.3 kb
Figure 7. PCR-based detection of EHEC O157:H7 by DAS kit in
artificially-seeded liquid milk samples. Lane (M) 1 kb plus ladder, (1) positive
control, EHEC O157:H7 genomic DNA B-10307, (2) no template control (3) MS-1,
uninoculated (4) MS-2, seeded 1 cfu/ml (5) MS-3, seeded 200 cfu/ml (6) MS-4, uninoculated,
(7) MS-5 seeded 1 cfu/ml (8) MS-6, seeded 200 cfu/ml (9)MS-7, uninoculated (10) MS-8,
seeded 1 cfu/ml (11) MS-9, seeded 200 cfu/ml (12) MS-10, uninoculated (13) MS-11,
seeded 1 cfu/ml (14) MS-12, seeded200 cfu/ml (15) gDNA B-10307 (16) no template (17)
MS-13, uninoculated (18) MS-14,seeded1 cfu/ml(19)MS-15,seeded 200cfu/ml
UHT Fresh Milk samples
Figure 8. PCR based detection of EHEC O157:H7 by DAS kit in
unseeded hamburger patties samples at three periods of
enrichment. Lane (1) positive control, EHEC O157:H7 gDNA (10311), (2) HP-1 (K),
(3) HP-4, (L) (4) HP-7, (M) (5) HP-10, (N) (6) HP-13, (O) (7) HP-1 (8) HP-4 (9) HP-7
(10) HP-10 (11) HP-13 (12) HP-1 (13) HP-4 (14) HP-7 (15) HP-10 (16) HP-13 (17) no
template
0.3 kb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
+ T0 T6 T24
Figure 9. PCR-based detection of EHEC O157:H7 by DAS kit in unseeded and
artificially-seeded hamburger patties samples. a. Lane (M) 1 kb plus ladder, (1) HP-1
uninoculated, (2) HP-2, seeded 30 cfu/g (3) HP-3, seeded 600 cfu/g (4) HP-4, uninoculated (5) HP-5,
seeded 30 cfu/g (6) HP-6, seeded 600 cfu/g (7) HP-7 uninoculated (8) HP-8, seeded 30 cfu/g (9) HP-9,
seeded 600 cfu/g (10) HP-10, uninoculated (11) HP-11, seeded 30 cfu/g (12) HP-12, seeded 600 cfu/g
(13) positive control,EHEC O157:H7gDNA [B-10307], (14) no template. b. Lane (M) 1 kb plus ladder, (1)
HP-13, uninoculated (2) HP-14, seeded 30 cfu/g (3) HP-15, seeded 600 cfu/g (7) positive control, EHEC
O157:H7 gDNA B-10307
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M M 1 2 3 4 5 6 7
0.3 kb
0.3 kb
a. b.
U U U U + U +
Batch 1 meat samples
M 1 2 3 4 5 6 7 8 9 10 11 M
Figure 10. PCR based detection of EHEC O157:H7 by
DAS kit in Batch 2 unseeded hamburger patties
samples. Lane (M) 1 kb plus ladder, (1) sample A, (2) sample B, (3)
sample C, (4) sample D, (5) sample E, (6) sample F, (7) sample G, (8)
sample H, (9) sample I, (10) no template, (11) positive control, EHEC
O157:H7 gDNA (10307)
0.3 kb
Batch 2 meat samples
Figure 11. PCR-based detection of E. coli O157:H7 in
various samples along the meat processing line. Lane (M) 1
kb plus labber, (1) C1, (2) C2, (3) C3, (4) C4, (5) C5, (6) MD1, (7) MD2, (8) E.
coli O157:H7 gDNA, 10307, (9) no template control, (10) B1, (11) B2, (12) B3,
(13) P1, (14) P2, (15) P3, (16) P4, (17) P5, (18) P6, (19) SC2, (20) FP1, (21)
FP2, (22) FP3, (23) FP4, (24) FP5, (25) FP6, (26) E. coli O157:H7 gDNA, 10307
1 2 3 4 5 6 7 8 M M 9 10 11 12 13 14 15 16 17 18 19 20 21 M 22 23 24 25 26 M
0.3 kb
Batch 3: carcass swabs, meat ingredients and
finished products in a meat processing plant
Sample No. of presumptive
colonies screened
by IMViC
E. coli ID No. of colonies
screened by
PCR
No. of
colonies
positive for 0.3
kb amplicon
White cheese
A 9 0 1 0
B 9 0 2 0
C 9 1 [9b] 1 [9b] 1 [9b]
D 0 0 4 0
E 9 0 3 0
Total 36 1 11 1
Table 9. Verification of isolates obtained from white cheese and hamburger
patties samples
Sample No. of presumptive
colonies screened
by IMViC
E. coli ID No. of colonies
screened by PCR
No. of colonies
positive for 0.3
kb amplicon
Frozen hamburger
patties
K 10 0 6 0
L 7 0 2 0
M 15 0 0 nd
N 10 0 4 0
O 6 0 9 0
Total 48 0 21 0
Control strains
E. coli B-1098 - positive - negative
E. coli O157:H7
B-10084
- positive - positive
Con’t… Table 9
0.3 kb
0.3 kb
[M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
Figure 12. Colony PCR screening of non sorbitol - fermenting isolates obtained
from the raw ingredients in the meat processing plant using primers for E. coli
O157:H7. Lane (M) 1 kb plus ladder, (1,) P1a, (2) P1b, (3) P1c, (4) P2a, (5) P2b, (6) P2c, (7) P3a, (8) P4b, (9) P5a,
(10) P5b, (11) P5c, (12) P5d, (13) B2a, (14) B2b, (15) E. coli O157:H7 10311, (16) B2c, (17) B2d, (18) B3b, (19) B3c,
(20) B3d, (21) Md1b, (22) Md1c, (23) Md1d, (24) Md2a, (25) Md2b, (26) Md2c, (27) Md2d, (28) E. coli O157:H7 10307,
(29) no template, (30) E. coli O157:H7 10311. Positive amplifications of 0.3 kb band are indicated in bold print.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 M
1 kb
Figure 13. Colony PCR of presumptive isolates
obtained from Batch 3 analysis using primers for E.
coli. Lane (M) 1kb plus ladder, (1) E. coli O157:H7, 10307, (2-3)
P3a,(4-5)P4b, (6-8)FP3-Ea, (9-11) FP3-Ee, (12) E. coli O157:H7, 10311,
(13) no template control. All isolates tested were positive.
Summary and Conclusion
Part 2. E. coli O157:H7 DAS kit
• In the analyses of dairy products, the results were in agreement with the expected in that uninoculated samples were negative for the target 300 bp amplicon, except for two uninoculated cheese samples coded as CS-7 and CS-10 wherein the target amplicon was observed.
• Batch 1 testing of 15 meat samples by conventional plating and by PCR using the E. coli O157:H7 DAS kit showed that all samples, including the uninoculated matrices, were positive for EHEC O157:H7. Results of the batch 2 experiment wherein nine uninoculated beef burger patties were analyzed showed that one sample was found to be positive for EHEC O157:H7 by PCR detection. In batch 3 analysis, 14 out of the total number of 23 samples were positive by PCR analysis. One carcass swab, raw meats, most of the meat ingredients and some of the finished products indicated the presence of O157:H7.
• Given the difficulty in retrieving and confirming isolates by
conventional tests from PCR positive samples, the
percentage agreement between the two methods employed
in the analyses of uninoculated meat samples could not be
computed. Likewise, the validation parameters such as
sensitivity, specificity, false positive rate and false negative
rate could not be determined.
• Improvement in the conventional tests and increasing the
number of colonies sampled for confirmatory tests would
have to be undertaken. Other colonies belonging to other
species or other strains should also be tested for
amplification of the 0.3 kb (300 bp) target band to rule out
false PCR positive reactions.
Thank you
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