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. Mercado

University Researcher
M.S. Food Science
 Studies and Study Leaders
• Optimization of conditions for enrichment of meat and dairy
samples
Study 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.
Staphylococcus aureus

• 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
• 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.
E. coli O157:H7
• enterohemorrhagic strain

• causes life-threatening
complications in children
& the immunocompromised

• produces Shiga-like toxin(s)

SLT-I /SLT-II or both

• responsible in foodborne
illness outbreaks with a
variety of foods
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 and

E. 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
 Enrichment of samples
Gathering of
samples

Preparation of samples Spiking of samples

with known levels of
S. aureus

Incubation for 20 h at Cultivation in enrichment broth

37o C 100 rpm
 Conventional plating method

Plating in BPA

Serial dilutions of Observation of

enriched samples presumptive S. aureus
colonies after 48 h

DNAse test results Coagulase test

after 24 h results after 30 h
 S. aureus DAS kit method

Preparation of cell Addition of diluted cell

lysates lysates to DAS tubes

Incubation of reaction
tubes in the thermal
Visualization of
cycler for 5 h
PCR products
Gel
electrophoresis
Comparison of analysis time using S.aureus DASTM kit
and conventional method
A. DAS Kit

Day 1 Day 2

Receive Pre-PCR
samples processing
PCR
Enrichment Result

B. Conventional Plating Method

Day 1 Day 2 Day 3 Day 4 Day 5

Plating Pick Coagulase

Receive
presumptive and other
samples
colonies ancillary
Enrichment
Incubation tests

Day 6 Day 7 Day 8

Research Accomplisments
S.aureus DASTM kit
Protocol improvement for fresh milk samples

1kb

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).
 UHT Fresh milk samples

1kb 1kb

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
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 DAS kit Remarks
(S. aureus colonies in BPA) (1.0kb amplification)

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 continuation
Sample Code Cultural Method S. aureus DAS kit Remarks
(S. aureus colonies in BPA) (1.0kb amplification)
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 - -
 Raw cow’s milk samples

Percent agreement: 31 x 100 = 77.5%

40
Percent disagreement: 2 x 100 = 5%
40
Percent indeterminate: 7 x100 = 17.5%
40
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.
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 + +
 White cheese samples

1kb

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.
 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

1kb

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.
 Raw cow’s milk samples
M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 M

1kb

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
Table 3. Different samples (N=171) obtained from five collaborators and analyzed for
the presence of S. aureus using PCR and cultural methods.
Collabo- Samples PCR , Cultural, S. aureus
rator
1.0 kb plating in BPA 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 - - 0
milk, lecithin
20- uninoculated swab samples - - 0
7- inoculated (103 CFU/g S.aureus 1526) liquid + + 107 - 108
blend, powdered filled milk, lecithin
2- inoculated (436 CFU/g S.aureus 1526) swab + + 107 - 108
samples
C3 9-raw cow’s milk, white cheese, pasteurized - - 0
fresh and choco milk, yoghurt
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 - - 0
handlers, hose
 Continuation…Table 3
Collabo Samples PCR , Cultural, S. aureus
rator 1.0kb plating in CFU/ml
BPA
C4 17 - sausage, smoked mortadella, lean pork, + + 107 - 108
ground pork, pork fat, ground pork fat, pork
and beef emulsion, ground beef, natural
casings
4 - mortadella, hotdog, beef sausage, lean beef - - 0
22 - swab from equipment, food handlers, - - 0
food-contact surfaces
3 - swab from manual stuffer, hands of butcher + + 107 - 108
C5 18 - inoculated (108 cells/g S. aureus 1526) + + 104 - 106
processed meat – longaniza slices, ham,
hotdog, corned beef, skinless longaniza,
cheesedog, deliburger beef, deliburger chicken
6 - uninoculated processed meat – corned - - 0
beef, deliburger beef, deliburger chicken
Table 4. Ancillary tests of 33 presumptive S. aureus from different
samples that exhibited 1.0 kb amplicon in PCR
No. of Origin Coagulase Mannitol DNAse Gram
Isolates Test Test Test reaction
10 Raw milk, pork, pork emulsion, ground 4+ + + (+) cocci
pork fat, sheep casing, manual stuffer,
swab from hand of butcher
2 Ground pork, pork sausage 4+ - + (+) cocci
9 Raw milk, swab from hand of butcher 3+ + + (+) cocci
and cow’s udder, pork fat, pork
emulsion plus fat cubes
6 Beef emulsion, pork sausage, 3+ - + (+) cocci
mortadella, swab from cow’s udder
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 5. Inclusivity and exclusivity of S. aureus DASTM kit
Target
No. of strains Source Amplicon
1.0 kb
S. aureus reference strains 3 PNCM +
S. aureus clinical isolates 13 CPH +
Closely-related bacteria 10 PNCM -
S. saprophyticus ; S. epidermidis ; Streptococcus bovis ;
Proteus vulgaris ; Enterococcus faecalis ; Corynebacterium
flavescens ; C. glutamicum ; Micrococcus luteus ;
Pediococcus acidilactici ; Enterobacter aerogenes
Escherichia coli 20 CPH/RITM/PNCM -
EIEC/EPEC/EHEC ; clinical isolates; BIOTECH
food isolates ; nonpathogenic strain
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 Identification Coagulase

DAS kit Method Test
S. aureus reference strain 1 + Conventional 3+
S. aureus clinical isolates 53 + Conventional 2+/ 3+ / 4+
S. cohnii ssp. cohnii 8 - BBL ID kit -
S. xylosus 6 - BBL ID kit -
E. coli 5 - Conventional -
Total no. of strains analyzed 73
 Summary and Conclusion
Part 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 Conclusion
Part 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 SOURCE/
Accession No. 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, UPLB
2 Research Institute for Tropical Medicine, Alabang MM
3 Michigan State University, East Lansing, Michigan USA
4 Cavite State University, Indang, Cavite
 Table 8. Inclusivity and exclusivity of E. coli O157:H7 DAS kit
Species/Strain No. of strains Target Amplicon
0.3 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, Klebsiella 10 -
oxytoca, K. pneumoniae, Enterobacter aerogenes,
Proteus vulgaris
Serratia liquefaciens, S. marcescens 2 -
Erwinia carotovora 1 -
Other foodborne pathogens: Listeria 12 -
monocytogenes, Yersinia enterocolitica,
Staphylococcus aureus, Bacillus cereus, Vibrio
cholerae
B. amyloliquefaciens 1 -
 E. coli O157:H7 Validation
Enrichment in modified trypticase soy broth (mTSB) +0.02 mg/ml
novobiocin for 20-24 h, 37°C, without shaking

Washing of cells
DNA Extraction
Set up PCR

Read-out

Thermal cycling Gel electrophoresis

 Cultural Method for detection of
E. coli O157:H7
Enrichment in modified trypticase soy broth (mTSB) +0.02
mg/ml novobiocin for 20-24h, 37°C, without shaking

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

O157 serotyping by latex agglutination

 White cheese samples
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
 UHT Fresh Milk 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
 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0.3 kb

+ T0 T6 T24

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
 Batch 1 meat samples
a. b.
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
U U U U + U +

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
Batch 2 meat samples

M 1 2 3 4 5 6 7 8 9 10 11 M

0.3 kb

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)
Batch 3: carcass swabs, meat ingredients and
finished products in a meat processing plant
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

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
Table 9. Verification of isolates obtained from white cheese and hamburger
patties samples
Sample No. of presumptive E. coli ID No. of colonies No. of
colonies screened screened by colonies
by IMViC PCR 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
Con’t… Table 9

Sample No. of presumptive E. coli ID No. of colonies No. of colonies

colonies screened screened by PCR positive for 0.3
kb amplicon
by IMViC
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 - positive - positive
B-10084
 [M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

0.3 kb

M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M

0.3 kb

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