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