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• 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
• 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
• 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 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
Plating in BPA
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
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
1A - -
1B - - multiband
1C - - multiband
1D - -
2A - - multiband
2B - -
2C - -
2D - - multiband
3A + +
3B + +
3C + +
3D + +
4A + +
4B - -
4C + + faint
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
Uninoculated, replicate 1 + +
Uninoculated, replicate 2 + +
21 cells/g, replicate 1 + +
21 cells/g, replicate 2 + +
1kb
1kb
1kb
• 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
Washing of cells
DNA Extraction
Set up PCR
Read-out
0.3 kb
0.3 kb
0.3 kb
+ T0 T6 T24
0.3 kb
0.3 kb
U U U U + U +
M 1 2 3 4 5 6 7 8 9 10 11 M
0.3 kb
0.3 kb
White cheese
A 9 0 1 0
B 9 0 2 0
D 0 0 4 0
E 9 0 3 0
Total 36 1 11 1
Con’t… Table 9
Control strains
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
• 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.