Chemical, Biological, and Physical Methods
259
b. For Staphylococcus aureus, the primers used were: entC (SEC gene) and huc (TNAse gene).
Amplification by RFLP could detect 10 cfu/ml in skim milk and 20 cfu/20 g in Cheddar
cheese with results in <6 hours.
205
c. For Listeria, used primer pairs within the 16S or rRNA that are specific for Listeria and
L. monocytogenes to simultaneously detect each. Could detect 1–5 cfu/25 g of food. Only
live cells detected.
192
d. Salmonella and Campylobacter capture probes used: invA (Salmonella) and ceuE (Campylobacter). ELISA used to detect the PCR products. Could detect 2 × 10
2 cfu/ml of salmonellae,
and 4 × 10
1 Campylobacter.
82
2. RT-PCR
a. For Cryptopsporidium parvum, dsRNA extracted and hybridized to the Xtra Bind Capture
System and amplified while on the Xtra Bind material. Could detect as few as 1 oocyte/l in
ca. 2 hours.
116
b. Norwalk virus detected by replacing RT and Taq polymerase with rTth polymerase in single
tube. The generated amplicons were detected using biotinylated oligoprobes in an ELISAbased format. Results from oysters and clams obtained in 1 day.
181
c. Polio and hepatitis A viruses added to oysters at 10
1 –10
5 pfu, concentrated with polyethylene
glycol. Could detect 10 pfu by RT-PCR.
96
d. One commercially available system (Genevision™, Warnex Diagnostics, Laval, Canada) employs RT-PCR to identify Salmonella spp. in 1 day, and Listeria spp. in about 2 days following
enrichments.
3. Probelia system
a. For Salmonella spp. in foods, method had threshold sensitivity of 10
2 cfu/ml. As few as
3 cfu/25 g could be detected after 18 hours pre-enrichment. With 285 contaminated food
samples, a 99.6% agreement was found in a collaborative study. Findings agreed with those
by ISO 6579 method.
53
b. Listeria monocytogenes, when compared to ISO culture method 11290 for recovery from
salmon, the two methods produced comparable results but Probelia detected 20 cfu/ml in
48–50 hours compared to 5+ days for ISO method.
216
4. BAX for screening E. coli
a. When compared to other methods for detecting levels of <3 cfu/g in ground beef, BAX
detected 96.5% of positives compared to 71.5% by immunoassay methods, and 39% for the
best culture method.
101
5. Molecular beacon PCR
a. For Escherichia coli 0157:H7, a fluorescently labeled oligonucleotide probe designed to hybridize with a region of the slt-II gene such that it fluoresces when the hairpin-stem conformation is linear to the target sequence. In PCR reactions containing DNA from contaminated skim
milk, fluorescence increased with increasing numbers of organisms, and method produced
results without need for electrophoresis or Southern blotting.
138
6. PCR-DGGE
a. For Listeria spp., used PCR amplification of a fragment of the iap gene from 5 Listeria spp.
PCR products analyzed by denaturing gradient gel electrophoresis (DGGE). Species specific
DGGE migrations allowed identifications of all listerial species. Can be used to detect the
listeriae and L. monocytogenes in foods.
35
A number of other PCR-based methods have been developed and tested, and this is an area of active
pursuit with those below being among some that have been used for foodborne organisms.
259
b. For Staphylococcus aureus, the primers used were: entC (SEC gene) and huc (TNAse gene).
Amplification by RFLP could detect 10 cfu/ml in skim milk and 20 cfu/20 g in Cheddar
cheese with results in <6 hours.
205
c. For Listeria, used primer pairs within the 16S or rRNA that are specific for Listeria and
L. monocytogenes to simultaneously detect each. Could detect 1–5 cfu/25 g of food. Only
live cells detected.
192
d. Salmonella and Campylobacter capture probes used: invA (Salmonella) and ceuE (Campylobacter). ELISA used to detect the PCR products. Could detect 2 × 10
2 cfu/ml of salmonellae,
and 4 × 10
1 Campylobacter.
82
2. RT-PCR
a. For Cryptopsporidium parvum, dsRNA extracted and hybridized to the Xtra Bind Capture
System and amplified while on the Xtra Bind material. Could detect as few as 1 oocyte/l in
ca. 2 hours.
116
b. Norwalk virus detected by replacing RT and Taq polymerase with rTth polymerase in single
tube. The generated amplicons were detected using biotinylated oligoprobes in an ELISAbased format. Results from oysters and clams obtained in 1 day.
181
c. Polio and hepatitis A viruses added to oysters at 10
1 –10
5 pfu, concentrated with polyethylene
glycol. Could detect 10 pfu by RT-PCR.
96
d. One commercially available system (Genevision™, Warnex Diagnostics, Laval, Canada) employs RT-PCR to identify Salmonella spp. in 1 day, and Listeria spp. in about 2 days following
enrichments.
3. Probelia system
a. For Salmonella spp. in foods, method had threshold sensitivity of 10
2 cfu/ml. As few as
3 cfu/25 g could be detected after 18 hours pre-enrichment. With 285 contaminated food
samples, a 99.6% agreement was found in a collaborative study. Findings agreed with those
by ISO 6579 method.
53
b. Listeria monocytogenes, when compared to ISO culture method 11290 for recovery from
salmon, the two methods produced comparable results but Probelia detected 20 cfu/ml in
48–50 hours compared to 5+ days for ISO method.
216
4. BAX for screening E. coli
a. When compared to other methods for detecting levels of <3 cfu/g in ground beef, BAX
detected 96.5% of positives compared to 71.5% by immunoassay methods, and 39% for the
best culture method.
101
5. Molecular beacon PCR
a. For Escherichia coli 0157:H7, a fluorescently labeled oligonucleotide probe designed to hybridize with a region of the slt-II gene such that it fluoresces when the hairpin-stem conformation is linear to the target sequence. In PCR reactions containing DNA from contaminated skim
milk, fluorescence increased with increasing numbers of organisms, and method produced
results without need for electrophoresis or Southern blotting.
138
6. PCR-DGGE
a. For Listeria spp., used PCR amplification of a fragment of the iap gene from 5 Listeria spp.
PCR products analyzed by denaturing gradient gel electrophoresis (DGGE). Species specific
DGGE migrations allowed identifications of all listerial species. Can be used to detect the
listeriae and L. monocytogenes in foods.
35
A number of other PCR-based methods have been developed and tested, and this is an area of active
pursuit with those below being among some that have been used for foodborne organisms.