The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities.
The TaqMan LS-50B PCR Detection System facilitates the automated and direct detection of polymerase chain reaction (PCR) products. The system employs the 5' nuclease activity of Taq DNA polymerase to hydrolyse a Salmonella specific internal fluorogenic probe for monitoring the amplification of a 287-bp region of the Salmonella invA gene. Using the fluorogenic 5' nuclease assay, 164 Salmonella strains representing all the subspecies of Salmonella enterica were detected while over 50 non-Salmonella strains were not detected. The detection limit of the assay was two colony forming units (cfu) per PCR reaction when a pure culture of S. typhimurium was used. Six protocols for the isolation of PCR-amplifiable DNA were evaluated using chicken carcass rinses, ground beef, ground pork and raw milk contaminated with Salmonella. Of the six DNA isolation protocols, a modified sample preparation protocol using the EnviroAmp kit was chosen for subsequent studies because it was reliable, easy to use and efficient for the isolation of PCR-amplifiable DNA from foods. A detection limit of 3-7 cfu per PCR reaction was obtained using food samples that were pre-enriched overnight and then inoculated with Salmonella. The detection limit was below 3 cfu/25 g or 25 ml when foods inoculated with Salmonella were pre-enriched overnight. Naturally contaminated foods (50 chicken carcass rinses and 60 raw milk samples) were examined using both the fluorogenic 5' nuclease assay and a modified semi-solid rappaport vassiliadis (MSRV) culture method. Thirty four of the 110 samples tested were Salmonella-positive and 74 were Salmonella-negative by both the 5' nuclease assay and the MSRV method. Two samples were Salmonella-positive by the 5' nuclease assay, but negative by the MSRV method. The correlation between the 5' nuclease assay and the MSRV method was over 98%.
Chen
S
S
University of Guelph, Department of Food Science, Ontario, Canada.
Yee
A
A
Griffiths
M
M
Larkin
C
C
Yamashiro
C T
CT
Behari
R
R
Paszko-Kolva
C
C
Rahn
K
K
De Grandis
S A
SA
D015720:5'-Nucleotidase; D000818:Animals; D002417:Cattle; D002645:Chickens; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005069:Evaluation Studies as Topic; D005506:Food Contamination / Q000517:prevention & control; D005516:Food Microbiology / Q000592:standards*; D005784:Gene Amplification; D006801:Humans; D008460:Meat / Q000032:analysis / Q000382:microbiology*; D008892:Milk / Q000737:chemistry / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012478:Salmonella Food Poisoning / Q000175:diagnosis / Q000517:prevention & control; D012486:Salmonella typhimurium / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013552:Swine
D004269:DNA, Bacterial; D015720:5'-Nucleotidase