The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities

Shu Chen, Arlene Yee, Mansel Griffiths, Carolyn Larkin, Carl Yamashiro, Richa Behari, Christine Paszko-Kolva, Kris Rahn, Stephanie A. De Grandis

Research output: Contribution to journalArticle

159 Citations (Scopus)

Abstract

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%.

Original languageEnglish (US)
Pages (from-to)239-250
Number of pages12
JournalInternational Journal of Food Microbiology
Volume35
Issue number3
DOIs
StatePublished - Apr 15 1997
Externally publishedYes

Fingerprint

commodity foods
Salmonella
Polymerase chain reaction
Assays
nucleases
polymerase chain reaction
Food
Polymerase Chain Reaction
assays
DNA
Limit of Detection
chicken carcasses
detection limit
Stem Cells
raw milk
Chickens
Milk
sampling
Taq Polymerase
ground pork

Keywords

  • DNA detection
  • DNA isolation
  • Food-borne
  • Polymerase chain reaction
  • Salmonella

ASJC Scopus subject areas

  • Microbiology
  • Food Science
  • Safety, Risk, Reliability and Quality

Cite this

The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. / Chen, Shu; Yee, Arlene; Griffiths, Mansel; Larkin, Carolyn; Yamashiro, Carl; Behari, Richa; Paszko-Kolva, Christine; Rahn, Kris; De Grandis, Stephanie A.

In: International Journal of Food Microbiology, Vol. 35, No. 3, 15.04.1997, p. 239-250.

Research output: Contribution to journalArticle

Chen, Shu ; Yee, Arlene ; Griffiths, Mansel ; Larkin, Carolyn ; Yamashiro, Carl ; Behari, Richa ; Paszko-Kolva, Christine ; Rahn, Kris ; De Grandis, Stephanie A. / The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. In: International Journal of Food Microbiology. 1997 ; Vol. 35, No. 3. pp. 239-250.
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