Abstract:

The objective of this study was to develop and apply a one-step real-time reverse transcription PCR (real-time RTPCR)
assay for the specific detection of Salmonella. A pair of primers was designed using a Salmonella spp. specific 284-bp
fragment of the invA gene (GeneBank ID: AE008832) as the target sequence. The reaction system and conditions of real-time
RT-PCR were optimized. The specificity, sensitivity and repeatability of the RT-PCR method were analyzed. Pork artificially
contaminated with Salmonella were detected using the new method and traditional method, and the results obtained were
compared. The optimal reaction conditions were 42 ℃ for 5 min, 95 ℃ for 10 s, 95 ℃ for 5 s, and then 67 ℃ for 34 s for 40
cycles; both the optimal concentrations of forward primer and reverse primer were 0.16 μmol/L. Results showed that the new
method was 100% specific. The sensitivity of the method was sufficient to detect 1.5 × 102 CFU/mL of Salmonella in pure
culture with good stability and repeatability. The results of detection of artificially contaminated foods were in accordance
with those obtained with the traditional method. This method could prevent RNAnase effectively and it was proved to be a
simple method for the detection of food-borne Salmonella.

Key words: Salmonella, RNA, real-time reverse transcription polymerase chain reaction (real-time RT-PCR)