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Real-Time Reverse Transcriptase PCR for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

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Abstract:

Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 108 to 100 CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37°C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (Tm ) analysis to determine specific Salmonella invA (Tm = 87.5°C) and IAC (Tm = 82°C) products. Improved Salmonella detection up to 101 CFU/25 g of pork and 100 CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 106 CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take ≥1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.

Document Type: Research Article

Affiliations: 1: Department of Food Science and Technology, University of Tennessee, 2605 River Drive, Knoxville, Tennessee 37996-4591, USA 2: Department of Food Science and Technology, University of Tennessee, 2605 River Drive, Knoxville, Tennessee 37996-4591, USA;, Email: ddsouza@utk.edu

Publication date: March 1, 2010

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