A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis

Abstract

A 5′-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene ( ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non- Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6 h) non-selective culture enrichment.