Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

Abstract

A real-time PCR assay was evaluated for the rapid detection (10 h) of Salmonella in meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated with Salmonella enterica serovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

Development of a High-Speed Real-Time Polymerase Chain Reaction System Using a Circulating Water-Based Rapid Heat-Exchange

“…Because PCR amplifies target regions of DNA, PCR can be used to analyze extremely small amounts of a sample. Moreover, as it can distinguish only small differences of nucleotides in a long sequence, even when only a trace amount of DNA is available for measurement, PCR is widely used in all life science fields not only for basic research such as molecular biology, but also for practical applications such as medical diagnosis of influenza, 1) food safety monitoring, 2) and countermeasures against bioterrorism. 3) Even after the progress of other DNA analysis methods such DNA chip methods, quantitative PCR methods are still the fastest in principle, most powerful and reliable for the estimation of the amount of a given sequence present in a sample, which is applied to determination of levels of gene expression.…”

Rapid diagnosis of typhoid fever by an in-house flagellin PCR

“…Apart from five cases, all the PCR-negative samples were sterile on blood culture following a week of incubation (Table 1). The sensitivity and specificity of this assay was assessed to be 1 2 3 4 5 6 7 8 9 10 500 bp 486 bp PCR has been used to detect Salmonella serovars in samples from different sources of animal origin (Oliveira et al, 2002;Patel & Bhagwat, 2008). A DNA-based assay based on alleles of genes encoding the three phases of flagellar H antigen to identify serovar-specific antigens has also been reported to show partial success (McQuiston et al, 2000).…”

Comparison of Bacterial Culture and Real‐Time PCR for the Detection ofSalmonellain Grow–Finish Pigs in Western Canada Using a Bayesian Approach

“…The need for selective enrichment of samples limits the usefulness of PCR as a ‘rapid’ assay for detecting Salmonella in faeces, though results may still be available 24–48 h sooner than when only traditional culture methods are utilized. Other studies have evaluated PCR for detecting Salmonella in pork and pork carcasses (Croci et al, 2004; Patel and Bhagwat, 2008; Lofstrum et al, 2009) with results often available the same day as the absence of high numbers of competing bacteria and inhibitors precludes a need for selective enrichment steps. A number of major Danish slaughterhouses have taken advantage of the rapid PCR, using it to screen meat and carcass swabs in order to shorten the cold storage time and facilitate the export of fresh pork (Lofstrum et al, 2009).…”