A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices

Abstract

Conventional culture methods have traditionally been considered the "gold standards" for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

Presence of Salmonella in retail grade a eggs determined by the International Organization for Standardization 6579 method and a LightCycler polymerase chain reaction system

“…The higher positivity in LCPCR had previously been observed in some 'poultry meat-related' LCPCR studies by Bohaychuk et al (3) and Eyigor et al (10), and was related factors such as high numbers of nonculturable/dead Salmonella cells in the sample, insufficient recovery of sub-lethally injured cells, despite specific enrichment in the culture-based method (3,9). However, correlating this high LCPCR rate in our egg samples only to the factors mentioned above seemed inconclusive.…”

“…In recent years, novel real-time PCR assays were developed for the rapid detection of Salmonella particularly from eggs (5). LightCycler PCR (LCPCR) is a specific real-time PCR system enabling rapid and reliable detection of the specific PCR product with probe-based technology and high sensitivity (3). Still, complementation of PCR by standard culture is required for the elimination of possible false negative and/or variable PCR results related to inhibitory substances within the process (29), and for avoiding false positive results due to amplification of target DNA from dead/non-culturable/injured Salmonella cells in the sample (20).…”

Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA

“…Furthermore, several studies have shown that the invA gene and its mRNA are good candidates for detection of Salmonella spp. in environmental samples by qPCR (5,26,30,31,35,41), quantitative reverse transcriptase real-time PCR (qRT-PCR) (10), or conventional reverse transcriptase PCR (RT-PCR) (21).…”

Multiplex TaqMan Real-Time PCR (qPCR) Assay Targeting prot6E and invA Genes for Fast and Accurate Detection of Salmonella Enteritidis

“…The inclusion of this internal control did not affect either the amplification or the detection limit of the qPCR assay. The qPCR developed here as opposed to an invA single target qPCR method (Feder et al, 2001;Malorny et al, 2004;Bohaychuk et al, 2007;Gonzalez-Escalona et al, 2009) is able to detect specifically SE strains by the use of an SE specific marker (prot6E). Additionally it is capable to detect other SE that might lack the prot6E gene (Malorny et al, 2007a), such as the case for SE-10.…”