In‐house validation of real‐time PCR methods for detecting the INV A and TTR genes of Salmonella spp. in food

Abstract

The microbiological standard method for the detection of Salmonella spp. is time‐consuming, and consequently there is a need for an alternative rapid methodology for its detection. In this study, two open‐formula real‐time PCR methods for detecting the inv A and ttr gene Salmonella spp. have been successfully optimized and in‐house validated. Different DNA extraction procedures were used (boiling, Chelex resin and standard I ‐ Biorad). The relative sensitivity and false positive ratio for the alternative methods were 100% and 0.0%, respectively. The relative trueness was in range from 96.8% to 99.2%. No false negative results were detected. The lowest Ct values were obtained using the protocol for detecting the ttr gene after DNA extraction by the Chelex. The results were compared in a large number of food and environmental samples to those of the SRPS EN ISO 6579:2008 standard method and commercial kit (IQ check® Salmonella II kit, Bio‐Rad, USA). Practical applications The whole procedure of real‐time PCR methods in this study was approximately 20 hr, in contrast to 4–5 days of analysis time for the standard method (SRPS EN ISO 6579:2008). The real‐time PCR method also provides a high level of sensitivity and specificity with a low risk of cross‐contamination. The implementation of the method for routine analysis will help improve safety in the food production chain, by providing results compatible with the ISO standard, but more rapidly.

Prevalence of Salmonella Pathogenisity Island (SPI1, SPI2, SPI3 and SPI5) Genes in Salmonella species Isolated from fresh Broiler Chicken Meat in Sri Lanka

“…Rahn et al (1992) confirmed that this invA gene (gene encoding the invasionassociated protein) contains sequences unique to Salmonella and demonstrated that the invA gene is an appropriate PCR target, with possible diagnostic applications. By then, this gene was used for identification of Salmonella in many studies as with the current study (Shabarinath et al, 2007;Bhowmick et al, 2011;Yang et al, 2016;Dmitric et al, 2018;Yang et al, 2018). All the Salmonella isolates of the present study harbor the invH gene as it was shown by positive PCR (Table 03, Figure 01).…”

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

“…More specifically, the ttr gene, which encodes for the tetrathionate reductase, was targeted for the detection of Salmonella spp. as tetrathionate respiration should be genetically stable within the genus [ 39 , 40 ]; the gene hly , encoding for the listeriolysin O, was chosen for the detection of L. monocytogenes , as it has been reported to be relatively well conserved in the species [ 41 , 42 ]; last, the gene rfbE was selected for the specific detection of E. coli O157 as this gene is the one encoding for the “O” antigen of E. coli , and as such, it is highly specific for this serogroup [ 43 , 44 ]. The Tt values obtained were directly compared.…”

Whole genome sequencing and protein structure analyses of target genes for the detection of Salmonella

“…In terms of the number of mutations present in ttr RSBCA, we speculated that the ttr R gene was the most highly conserved, followed by the ttr B and ttr C genes, which have been successfully used in real-time PCR detection of Salmonella in food 10 , 34 . Differences in the prevalence of mutations in the ttr RSBCA cluster may be related to the distinct functions and loci of these genes.…”