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 C-t 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((R))Salmonella II kit, Bio-Rad, USA). Practical applicationsThe 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.