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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.

Authors:   Marko Dmitric, Dejan Vidanovic, Kazimir Matovic, Milanko Sekler, Ljubisa Saric, Milos Arsic, Nedjeljko Karabasil
Journal:   Journal of Food Processing and Preservation
Year:   2017
Pages:   n/a
DOI:   10.1111/jfpp.13455
Publication date:   18-Aug-2017
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