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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
Author(s) -
Dmitric Marko,
Vidanovic Dejan,
Matovic Kazimir,
Sekler Milanko,
Saric Ljubisa,
Arsic Milos,
Karabasil Nedjeljko
Publication year - 2018
Publication title -
journal of food processing and preservation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.511
H-Index - 48
eISSN - 1745-4549
pISSN - 0145-8892
DOI - 10.1111/jfpp.13455
Subject(s) - salmonella , real time polymerase chain reaction , dna extraction , chromatography , polymerase chain reaction , food safety , extraction (chemistry) , food science , contamination , computational biology , biology , gene , chemistry , genetics , bacteria , ecology
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 ® 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.