Identifi cation of Salmonella spp and serovars Typhimurium, Enteritidis by qPCR

Abstract

This article presents the results of the identifi cation of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fl uorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specifi city of the primers was evaluated on Salmonella strains of diff erent serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientifi c Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specifi city of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confi rmed the specifi city of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecifi c reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specifi city. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and diff erentiate Enteritidis and Typhimurium serovars.

Keywords

• salmonella
• bacteria
• polymerasechainreaction
• dna
• qpcr