Test-System for Identification of Typical and Genetically Altered Variants of Cholera Vibrios, Biovar El Tor, Using Real-Time PCR

Abstract

The aim of the work was to analyze and assess the results of using PCR test-system “V. cholerae variant ctxB-rtxC FL genes” for identification of Vibrio cholerae O1 with differentiation between typical toxigenic and genetically modified variants in a multiplex format with a real-time hybridization-fluorescent recording of results. Materials and methods. To achieve this goal, a set of reagents for the PCR test-system “V. cholerae O1 variant ctxB-rtxC FL genes”, as well as a method for identifying V. cholerae O1 with differentiation between typical toxigenic and genetically altered variants were utilized. The specificity, specific activity and sensitivity of the developed test-system by the example of 35 V. cholerae O1 strains, 6 V. cholerae non-O1 strains, 5 heterologous strains (Shigella zonnei – 2 strains, one Salmonella typhimurium strain, S. enteritidis, Escherichia coli) were investigated. Results and discussion. The panel of reagents for the PCR test system “V. cholerae variant ctxB-rtxC FL genes” detects DNA fragments of the ctxBCl, ctxBEl, rtxC genes in the genome of toxigenic V. cholerae О1 (has specific activity, analytical sensitivity 1·103 GE/ml) and does not detect these genes in non-toxigenic V. cholerae O1 and non-O1, as well as in heterologous strains of microorganisms (100 % specificity). RF Patent No. 2732448 was granted for the PCR test-system “V. cholerae variant ctxB-rtxC FL genes”. It can be used to increase the efficiency of the epidemiological surveillance system and to carry out a justified scope of anti-epidemic measures in the event of cholera importation into the territory of the Russian Federation.