FIELD: genetics.
SUBSTANCE: invention relates to microbiology and genetic engineering and can be used in laboratory diagnostics when monitoring cholera, planning anti-epidemic measures and in studies related to the examination of the biology of the pathogen. A method for molecular typing of typical (O1) and atypical (RO) non-toxigenic V. cholerae El Tor strains using real-time PCR is described, including DNA extraction from the test strain, PCR with specific primers, and subsequent consideration of the amplification reaction. Amplification of the studied DNA is carried out simultaneously with that of five pairs of primers, while 0.5 mcl of 50X SYBR Green I intercalating dye is added to the incubation mixture to identify amplification products by the dynamics of changes in the fluorescence index through the FAM channel of the amplifier. The results are recorded according to the geometric method, by registering the Delta C p reflected on the monitor screen, namely: in the presence of the target product of the ACD-rtxA, tcpA, vce, vspD, vgrG3 genes, an amplicon with a low Cp indicator is formed within (8-15) cycles, confirming the presence of the tested gene of typical O1 and atypical (RO) non-toxigenic V. cholerae O1 El Tor strains; which confirms the absence of the tested gene.
EFFECT: creating a simple, fast and reliable method for molecular typing of the strains using PCR, followed by electrophoretic accounting of the results of typical and atypical (RO) non-toxigenic Vibrio cholerae El Tor strains.
11 cl, 6 tbl, 4 ex
