METHOD FOR IDENTIFICATION OF VIBRIO-CHOLERAE O1 STRAINS, DETERMINATION OF THEIR TOXIGENICITY AND BIOVAR WITH DIFFERENTIATION OF EL TOR BIOVAR ON TYPICAL AND GENETICALLY CHANGED VERSIONS BY MULTIPLEX POLYMERASE CHAIN REACTION AND TEST SYSTEM FOR IMPLEMENTATION THEREOF TAKING INTO ACCOUNT RESULTS IN REAL TIME MODE

摘要

FIELD: biotechnology.;SUBSTANCE: invention relates to the field of biotechnology. Invention represents a method for identification of Vibrio cholerae O1 strains, determination of their toxigenicity and biovar with differentiation of the biologist El Tor into typical and genetically modified versions by the method of multiplex polymerase chain reaction (PCR) in real time mode, including: recovering DNA from the analysed Vibrio cholerae O1 culture; components for PCR: 10x buffer for TaqPol, MgCL₂ (50 mM), a mixture of dNTPs (5 mM), TaqPol (5 units/mql), deionised sterile water, a mixture of primers 1 - (F+R) ctxB primers (10 mM), probe G-FAM (10 mM) and A-VIC probe (10 mM), mixture of primers 2 - (F + R) rtxC primers (10 mcM), ROX probe on rtxC gene (10 mM), two positive (PKO + Classical and PKO + E1 Tor) and one negative (H₂O) control; carrying out polymerase chain reaction in one step in two reaction mixtures of different composition in volume of 25 mcl in real time mode: 1 cycle 95 °C - 3 min, 40 cycles 95 °C - 20 s, 57 °C - 30 s (fluorescence measurement), 72 °C - 20 s; recording results on specific curve bands in Green channels (ctxB^CL gene), Yellow (ctxB^EL gene), Orange (rtxC gene) at level of threshold line on these accounting channels is 0.05; Ct ≤ 30.;EFFECT: method using a kit enables to quickly and reliably identify cholera vibrios of a classical biovar or El Tor biovar and differentiate the detected toxic strains of V_ cholerae biovar El Tor into typical and altered versions.;2 cl, 2 tbl, 18 dwg