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首页> 外国专利> METHOD FOR IDENTIFICATION OF VIBRIO-CHLERAE 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

METHOD FOR IDENTIFICATION OF VIBRIO-CHLERAE 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

机译：多元聚合酶链反应和测试系统鉴定玻璃藻-霍乱弧菌O1菌株的方法，确定其毒性和生物毒性的方法

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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 (ctxBCL gene), Yellow (ctxBEL 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
机译：技术领域本发明涉及生物技术领域。本发明代表一种用于鉴定霍乱弧菌O1菌株，通过生物学家El Tor通过实时模式的多重聚合酶链反应（PCR）的方法分化为典型的和遗传修饰的形式来确定其毒理性和生物变量的方法，包括：恢复来自霍乱弧菌O1培养物的DNA; PCR的成分：TaqPol的10x缓冲液，MgCL 2 （50 mM），dNTPs的混合物（5 mM），TaqPol（5个/ mql），去离子无菌水，引物1-的混合物（F + R）ctxB引物（10 mM），探针G-FAM（10 mM）和A-VIC探针（10 mM），引物2-（F + R）rtxC引物（10 mcM）的混合物，ROX探针rtxC基因（10 mM），两个阳性（PKO + Classical和PKO + E1 Tor）和一个阴性（H 2 O）对照;在实时模式下，以25 mcl的体积在两种不同组成的反应混合物中一步一步进行聚合酶链式反应：1个循环95°C-3分钟，40个循环95°C-20 s，57°C-30 s（荧光测量），72°C-20 s;在这些会计渠道的阈值线水平上，在绿色通道（ctxB CL 基因），黄色（ctxB EL 基因），橙色（rtxC基因）的特定曲线带上记录结果是0.05; Ct≤30 。;效果：使用试剂盒的方法能够快速可靠地鉴定经典生物变种或El Tor生物变种的霍乱弧菌，并将检测到的V_霍乱生物变种El Tor的毒株区分为典型的和变更的版本。; 2 cl，2 tbl，18 dwg

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