3组套式PCR用于检测及区分霍乱弧菌O1群古典型,埃尔托型和O139群

针对霍乱弧菌肠毒素Ａ亚单位（ｃｔｘＡ）基因设计两对引物，建立套式ＰＣＲ，可特异扩增霍乱弧菌Ｏ１群古典型、埃尔托型和Ｏ１３９群；针对霍乱弧菌毒力协调菌毛Ａ亚单位（ＴｃｐＡ）基因设计两对引物，外引物可特异扩增霍乱弧菌Ｏ１群古典型、埃尔托型和Ｏ１３９群，内引物只能特异扩增霍乱弧菌Ｏ１群埃尔托型和Ｏ１３９群；针对Ｏ１３９群特异基因设计两对引物，建立套式ＰＣＲ，可特异扩增Ｏ１３９群。先利用针对ｃｔｘＡ基因设计的套式ＰＣＲ进行初筛，再进一步用另外两组套式ＰＣＲ，可检测及区分霍乱弧菌Ｏ１群古典型、埃尔托型和Ｏ１３９群。对４８株ＥＶＣ，２株ＣＶＣ，６株Ｏ１３９群和３３株非Ｏ１非Ｏ１３９群进行检测，扩增结果与设计均一致。套式ＰＣＲ检测的敏感性可达到１～１０ＣＦＵ。本文建立的方法简单、快速、特异和敏感，有较大的应用价值。

Detection and differentiation of O1 classical,El Tor and O 139 stains of Vibrio cholerae by using three groups of nested PCR

Two pairs of primers were dsigned from cholera toxin subunit A(ctx A)gene and a nested PCR that can detect O1 classical (CVC),El Tor (EVC)and O 139 especially was established.Two pairs of primers were designed from toxin coregulated pilus subunit A(tcp A)gene.PCR with tcp A outer primers can detect CVC,EVC and O 139 especially.PCR with tcp A inner primers can only detect EVC and O 139 especially.Two pairs of primers were designed from O 139 special gene and a nested PCR that can detect O 139 especially was established.First the primers that were designed from ctx A were used to detect specimens,then the other primers were used to differentiate CVC,EVC and O 139 .48 EVC,2 CVC,6 O 139 and 33 non O1 non O 139 strains were detected,the results were all correct.The senstitivity of nested PCR reached 1 10 CFU.This method is simple,rapid,specific,sensitive,and economical.This method possessed great value for practical application.