传染性支气管炎病毒地方分离毒株S1基因的RT-PCR方法研究

Research on RT-PCR method for amplifying S1 gene of infectious bronchitis virus isolates

以传染性支气管炎病毒 (IBV)标准毒株M41为材料 ,根据Genbank公开序列自行合成一对特异引物 ,用于扩增IBV的完整S1基因。建立了针对IBV基因组RNA特点的RT PCR方法。对反应体系中的重要反应物Mg2 +、dNTP和引物浓度进行优化 ,确定其浓度值分别为 1 5mmol/L ,2 0 0 μmol/L和 40 μmol/L。使用此反应体系对国内IBV地方分离毒株JS/95 / 0 3 ,SD/ 97/ 0 1等 10多个毒株的S1基因进行扩增 ,均扩增出预期 1 7kb的完整S1基因 。

According to the Genbank open sequences,a pair of special primers was designed to amplify the whole S1 gene of IBV isolates.M41,the reference strain of IBV,was chosen to optimize the main elements in RT PCR.The concentrations of Mg 2+ ,dNTP and primers were defined to be 1 5 mmol/L,20 μmol/L and 40 μmol/L respectively after the experimentation.Based on the optimized system,JS/95/03 ,SD/97/01 and other isolates of IBV in China were amplified.The expected 1 7 kb cDNA fragments of whole S1 gene were obtained.It is conclude that the established method is general to some extent for IBV S1 gene amplifying.