聚合酶链反应技术用于汉坦病毒的基因分型

Typing of Hantavirus genes by reverse transcription polymerase Chain reaction

应用反转录ＰＣＲ技术对国内外分离的１８株汉坦病毒基因进行了检测，结果１０株血清Ⅰ型病毒仅能用Ⅰ型引物扩增，８株血清Ⅱ型病毒仅能用Ⅱ型引物扩增，与既往空斑减少中和试验的分型结果完全一致，两种ＰＣＲ产物经内引物再次扩增及酶切分析鉴定证明具有特异世，表明ＲＴ－ＰＣＲ是一项比较特异、敏感和简便实用的汉坦病毒基因分型方法。

Eighteen strains of Hantaviruses,including serotype Ⅰ and Ⅱ,were inoculated in vero-E6 cells.After viral titer increasedthe cells total RNA was extracted by guanidinum thiocyanate-phenol-chloroform method and the purified RNA was added to reverse transcription system for synthesizing cDNA under the guide of general primer.Then,typing PCR was carried out using two sets of primer pairs:type Ⅰ is comple-mentary to M fragment of serotype Ⅰ virus,76～118;type Ⅱ is specific for M fragment of R22.The specifi-cation of PCR products were verified by agarose electrophoresis,digestion of restriction enzyme and second amplification with inner primer pairs.The results showed that all virus strains of type Ⅰ were amplified only by type Ⅰ primer and all virus strains of type Ⅱ amplified by type Ⅱ primer.It is in agreement with the re-port on typing of Hantaviruses with plaque reduction nutralization test.The resuIts suggested that RT-PCR is a sensitive,specific and reliable method for typing of Hantaviruses.