甲肝病毒减毒株(H_2)RNA的全长RT-PCR扩增

The RT PCR Amplification of Full Length RNA of Hepatitis A Virus H 2 Strain.

通过ＲＴＰＣＲ技术扩增了甲肝病毒减毒株（Ｈ２）全长ＲＮＡ，并对长片段ＲＴＰＣＲ扩增进行了方法学上的探讨．采用抗血清特异沉淀病毒；盐酸胍酸性酚、氯仿一步法分离纯化病毒ＲＮＡ，可得到高质量的ＲＮＡ样品；以此ＲＮＡ为模板，在无ＲＮＡ酶的逆转录酶作用下，合成单链ｃＤＮＡ；继续以此ｃＤＮＡ为模板，利用３２ｍｅｒ寡核苷酸引物，在Ｔａｑ和ＤｅｅｐＶｅｎｔＤＮＡ多聚酶的作用下进行ＰＣＲ扩增。

A method for RT PCR amplifying long fragments cDNA was established and the 7 4 kb full length cDNA of Hepatitis A virus(HAV) H 2 strain was amplifyed by reverse transcription and polymerase chain reaction (RT PCR).HAV H 2 was precipitated by anti H 2 serum specifically, then the RNA of HAV H 2 was isolated from this precipitation by acid guanidinium hydrochloride phenal chloroform extraction. The first strand of HAV H 2 cDNA was synthesised by reverse transcriptase without RNase H activity,then was ampliflied by PCR using the 32mer primer and the Taq DNA polymerase with Deep Vent DNA polymerase. For obtaining longer PCR products,it is necessary to prepare high quality RNA and employ the longer primers and special Taq DNA polymerase .