甲肝病毒减毒株(H2)RNA的全长RT-PCR扩增
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The RT-PCR Amplification of Full Length RNA of Hepatitis A Virus H2 Strain
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    摘要:

    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32 mer寡核苷酸引物, 在Taq和Deep Vent DNA多聚酶的作用下进行PCR扩增,得到7.4 kb的扩增产物.

    Abstract:

    A method for RT-PCR amplifying long fragments cDNA was established and the 7.4 kb full-length cDNA of Hepatitis A virus(HAV) H2 strain was amplifyed by reverse transcription and polymerase chain reaction (RT-PCR).HAV H2 was precipitated by anti-H2 serum specifically, then the RNA of HAV H2 was isolated from this precipitation by acid guanidinium hydrochloride-phenal-chloroform extraction. The first-strand of HAV H2 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.

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周翔,谭德勇,赖建华,昝瑞光.甲肝病毒减毒株(H2)RNA的全长RT-PCR扩增[J].生物化学与生物物理进展,1998,25(5):472-475

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  • 收稿日期:1997-08-04
  • 最后修改日期:1998-02-04
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