用生物素标记CVB_3 cDNA制备探针检测病毒核酸

Preparation of CVB_3 cDNA Probe Labeled with Biotin and Detection of Virous Nucleic Acid

本文报道用含CVB_3 cDNA PstI酶切片段(530bp)的重组质粒pGP_(51)B转化大肠菌;经扩增后提取,纯化重组质粒DNA;用生物素标记制备探针。通过原位杂交技术检测不同的肠道病毒和非肠道病毒感染的HeLa细胞。实验结果表明,该探针只与肠道病毒RNA杂交,而不与非肠道病毒核酸杂交,且可检测出病毒感染后3h,5h(CPE-)的病毒核酸。可见该探针具有良好的特异性和敏感性,且可用于肠道病毒感染引起的心脏疾病的活检标本诊断。

pGP_(51) B, a recombinant plasmid of CVB_3 cDNA fragment and plasmid PGEM digested with restriction endonuclease pstⅠ, was transformed into E. coll HB_(101), then amplified, isolated and purified. The purified PGP_(51)B was labeled with bio-11-dUTP by nick translation. The cells infected with enterovirus and other virus (non-enterovirus)were detected with the probe by in situ hybridization. The result showed that the probe hybridized with enteroviruses' RNA, but not with other viruses' and HeLa cells' nucleic acid. And the enterovirus nucleic acid coudl be identified in cells infected with the virus within 3 and 5 hours (CPE-). It showed that the probe was sensitive and enterovirus specific, and could be applied in biopsy diagnosis of heart diseases induced by enteroviruses.