一管式多引物焦磷酸测序技术在HPV分型检测中的应用

Genotyping of human papillomaviruses by pyrosequencing with multiple primers in one tube

目的探讨一管式多引物焦磷酸测序技术在人类乳头瘤病毒(HPV)分型检测中的临床应用价值。方法设计针对7个不同HPV亚型的高度特异性测序引物,采用一管式多引物测序法,通过半巢式聚合酶链反应(PCR)扩增获得HPV基因片段,并进行焦磷酸测序分析。结果通过半巢式PCR扩增和一管式多引物焦磷酸测序,成功区分了7种不同的HPV亚型,且测序结果与PCR-反向点杂交法检测结果的符合率是100%。结论一管式多引物焦磷酸测序技术是一种简便、高效、高通量、高灵敏度、高准确度的HPV分型检测方法,值得推广应用。

Objective To investigate the significance of pyrosequencing in the genotyping of human papilloma-viruses （HPV） .Methods Seven highly specific sequencing primers for HPV subtypes were designed and used to am-plify HPV DNA fragments by semi-nested polymerase chain reaction （PCR） .Then ,the PCR products were se-quenced by pyrosequencing .Results Seven different HPV subtypes were successfully identified by semi-nested PCR and pyrosequencing with seven sequencing primers mixed in one well .Conclusion Pyrosequencing using multi-prim-ers mixed in one well could be an efficient way to detect different HPV subtypes .