宫颈肿瘤组织高危型HPV新分型检测方法的建立及应用

The Establishment and Application of A New Method for Detecting the High-risk Human Papillomavirus in Cervical Tumor Tissues

[目的]建立一种新型宫颈肿瘤组织高危型人乳头瘤病毒(HPV)检测及分型方法,并评价其临床应用。[方法]基于我国人群流行的5种高危型HPV(HPV 16、18、31、33、51)的E6/E7早期基因序列,设计特异性引物,以明确HPV型别的宫颈癌细胞株及宫颈肿瘤组织为模板,进行PCR检测,结合基因测序验证引物的特异性。进一步优化条件,建立高危型HPV多重PCR检测方法,检测65例宫颈肿瘤组织标本,与临床上使用的流式荧光杂交HPV检测法比较,评价其敏感性和特异性。[结果]成功建立可同时检测5种高危型HPV的多重PCR检测方法,对65例宫颈肿瘤组织进行高危型HPV检测,检出率为72.3%,明显高于临床流式荧光杂交检测法的检出率(32.31%)(χ2=3.86,P<0.05)。[结论 ]基于HPV E6/E7早期基因的高危型HPV多重PCR检测方法是特异、灵敏的高危型HPV检测方法。

[Purpose] To establish a new method for typing the high-risk human papillomavirus（HPV） in the cervical cancer tissues and to evaluate its clinical application. [Methods] According to the sequences of E6/E7 genes of 5 high-risk HPV types（HPV 16,18,31,33 and 51） prevailing in China,the type-specific primers were designed. DNA templates from the tissues and cells of cervical cancer with known HPV genotypes were amplified with the type-specific primers by PCR. Gene sequencing verified their specificity of these type-specific primers. After optimized the conditions,a high-risk type HPV multiplex PCR assay was established. Then,the established HPV multiplex PCR assay was used to detect the HPV genotypes of 65 cases of cervical tumor tissues. The sensitivity and specificity were further evaluated when compared with the x MAP technology which was widely used in clinic. [Results] The multiplex PCR method detecting 5types of high-risk HPVs was successfully established. In 65 cases of cancer tissues,HPV was detected in 72.3% cervical tumor tissues by multiplex PCR method which was significantly higher than that in Kit detection（32.31%）（χ2=3.86,P〈0.05）. [Conclusion] The established HPV multiplex PCR assay based on the design of E6/E7 genes is a specific and sensitive method for the detection and genotyping of high-risk HPVs.