多重PCR法和HPV分型基因芯片法在高危型人乳头瘤病毒感染检测中的应用

目的评价高危型人乳头瘤病毒（HPV）多重核酸扩增荧光检测法和HPV分型基因芯片检测法在HPV感染女性患者标本基因分型的临床应用效果。方法应用13种高危型HPV多重PCR荧光检测法对在深圳市人民医院进行宫颈癌筛查的653例疑似HPV感染女患者的宫颈细胞样本进行检测，并与HPV分型基因芯片检测法的检测结果进行比较，2种方法检测13种高危型HPV不一致的样本经序列分析方法进一步验证。结果13种高危型HPV多重PCR荧光检测法检测HPV阳性样本，阳性检出率为21．5％（140／653）；用HPV分型基因芯片检测法验证，与13种高危型HPV多重PCR荧光检测法一致的阳性样本占20．4％（133／653），总一致率为98．2％。2种方法的检测结果具有高度一致性（kappa值一o．945）；用HPV分型基因芯片检测法检出：HPV单一型别感染占59．4％（79／133），主要的高危HPV型别为HPV16、52、39、68、33和59型，6种高危型占总数的87．3％（69／79），其中HPV16和HPV52为主要感染，占44．9％。结论高危型HPV多重PCR荧光检测法和HPV分型基因芯片检测法在13种高危型HPV的检结果具有高度一致性；多重PCR荧光检测法覆盖主要的13种高危型HPV，分型基因芯片检测法可进行具体的单一型别分型。2种检测方法的联合应用，对宫颈癌筛查和预防具有较高的临床应用价值，同时可为HPV分子流行病学和HPV疫苗的应用研究提供依据。

Application of multiplex PCR and HPV genotyping assay in the detection of high-risk HPV infection

Objective To evaluate the applicable value of multiplex PCR and HPV genotyping assay in the detection of high-risk human papillomavirus (HPV) infection in female patients. Methods Total 653 samples obtained from women aged 19 to 74 who undertaken the cervical cancer screening tests in Shenzhen People's Hospital. The exfoliated cervical cell samples were collected from each woman and tested by fluorescence real-time multiplex PCR assay and cytological examination, respectively. The HPV infection in all samples was validated by HPV genotyping assay. The 13 discordant samples between the two assays were further analyzed by direct sequence analysis. Results The positive detection rate of fluorescence real-time multiplex PCR assay was 21.5% (140/653). The results of high-risk HPV detection were confirmed by HPV genotyping assay in 639 cases, showing an absolute agreement of 98.2% with a Cohen's kappa of 0. 945 between the two HPV DNA tests, indicating almost complete similarity of the two tests. The thirteen high-risk HPV genotypes were detected by both fluorescence real-time multiplex PCR assay and HPV genotyping assay in 133/653 cervical samples (20.4%). The infection with single high-risk HPV genotype was detected in 79/133 cases (59.4%). The HPV16, 52, 39, 68, 33 and 59 accounted for 87. 3% of the single HPV infections, with HPV type 16 and 52 being the most common (accounting for 44.9% of all single infection). Conclusion Multiplex PCR and HPV genotyping assay shows high consistency in detecting high-risk HPV infection. The 13 kinds of high-risk HPV genotypes detected with multiplex PCR can be further genotyped by using genotyping gene chip assay. The combined application of two assays contributes to screening and prevention of uterine cervix cancer, and provides basis for application study on HPV molecular epidemiology and HPV vaccines.