基因芯片对人乳头瘤病毒的快速检测和分型

Rapid Detection and Classification of HPV Genotype with Gene-chips

目的 针对HPV DNA亚型的异质性,建立HPV DNA亚型的基因芯片快速检测方法,为HPV感染病例提供诊断依据。方法 18 种HPV DNA亚型(HPV6、16、18、31、33、35、39、11、45、51、52、53、56、58、42、59、66、68)特异性探针,固定于硝酸纤维素膜制备成基因芯片,生物素标记引物经通用引物介导PCR(GD PCR)扩增HPV DNA, PCR产物与基因芯片经反向点杂交检测HPV亚型;同时采用荧光定量PCR检测HPV6、11、16 和18亚型。结果 31 例标本中,基因芯片的阳性检出率为74.2%,其中HPV6/18、HPV11/16、HPV33/58 和HPV6/11/33多重感染各1 例(3.2%);荧光定量PCR 阳性检出率为67.7%,与前种方法相比较,漏诊率为6.5%。结论 HPV分型基因芯片可1次检测HPV多种亚型,灵敏度高和特异性强,有利于对HPV多重感染的诊断。

OBJECTIVE To establish gene-chips rapidly detecting different genotype of human papillomavirus (HPV) in order to provide valuable laboratory data for the HPV diagnosis. METHODS Gene-chips constructed with probes targeting 18 different genotypes of HPV were immobilized on nitrocellulose. General primer mediated PCR using biotin labeled primers was used to amplify the HPV DNA and then it was used to hybridize with gene-chips with reverse dot blot hybridization. HPV genotype could be read via the color of gene-chips. Real-time PCR targeting HPV 6, 11, 16 and 18 was also used, as a parallel method. RESULTS In 31 clinical samples, the total positive percent was 74.2% (23/31) for gene-chips, including one case of each HPV6/18, HPV11/16, HPV33/58 and HPV6/11/33 multiple infection (3.2%, 1/31). And the total positive percent was only 67.7% (21/31) for real-time PCR, and the false negative result was 6.5% (2/31) compared with gene-chips. CONCLUSIONS Gene-chips can be used as in this study to simultaneously detect and classify different HPV genotype with high sensitivities and specificities, which should be much more easily accepted by patients compared with traditional laboratory methods.