基于PCR的反向线杂交技术检测外阴上皮内瘤样病变中的HPV DNA

Identification of human papillomavirus in vulvar intraepithelial neoplasia

目的了解人乳头瘤病毒(HPV)在外阴上皮内瘤样病变中的感染状况和病毒型别。方法在PCR基础上用反向线杂交和测序法检测40例外阴上皮内瘤样病变患者的67处病灶。应用GP5/GP6引物,PCR扩增HPV的L1区长度为150bp的DNA片段,如结果为阴性,则采用SPF1/SPF2引物扩增同区域长度为65bp的短片段。结果采用通用引物GP5/GP6,病灶HPV DNA阳性率为52.2%(35/67);阴性的32处病灶用SPF引物扩增短片段的阳性率为81.2%(26/32),总阳性率为91.0%(61/67)。检出的HPV90%为高危类型。35例GP引物PCR扩增产物作了基因测序,其中31例单一感染病灶的测序结果与反向线杂交的结果一致,4例多重感染病灶的基因测序结果分析不成功。用SPF引物PCR扩增的26例产物中,24例成功进行基因测序,2例测序结果不能分析。结论高危型HPV与外阴上皮内瘤样病变相关,可能在浸润性外阴癌的发生中起重要作用。

Objective To evaluate the rate and types of human papillomavirus （HPV） in vulvar intraepithelial neoplasia （VIN）. Methods We detected HPV DNA in 67 lesion tissues collected from 40 VIN patients with PCR based reverse line blot hybridization and DNA sequencing. The PCR using GP5/GP6 primers in HPV L1 region resulted in a 150-bp fragment. When the PCR did not amplify the target DNA, another PCR using SPF1/SPF2 primers was performed to amplify the 65-bp fragment. Results HPV DNA amplified with primers GP5/GP6 was positive in 52.2% （35/67） of the lesions. Of the 32 negative lesions, 26 （81.2%） were positive for HPV DNA amplified with SPF1/SPF2 primers. The total positive rate was 91.0% （ 61/67 ）. Ninety percent of the HPV belonged to high risk types. Sequencing data showed that the genotypes of 31 mono-infection lesions were the same as those of the reverse line blot results, yet the sequences in 4 multi-infection samples could not be determined. Of the 26 SPF PCR products, 24 could be sequenced. 80.6% （25/31） patients with multiple lesions displayed the same genotype, suggesting HPV in the different lesions from the same patient was monoclonal. Conclusion The high risk type of HPV is associated with vulvar intraepithelial neoplasia and may play an important role in the development of invasive vulvar carcinoma.