单管多重PCR鉴定HPV基因型方法建立及临床评价.docVIP

单管多重PCR鉴定HPV基因型方法建立及临床评价 　作者：曾凡杞，张志云，李永双，冉灵芝 【摘要】 目的 建立单管多重PCR鉴定低危和高危乳突瘤病毒(HPV)的方法以及用临床标本对该方法进行评价。方法 设计HPV基因型特异的引物，在一个PCR反应管中PCR扩增来自上皮细胞提取的DNA，然后用毛细管电泳对PCR产物进行分离，根据扩增产物的大小确定相应的HPV基因型。对不同病理期的1094份临床宫颈标本和对照进行了分析。结果 一共鉴定了16个HPV基因型，即HPV16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45和11。该方法具有高度的灵敏性和可重复性。同对照相比，未知重要意义的非典型鳞状细胞(ASCUS)、低级别表皮内损伤(LSILs)、高级别的表皮内损伤(HSILs)标本HPV检出阳性率分别为60.5%、77.1%和82.2%，而对照为14.4%，其中在HSILs标本HPV16和HPV58检出率最高，并与细胞恶性程度呈正相关。结论 单管多重PCR鉴定HPV的方法是稳定的，且可以用于HPV的流行病学的调查。 【关键词】 乳突瘤病毒;基因型;多重PCR;毛细管电泳 　　ABSTRACT：Objective To develop a method for identification of human papillomaviruses by multiplex PCR in a single PCR tube and to evaluate this method.Methods Multiplex PCR was based on the amplification of HPV DNA by sets of HPV genotypespecific primers,and the genotypes of HPV were visually identified by the sizes of amplicons after they were separated by capillary electrophoresis.We conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 1094 cervical samples.Results We detected all 16 HPV genotypes (types 16,58,52,51,56,31,18,39,66,59,6,33,30,35,45and11) with a high sensitivity and a high degree of reproducibility.Compared with the group of samples considered normal(14.4%),there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance(ASCUS)(60.5%),lowgrade intraepithelial lesions(LSIL)(77.1%),and highgrade intraepithelial lesions(HSILs)(82.2%).The prevalence and distribution of type 58 were correlated with cytological malignancies,with the highest prevalence in women with HSILs.Conclusion The novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in largescale epidemiological studies 　　KEY WORDS：Human papillomaviruses;Genotype;Multiplex PCR;Capillary electroph