人乳头瘤病毒PCR产物酶标-微孔板杂交分型

目的建立聚合酶链反应（ＰＣＲ）产物直接酶标记微孔板反向分子杂交法用于人乳头瘤病毒（ＨＰＶ）型别鉴定。方法利用ＨＰＶ通用引物介导ＰＣＲ（ＧＰＰＣＲ）扩增靶ＤＮＡ，然后对产物直接标记辣根过氧化物酶复合物（ＨＲＰＰＥＩＱＩ），与预先包被在微孔板上的ＨＰＶ寡核苷酸探针杂交后酶显色法检测。结果ＨＰＶ各型之间无交叉杂交；杂交灵敏度可达１３～７６个病毒ＤＮＡ拷贝，比ＰＣＲ琼脂糖凝胶电泳检测高１０倍；该法重复性好，阳性孔批内ＣＶ为６．３４％，批间ＣＶ为１０．５３％，阴性孔批内ＣＶ为１％，批间ＣＶ为５．７９％；而且杂交影响因素较少。结论该法特异性强、灵敏度高、操作简便，无放射性和ＥＢ染料污染，杂交时间短、仪器读取结果，便于大量常规临床样本定性或定量检测。

A novel method of directly labeling PCR products and hybridization in microplate for typing human papillomaviruses

Objective To develop a easy method for detecting and typing HPVs.Method General primer mediated PCR (GP PCR) was applied successfully to detect a broad spectrum of HPV in cervical scrapes and paraffin wax embedded specimens diagnosed pathologically as squamous cell carcinoma of the cervix uterine. To facilitate HPV typing, a calorimetric microplate base hybridization assay was developed. The PCR products were directly labeled with HRP complex (HRP PEI PBQ). The microplate was previously covered with avian which can bind a biotinylated HPV probe, then fixed with the biotinylated HPV genomic probes mix and type specific probes respectively. Hybridization was completed in a short time and the hybridized signal was detected by ELISA like assay.Results Since this method is highly specific, no cross hybridization was noted between all types of HPVs. The sensitivity was ten times higher than that of agrose electrophic the lowest concentration of template was 13 76 copies of HPV DNA.Conclusion The method is of potential use for routine HPV examination of clinical specimens.