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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（ＧＰＰＣＲ）扩增靶ＤＮＡ，然后对产物直接标记辣根过氧化物酶复合物（ＨＲＰＰＥＩＱＩ），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ＥＢ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。
Objective To establish a polymerase chain reaction (PCR) product direct enzyme labeling microplate reverse molecular hybridization for human papillomavirus (HPV) type identification. Methods The target DNA was amplified by polymerase chain reaction (PCR) using HPV universal primers. The product was directly labeled with HRP-PEI-QI, and then mixed with HPV pre-coated on microplate Oligonucleotide probe hybridization enzymatic colorimetric assay. Results There was no crossover between HPV types. The sensitivity of hybridization was 13 ~ 76 copies of virus DNA, which was 10 times higher than that of PCR-agarose gel electrophoresis. The reproducibility of this method was good and the CV in positive wells was 6.34 %, Inter-assay CV was 10.53%, intra-assay CV was 1%, inter-assay CV was 5.79%, and hybridization was less affected. Conclusion The method is of high specificity, high sensitivity, simple operation, no radioactivity and contamination of E B dye, short hybridization time, and the results of instrument reading. It is convenient for qualitative or quantitative detection of a large number of routine clinical samples.