METHODS: HPV-DNA containing plasmids and cervical scrapings from histologically confirmed cervical cancer cases were used to evaluate the NM-PCR. In the first round PCR, a set of consensus primers were used to amplify 38 mucosal HPV types. HPV Type-specific primers were used in the second-round polymerase chain reaction (PCR) to amplify 15 HPV types in three multiplex cocktails. The assay sensitivity was determined with the control panel containing one to 1010genome equivalents (GE). DNA sequencing was done to confirm the PCR results.
RESULTS: The assay was able to amplify all HPV types and detected as few as 50GE per reaction. A total of 23 endo-cervical samples obtained from healthy, HPV negative subjects and 52 histologically confirmed cervical scrapings were processed for HPV genotyping by NM-PCR. HPV DNA was detected in all histologically confirmed samples. DNA sequencing results showed complete concordance with PCR results.
CONCLUSIONS: The designed nested PCR based assay had good concordance with clinical histology and sequencing results and appears to be a promising tool for HPV genotyping especially in resource-constrained settings.
方法:使用包含HPV-DNA的质粒和组织学证实的宫颈癌病例的宫颈刮片来评估NM-PCR。在第一轮PCR中,一组共有引物用于扩增38种粘膜HPV类型.在第二轮聚合酶链反应(PCR)中使用HPV类型特异性引物在三种多重混合物中扩增15种HPV类型。用含有1至10个基因组当量(GE)的对照组测定测定灵敏度。进行DNA测序以确认PCR结果。
结果:该测定能够扩增所有HPV类型,并且每个反应检测到少至50GE。总共从健康的子宫颈样本中获得23个,HPV阴性受试者和52个组织学证实的宫颈刮片通过NM-PCR进行HPV基因分型。在所有组织学证实的样品中检测到HPVDNA。DNA测序结果与PCR结果完全一致。
结论:设计的基于巢式PCR的测定与临床组织学和测序结果具有良好的一致性,并且似乎是用于HPV基因分型的有希望的工具,尤其是在资源受限的环境中。