Abstract：
OBJECTIVE: Due to a wide range of Human Papillomavirus (HPV) types associated with genital cancers; HPV genotyping remains important for the introduction of an appropriate vaccine, disease diagnosis, follow-up and epidemiological surveys. Currently, available molecular genotyping assays are not only expensive but also requires dedicated and expensive equipment which is not feasible in the majority of low-and-middle-socioeconomic countries. The purpose of the study was to develop and evaluated a cost-effective nested-multiplex polymerase chain reaction (NM-PCR) assay for HPV genotyping.
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.
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.
摘要：
目的：由于与生殖器癌症相关的人乳头瘤病毒（HPV）类型广泛，HPV基因分型对于引入适当的疫苗仍然很重要，疾病诊断,随访和流行病学调查。目前,现有的分子基因分型分析不仅昂贵，而且需要专用和昂贵的设备，这在大多数中低社会经济国家是不可行的。该研究的目的是开发和评估用于HPV基因分型的具有成本效益的嵌套多重聚合酶链反应（NM-PCR）测定。
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。
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。
