Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
Jawaharlal Nehru Medical College and Hospital, Aligarh, India.
J Med Microbiol. 2010 Nov;59(Pt 11):1303-1310. doi: 10.1099/jmm.0.019240-0. Epub 2010 Aug 5.
A simple paper smear (PS) method for dry collection and storage of cervical specimens was employed to develop an easy multiplex (MPX) PCR for simultaneous detection of generic human papillomaviruses (HPVs) as well as typing of the high-risk HPV-16 and -18, the two clinically most important HPV genotypes, which are responsible for more than 80 % of cervical cancers. Multiplexing was performed with a small amount of DNA eluted by boiling from a single PS punch in a single tube and using a mixture of four pairs of primers specific for the HPV L1 consensus sequence, HPV-16, HPV-18 and the β-globin gene. Sixty HPV-positive biopsies and corresponding PS specimens from cervical cancer patients as well as cervical smears from 100 healthy women with or without abnormal cytology were collected both as PSs and in PBS. Detection of HPV DNA from cervical biopsies collected in PBS and corresponding cervical scrapes on a PS or in PBS by conventional and MPX-PCR showed a concordance of 100 % and adequacy of 93 %. A similar comparative study in cervical scrapes from normal women also revealed 100 % concordance. The technique was validated in a multicentric study at four different national laboratories. PSs collected by different centres showed variable adequacy (73-82 %) but the use of multiple PS discs for DNA extraction significantly increased the adequacy. Integration of PSs with MPX-PCR for the detection and typing of HPVs is a highly convenient, efficient, simple and cost-effective method for large-scale clinico-epidemiological studies and is also suitable for HPV vaccine monitoring programmes in resource-poor settings.
采用一种简单的拭子干收集和储存方法（PS），开发了一种简单的多重（MPX）PCR 方法，用于同时检测通用型人乳头瘤病毒（HPV），以及对两种临床上最重要的 HPV 基因型 - 高危型 HPV-16 和 -18 进行分型，这两种 HPV 基因型负责超过 80%的宫颈癌。在单个管中，通过煮沸从单个 PS 打孔中洗脱的少量 DNA 进行多重化，并使用针对 HPV L1 保守序列、HPV-16、HPV-18 和 β-球蛋白基因的四对引物混合物进行。收集了 60 例 HPV 阳性活检和相应的宫颈癌患者 PS 标本，以及 100 例健康女性的宫颈涂片，这些女性无论细胞学是否异常，均同时采集 PS 和 PBS 标本。通过传统和 MPX-PCR 检测 PBS 中采集的宫颈活检和 PS 或 PBS 中的相应宫颈刮片 HPV DNA，显示出 100%的一致性和 93%的充分性。在正常女性的宫颈刮片中进行的类似比较研究也显示出 100%的一致性。该技术在四个不同的国家实验室进行了多中心验证研究。不同中心采集的 PS 显示出不同的充分性（73-82%），但使用多个 PS 盘进行 DNA 提取可显著提高充分性。PS 与 MPX-PCR 相结合用于 HPV 的检测和分型，是一种非常方便、高效、简单且具有成本效益的方法，适用于大规模的临床流行病学研究，也适用于资源匮乏地区的 HPV 疫苗监测计划。
