Flores-Munguia Roberto, Siegel Erin, Klimecki Walter T, Giuliano Anna R
Arizona Cancer Center, The University of Arizona, Tucson, USA.
J Mol Diagn. 2004 May;6(2):115-24. doi: 10.1016/S1525-1578(10)60499-0.
Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.
嗜黏膜人乳头瘤病毒（HPV）感染是宫颈上皮内瘤变的必要病因。多项流行病学研究表明，HPV病毒载量可能是宫颈发育异常的一个危险因素。本研究的目的是评估一种测定8种致癌性HPV类型（16、18、31、39、45、51、52和58型）病毒载量的方法。定量分析基于高通量实时PCR。使用HPV 16、18、45和51型的E6-E7区域扩增特定DNA序列，并克隆到质粒载体中。使用有限稀释分析法对HPV 16、18、45和51型的构建体以及HPV 31、39、52和58型的全基因组进行定量，并用于创建标准曲线。使用型特异性HPV克隆来确定特异性、线性以及分析内和分析间的变异。我们检测方法的灵敏度覆盖高达7个数量级的动态范围，分析内变异系数小于6%，分析间变异小于20%。当使用亲缘关系相近的HPV类型作为模板时，在任何型特异性标准曲线上均未观察到交叉反应。我们的实时PCR方法在7倍数量级的动态范围内具有高度的可重复性、灵敏性和特异性。
