Broccolo Francesco, Cocuzza Clementina E
Department of Clinical Medicine and Prevention, University of Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy.
J Virol Methods. 2008 Mar;148(1-2):48-57. doi: 10.1016/j.jviromet.2007.10.003. Epub 2007 Nov 28.
Accurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimens.
准确的实验室检测方法对于诊断持续性致癌性人乳头瘤病毒（HPV）感染越来越被认为是宫颈上皮内瘤变女性临床管理的关键。HPV病毒载量被认为是持续性感染的替代标志物。针对HPV-16、-31、-18和/或-45以及-33和/或-52、-58、-67开发了四种独立的实时定量TaqMan PCR检测方法。这些检测方法具有广泛的检测动态范围以及高度的准确性、重复性和再现性。为了尽量减少材料使用和实际操作时间，使用集成到机器人液体处理工作站中的96孔板形式进行自动核酸提取。通过将TaqMan检测HPV的结果与使用通用引物（GP5+/GP6+）进行PCR和测序（296个样本）以及INNO-LiPA分析（31个样本）获得的结果进行比较，评估TaqMan检测方法对HPV鉴定的性能。实时PCR检测结果与GP(+)-PCR系统获得的结果总体上具有良好的一致性（kappa统计量=0.91）。总之，本研究描述了四种新开发的实时PCR检测方法，这些方法为检测宫颈标本中最常见的致癌性HPV类型的HPV DNA以及HPV活性提供了一种可靠且高通量的方法。
