Automated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system

被引:17
作者
Broccolo, Francesco [1 ]
Cocuzza, Clementina E. [1 ]
机构
[1] Univ Milan, Dept Clin Med & Prevent, I-20052 Monza, Italy
关键词
HPV; viral load; real-time PCR; quantitation; automated extraction;
D O I
10.1016/j.jviromet.2007.10.003
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 handier 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. (C) 2007 Elsevier B.V. All rights reserved.
引用
收藏
页码:48 / 57
页数:10
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