Li Jianduan, Gerhard Daniela S, Zhang Zhengyan, Huettner Phyllis C, Wright Jason, Nguyen Loan, Lu Danielle, Rader Janet S
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Clin Microbiol. 2003 Dec;41(12):5563-71. doi: 10.1128/JCM.41.12.5563-5571.2003.
Human papillomaviruses (HPVs) are important in the development of human cancers, including cervical and oral tumors. However, most existing methods for HPV typing cannot routinely distinguish among the more than 100 distinct types of HPV or the natural HPV intratypic variants that have also been documented. To address this problem, we developed a novel method, general primer-denaturing high-performance liquid chromatography (GP-dHPLC), for the detection and typing of genital HPV using an automated 96-well plate format. GP-dHPLC uses general primer PCR (GP-PCR) to amplify the viral DNA and then analyzes the GP-PCR products by denaturing high-performance liquid chromatography (dHPLC). A number of different primer pairs with homology to most known genital HPV types were tested, and the L1C1-L1C2M pair specific for the L1 region of the viral genome was chosen. A set of HPV standard control patterns, consisting of those for HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59, 6, and 11, was established for genital HPV typing. One hundred eighty-six frozen and formalin-fixed cervical cancer tissue samples were analyzed for the presence of HPV and the HPV type by this method, and 95.8% of them were found to contain HPV DNA. GP-dHPLC accurately discriminated among HPV variants that differed by as little as one nucleotide. Several new variants of HPV types 16, 18, 39, 45, 52, and 59 were identified. Moreover, multiple HPV infections were detected in 26.6% of the samples. Our results indicate that HPV typing by GP-dHPLC permits discrimination of common genital HPV types, detection of multiple HPV infections, and identification of HPV variants in clinical samples.
人乳头瘤病毒（HPV）在人类癌症的发生发展中起着重要作用，包括宫颈癌和口腔肿瘤。然而，大多数现有的HPV分型方法无法常规区分100多种不同类型的HPV，也无法区分已被记录的天然HPV型内变体。为了解决这个问题，我们开发了一种新方法，即通用引物-变性高效液相色谱法（GP-dHPLC），用于以自动化96孔板形式检测和分型生殖器HPV。GP-dHPLC使用通用引物PCR（GP-PCR）扩增病毒DNA，然后通过变性高效液相色谱法（dHPLC）分析GP-PCR产物。测试了许多与大多数已知生殖器HPV类型具有同源性的不同引物对，并选择了对病毒基因组L1区域特异的L1C1-L1C2M引物对。建立了一套HPV标准对照模式，包括HPV 16、18、31、33、39、45、51、52、56、58、59、6和11型的对照模式，用于生殖器HPV分型。通过该方法对186份冷冻和福尔马林固定的宫颈癌组织样本进行了HPV检测和HPV类型分析，发现其中95.8%含有HPV DNA。GP-dHPLC能够准确区分仅相差一个核苷酸的HPV变体。鉴定出了HPV 16、18、39、45、52和59型的几种新变体。此外，在26.6%的样本中检测到多重HPV感染。我们的结果表明，通过GP-dHPLC进行HPV分型能够区分常见的生殖器HPV类型，检测多重HPV感染，并鉴定临床样本中的HPV变体。
