基于实时聚合酶链反应的荧光检测法用于定量检测人乳头瘤病毒6型、11型、16型和18型
Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18.
作者信息
Tucker R A, Unger E R, Holloway B P, Swan D C
机构信息
Human Papillomavirus Section, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA.
出版信息
Mol Diagn. 2001 Mar;6(1):39-47. doi: 10.1054/modi.2001.21899.
BACKGROUND
Quantitation of human papillomavirus (HPV) DNA in clinical samples may yield important clinical information.
METHODS AND RESULTS
We developed a 5' exonuclease fluorescent probe assay for HPV quantitation that uses real-time PCR. The assay was optimized for HPV types 6 (HPV-6), -11, -16, and -18. A multiplex format was developed to quantify a cellular target of known iteration simultaneously with HPV quantitation, which controls for the amount of input DNA. Dilution series of target and heterologous templates were used to verify the assay. The assay was successfully used on fresh and PreservCyt-fixed cell lines, as well as cervical samples. The linear range of the assay is from 10 to 10 million copies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures.
CONCLUSION
The method is accurate over a large copy number range, reproducible, type specific, normalized for input DNA quantity, and applicable to PreservCyt-fixed material.
背景
对临床样本中的人乳头瘤病毒（HPV）DNA进行定量分析可能会产生重要的临床信息。
方法与结果
我们开发了一种用于HPV定量分析的5'核酸外切酶荧光探针检测法，该方法采用实时PCR技术。该检测法针对HPV 6型（HPV-6）、11型、16型和18型进行了优化。我们还开发了一种多重检测法，用于在定量HPV的同时对已知重复序列的细胞靶点进行定量分析，以此来控制输入DNA的量。通过对目标模板和异源模板进行系列稀释来验证该检测法。该检测法已成功应用于新鲜的和经PreservCyt固定的细胞系以及宫颈样本。该检测法的线性范围为10至1000万拷贝。HPV、肌动蛋白和球蛋白检测法的组内相关性在0.95至0.99之间，表明重复测量具有分析精度。
结论
该方法在较大的拷贝数范围内准确、可重复、具有型特异性、针对输入DNA量进行了标准化，并且适用于经PreservCyt固定的样本。
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