Department of Clinical Chemistry, Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
J Virol Methods. 2011 Dec;178(1-2):22-30. doi: 10.1016/j.jviromet.2011.08.007. Epub 2011 Aug 27.
Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for development of cervical cancer. At present, polymerase chain reaction (PCR)-based methods, the most widely molecular tools used for HPV detection, are time-consuming and require expensive instruments. In this study, loop-mediated isothermal amplification (LAMP) was established for detection of HPV types 16, 18, 45 and 58 which are frequently found in Thailand. The optimal condition for detection of these high risk HPVs was 63°C for 60min. Since a white magnesium pyrophosphate precipitate is a characteristic by product of the LAMP reaction which can be visualized directly by the naked eye, the entire assay time of LAMP is 1h compared to 6-8h of for a nested PCR detection. The detection limit of LAMP assay was shown to be equivalent to nested PCR that could amplify 10(2) copies of HPV-18 and 10(3) copies of HPV 16, 45 and 58, as determined by either turbidity detection or agarose gel electrophoresis. No cross-reaction was observed, indicating that LAMP assay has high type-specificity. The assay showed successful detection of HPV in 56 clinical specimens. Using nested PCR as the gold standard, the sensitivity, specificity, negative predictive values and positive predictive values of LAMP assay were 100%. In conclusion, LAMP assay is a high efficiency, low cost diagnostic tool, useful for rapid, accurate, direct detection of HPV for clinical diagnosis.
高危型人乳头瘤病毒（HPV）持续感染是宫颈癌发展的主要危险因素。目前，聚合酶链反应（PCR）是最广泛用于 HPV 检测的分子工具，但耗时且需要昂贵的仪器。在这项研究中，建立了用于检测泰国常见的 HPV 16、18、45 和 58 型的环介导等温扩增（LAMP）方法。这些高危型 HPV 的最佳检测条件为 63°C 60min。由于 LAMP 反应的特征副产物是白色焦磷酸镁沉淀，可以直接通过肉眼观察到，因此与嵌套 PCR 检测的 6-8h 相比，LAMP 检测的整个时间为 1h。LAMP 检测的检测限与嵌套 PCR 相当，可扩增 10(2)个拷贝的 HPV-18 和 10(3)个拷贝的 HPV 16、45 和 58，通过浊度检测或琼脂糖凝胶电泳均可确定。没有观察到交叉反应，表明 LAMP 检测具有高的型特异性。该检测方法成功地检测了 56 份临床标本中的 HPV。使用嵌套 PCR 作为金标准，LAMP 检测的灵敏度、特异性、阴性预测值和阳性预测值均为 100%。总之，LAMP 检测是一种高效、低成本的诊断工具，可用于临床诊断中 HPV 的快速、准确、直接检测。
