Rapid detection of the most common high-risk human papillomaviruses by loop-mediated isothermal amplification

Abstract

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 102 copies of HPV-18 and 103 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.