Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

Abstract

Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.