Loop-mediated isothermal amplification in detection of West Nile virus genome / 第二军医大学学报

ABSTRACT

Objective To establish a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the West Nile virus (WNV).Methods WNV genome (position nt 1 021 to nt 1 240) was synthesized by a PCR-based gene synthesis method.The synthetic fragments included 6 pairs of LAMP primer recognizing 8 primer sites of WNV genome.The LAMP gene amplification was carried out using a real-time PCR system at 63℃ for 60 min,then the amplification was terminated at 80℃ after 2 min.The amplification products were observed by agarose gel electrophoresis.The sensitivity and specificity of LAMP assay were compared with those of conventional PCR.Results The LAMP assay took less than 20 min,and the amplification product took on a ladder-like electrophoresis pattern.The sensitivity of LAMP assay was 10-fold higher than that of conventional PCR,and the detection limit of LAMP was 9.23 copies/?l.The specificity of WNV-specific LAMP assay was demonstrated by the negative amplification results from dengue virus and Japanese encephalitis virus,both were closely related members of the Flavivirus family.Conclusion LAMP assay is rapid,cost-effective,highly sensitive and specific in detecting genes of interest,and is of great significance for WNV surveillance,especially for grass root units and on-sport surveillance.