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°C for 60 min, then the amplification was terminated at 80°C 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/μl. 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.