关键词: 西尼罗病毒, 基因检测, 逆转录酶促重组, 核酸等温扩增, 荧光探针

Abstract:

Objective To establish a reverse transcription-enzymatic recombinase isothermal amplification (RT-ERA) assay for detection of specific gene segment of West Nile virus. Methods The highly conserved region of NS5 gene (120 bp) was selected as the target gene fragment to be detected. The primers and fluorescence probes were designed and synthesized based on the isothermal amplification principle to establish a fluorescence RT-ERA assay system. The fluorescence RT-ERA assay was performed to detect serial diluted recombinant plasmids (10⁴, 10³, 10², 10, 1 copy/μl) containing target gene fragments and West Nile virus RNA at different concentrations (10, 1, 10^-1, 10^-2, 10^-3 ng/μl) to determine the sensitivity. Further, this assay was applied to detect the genomic RNA of Tick-Borne Encephalitis virus, Chikungunya virus, Yellow fever virus, Dengue virus type I, Japanese Encephalitis virus and Influenza A virus (H2N1) to evaluate the specificity. Results A fluorescence RT-ERA assay was successfully established, which was effective in amplifying the specific gene fragments of West Nile virus within 20 min at 39 ℃. The minimum detectable limit of the fluorescence RT-ERA assay was 1 copy/μl using recombinant plasmids as templates and 10^-3 ng/μl using West Nile virus RNA samples as templates. The results of the fluorescent RT-ERA assays were all negative for detecting the genomic RNA from tick-borne encephalitis virus, chikungunya virus, yellow fever virus, dengue virus type I, Japanese encephalitis virus, and Influenza A virus (H2N1). Conclusion A fluorescence RT-ERA assay for detection of West Nile virus RNA is successfully established, which is easy to use, sensitive and specific.

Key words: West Nile virus, Gene detection, Reverse transcription-enzymatic recombinase, Isothermal amplification of nucleic acid, Fluorescent probe