摘要
目的建立一种早期快速检测西尼罗病毒的方法。方法以人工合成西尼罗病毒基因(1021～1240,NY99)作为模板,利用环介导等温扩增技术(LAMP)原理,设计合成3套6对引物,特异性识别人工合成西尼罗病毒基因的8个位点,在恒温63℃60min条件下扩增,80℃2min终止反应,通过real-timePCR仪实时监测反应过程,最终产物经凝胶电泳观察。同时将该方法的灵敏度及特异性与常规PCR进行比较。结果 LAMP反应在20min内即可完成,最终产物经电泳观察可见大小片段不等的呈梯度扩增条带,而同为黄病毒属的登革热病毒、流行性乙型脑炎病毒及阴性对照等均无扩增。灵敏性是常规PCR的10倍,可以检测到9.23copies/μl的病毒。结论该方法具有灵敏、特异、快速、简便、成本低等优点,适合基层和现场检测使用,对西尼罗病的早期诊断和流行病学筛查具有重要意义。
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/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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2010年第6期590-594,共5页
Academic Journal of Second Military Medical University
基金
第43批中国博士后科学基金(20080431367)
军队"十一五"科技攻关计划课题基金(06G65)
上海市自然科学基金(07ZR14141)~~
关键词
西尼罗病毒
环介导等温扩增
聚合酶链反应
West Nile virus
loop-mediated isothermal amplification
polymerase chain reaction
