基于LAMP法快速检测天花病毒

摘要

Objective To establish the loop-mediated isothermal amplification ( LAMP ) method for detection of variola virus. Methods One set of primers were designed for recognizing 5 distinct sequences on variola virus-specific gene HA. To optimize the reaction temperature and primers screening, and the sensitivity and specificity of this method for variola virus ( VARV) detection were evaluated. Results Rapid detection of variola virus by LAMP assay was completed within 60 min at 63 ℃. The sensitivity of LAMP with detection limits of 1 pg/μl was 10 times higher than that of PCR, that is, the LAMP sensitivity was 3. 37×105 copies/μl, and the PCR sensitivity was 3. 37×106 copies/μl. and the result of 2 kinds of other virus were negative, showing that it had a good specificity. Conclusions The method reported here demonstrates a potential and valuable means for detection of VARV. The LAMP assay is suitable for wide-area sample screening and on-site detection of variola virus in grassroots units, for on-site and primary quarantine, medical units for rapid diagnosis.%目的 建立天花病毒(Variola)环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测方法,并进行温度优化,从灵敏度、特异性等方面进行评价.方法 基于天花病毒的HA基因特异序列片段,设计5套引物.通过引物筛选及温度的优化,建立天花病毒的LAMP检测法,并对此方法的灵敏度、特异性进行评价.结果 LAMP法快速检测天花病毒在63℃条件下60 min内完成,该方法的检测灵敏度是PCR的10倍,即LAMP灵敏度为3.37×105拷贝/μl,PCR灵敏度为3.37×106拷贝/μl.对痘病毒和腺病毒两种非天花病原结果呈阴性,表明引物设计具有良好的特异性.结论 本方法用于快速检测天花病毒,具有操作简单、装置简便,灵敏度高、特异性强的特点,适合广泛应用于大批量样本初筛及基层单位现场检测天花病毒.