恙虫病东方体环介导等温扩增可视化快速检测方法的建立

Establishment of an assay for rapid visual detection of the Orientia tsutsugamushi using a loop-mediated isothermal amplification

目的利用环介导等温扩增(LAMP)可视化检测技术,建立一种灵敏、特异和简便的恙虫病检测方法。方法利用LAMP在线引物设计软件Primer Explorer V4,以恙虫病东方体热休克蛋白groEL基因作为靶序列,设计扩增引物,优化反应条件。利用恙虫病东方体标准株评估灵敏度和特异度;收集42份恙虫病血样,分别采用LAMP、普通PCR和实时荧光定量PCR方法进行验证。结果建立的LAMP方法可在61~65℃80 min内完成恙虫病东方体的快速检测,最低检测限为10拷贝/μL,高于普通PCR方法2个数量级,高于荧光定量PCR方法1个数量级;对8种常见的自然疫源性疾病病原体检测均为阴性,特异度为100%;对42份恙虫病血样采用3种方法检测,阳性率一致,均为21.4%(9/42)。结论本研究建立的恙虫病东方体LAMP可视化检测反应快速,操作简便,灵敏特异,可在基层医疗卫生机构运用推广。

Objective To establish a loop-mediated isothermal amplification(LAMP)technology to provide a sensitive,specific and simple detection assay for Orientia tsutsugamushi.Methods Primers were designed with online software Primer Explorer V4 based on groEL gene of Orientia tsutsugamushi.The reaction conditions were optimized.The standard Orientia tsutsugamushi strains was used to evaluate the sensitivity and specificity.A total of 42 blood samples of scrub typhus cases were evaluated using LAMP,conventional PCR and real time fluorescence quantitative PCR.Results Rapid detection of Orientia tsutsugamushi by colorimetric LAMP assay was completed within 80 min at temperature 61℃-65℃.The detection limit for LAMP was 10 copies/μL,which was two order of magnitude higher than conventional PCR and one order of magnitude higher than real time fluorescence quantitative PCR.The detection results for 8 pathogens of common natural foci diseases were all negative with a specificity of 100%.A total of 42 blood samples of scrub typhus cases were detected by LAMP,conventional PCR and real time fluorescence quantitative PCR,with the same positive rates of 21.4%(9/42).Conclusion The visual LAMP assay to detect Orientia tsutsugamushi is a rapid,simple method with high sensitivity and specificity.The method can be suggested to use in primary medical and health institutions.