恙虫病东方体环介导恒温扩增可视化快检方法的建立  

Rapid detection of Orientia tsutsugamushi causing scrub typhus using a loop-mediated isothermal amplification assay

作　　者：耿美玲[1] 操敏[2] 张锦海[2] 吕恒[2] 胡丹[2] 郑峰[2] 朱进[2] 潘秀珍[2] 王长军[1,2] 

机构地区：[1]南京师范大学生命科学院,江苏南京210046 [2]南京军区军事医学研究所,江苏南京210002

出　　处：《中国病原生物学杂志》2013年第6期489-492,共4页Journal of Pathogen Biology

基　　金：科技部传染病专项基金项目(No.2013ZX10004103-004;2013ZX10004801-004-017;2013ZX10004218-008);军队"十二五"项目(No.AWS11L001;No.AWS11L009;CWS11J258);南京军区医学创新课题(No.10MA129;10Z038;11Z040)

摘　　要：目的建立快速检测恙虫病东方体(Orientia tsutsugamushi,Ot)的环介导恒温扩增技术(Loop-mediated iso-thermal amplification,LAMP),以期用于基层医院及疫区恙虫病快速筛查。方法以恙虫病东方体56ku外膜蛋白基因为检测靶序列,设计2组恒温扩增引物,筛选最佳引物,优化反应条件,对多种培养物中的Ot标准株及地方株进行验证检测,以经典PCR检测为对照,评估LAMP检测方法的敏感性和特异性。结果建立的LAMP法可快速检测鸡胚培养物、动物脏器及病人血样中的Ot DNA,经63℃恒温扩增1h之后,即可肉眼或用浊度仪观察结果,其他5种立克次体DNA模板对照,均无特异性扩增。LAMP法测定Ot DNA的最低限为5.3×101拷贝/反应,普通PCR法为5.3×104拷贝/反应,LAMP法比普通PCR提高约3个数量级。结论建立的LAMP法能快速检测多种样品中的Ot 56ku基因,方法的灵敏度和特异性高,实现了检测的可视化,适用于基层医院及疫区恙虫病筛查。Objective A loop mediated isothermal amplification （LAMP） assay was established for rapid detection of Orientia tsutsugamushi in rural areas and/or endemic loci of scrub typhus. Methods Two sets of LAMP primers targeting the 56 kd outer membrane protein gene of O. tsutsugamushi were designed. The more effective set was selected and reaction conditions were optimized. Evaluation included testing the standard O. tsutsugamushi strains from chick embryo cultures and O. tsutsugamushi isolates from animal organs and patient blood samples. Specificity was evaluated using five members of the genus Rickettsia, which are closely related to O. tsutsugamushi. The detection limit for LAMP was assessed via serial dilution using the same target gene and results were compared to those of conventional PCR. Results LAMP rapidly detected （within an hour） the 56 kd gene specific to O. tsutsugamushi in various types of samples, provi ding results that could be observed visually or based on turbidity. No corresponding amplification was noted when using DNA of the five rickettsiae as a template, indicating that LAMP was highly specific. The detection limit for LAMP was 5.3×10^1 copies while it was 5.3×10^4 copies for conventional PCR, indicating a 3-fold increase. Conclusion LAMP proved to be a simple, rapid, sensitive, and specific way to detect O. tsutsugamushi and may be a useful way to diagnose scrub typhus in poor rural areas.

关 键 词：恙虫病东方体 环介导恒温扩增技术 诊断 56ku外膜基因 灵敏度 特异性 可视化 

分 类 号：R376.2[医药卫生—病原生物学]