山羊无形体可视化等温扩增法的建立与应用

Establishment and application of visual loop-mediated isothermal amplification of Anaplasma capra

目的 山羊无形体(Anaplasma capra)是无形体属中近年来新发现的一种人兽共患无形体,可引起无形体病,严重威胁人类的健康。建立一种适用于现场快速检测山羊无形体的可视化环介导等温扩增(LAMP)方法,对无形体病的诊断与防控有重要意义。方法 用聚合酶链式反应(PCR)扩增山羊无形体的msp2基因,构建重组质粒。利用在线引物设计软件设计LAMP引物,优化其反应条件和反应体系;加入一定浓度的钙黄绿素,建立可视化LAMP方法,评价其敏感性和特异性,并与PCR方法比较。结果 建立的可视化LAMP反应可准确检测山羊无形体的重组质粒和样品中的山羊无形体基因,且不与其他无形体发生交叉反应,最低检测限为1 copy/μL,比PCR方法的灵敏度高100倍。用该方法检测已知山羊无形体阳性蜱的总DNA,结果均为阳性;检测承德市野外采集的蜱标本,山羊无形体阳性率为16.1%,PCR方法的阳性率为12.5%。结论 建立的可视化LAMP方法敏感性、特异,操作简单、快捷,可用于人感染山羊无形体的早期检测与蜱携带无形体的监测。

Objective Anaplasma capra is a newly discovered zoonotic Anaplasma species in recent years,which can cause human anaplasmosis,and seriously threaten human health.Hence,it’s important to develop a visual loop-mediated isothermal amplification(LAMP) method for the rapid and on-site detection of A.capra,which is very significant for the diagnosis,prevention and control of anaplasmosis.Methods The msp2 gene of A.capra was amplified by polymerase chain reaction(PCR) to construct the recombinant plasmid.LAMP primers were designed by primer explorer software 5.0.The LAMP reaction conditions and reaction system were optimized,and calcein was added to establish the visual LAMP method.All available msp2 gene sequences of A.capra were downloaded from the GenBank database.All the LAMP primer sequences were aligned with the msp2 gene sequences to ensure that the primers were located at the conserved region.The sensitivity was evaluated using a 10-fold ratio dilution plasmid as a template,and the specificity was evaluated by detecting A.capra in collected ticks.Results The established visual LAMP reaction can accurately detect the recombinant plasmid containing the msp2 gene.The detection limit of the LAMP assay established in this study can reach up to 1 copy/μL,which is 100 times more sensitive than the PCR method.The established visual LAMP can accurately detect the positive tick sample of A.capra without cross-reaction with other members in the genus.The total DNA of forty known A.capra-positive ticks was detected by this LAMP method,and the results showed that all the samples tested positive for A.capra.This method was used to detect the A.capra infection in ticks collected in Chengde,the results showed that the positive rate was 16.1%(31/192),higher than the positive rate of 12.5%(24/192) detected by the PCR method.Conclusion Totally,the established visual LAMP method can detect A.capra sensitively and specifically,which is simple and rapid to use for early detection of human infection with A.capra and surveillance of its infection in ticks.