贝氏柯克斯体可视化等温扩增法建立

A loop-mediated isothermal amplification for visual detection of Coxiella burnetti

目的建立针对贝氏柯克斯体的快速可视化环介导等温扩增(LAMP)方法。方法体外合成贝氏柯克斯体的IS1111a基因,构建重组质粒。利用在线引物设计软件设计3组LAMP引物,用实时浊度仪筛选出最佳引物,同时对羟基萘酚蓝浓度进行优化,建立可视化LAMP反应体系,并评价其敏感性和特异性。结果建立的可视化LAMP反应可准确检测出贝氏柯克斯体的重组质粒和新桥株,不与其他立克次体产生交叉反应,最低可检测到3.6×10~2拷贝/反应,较普通聚合酶链反应(PCR)法的灵敏度提高了一个数量级,等同于实时浊度法和实时荧光定量PCR方法的检测灵敏度。结论建立的可视化LAMP方法可敏感、特异地检测出贝氏柯克斯体感染,操作简单快捷,适用于基层卫生防疫和疫情现场的病原筛查。

Objective To develop a loop-mediated isothermal amplification （LAMP） assay for rapid and visual de- tection of Coxiella burnetti. Methods The recombinant plasmid containing ISl111a gene of Coxiella burnetti was con- structed using in vitro gene synthesis. Three sets of LAMP primers targeting ISl111a gene were designed with Primer Ex- plorer software 4.0. The most effective primer set was selected and the concentration of hydroxynaphthol blue （HNB） was optimized using real-time turbidimetry. Color change of the reaction tube was used to indicate the result. The detec- tion limit of the optimized visual LAMP assay was evaluated using serially diluted recombinant plasmids of Coxiella bur- netti. The specificity of the LAMP assay was tested using recombinant plasmid and XinQiao strain of Coxiella burnetti, together with other five members of genus Rickettsia. Results The optimized final concentration of HNB in the visual LAMP was 150 gM. The visual LAMP could specifically detect the recombinant plasmid and XinQiao strain of Coxiella burnetti, with no cross reactions with O. tsutsugamushi, Rickettsia rickettsii, spotted fever group Rickettsia, Rickettsia prowazekii,and Rickettsia Canada. The detection limit of this method was 3.6×10^2 copies/reaction, which was 10-fold higher than that of the conventional polymerase chain reaction （PCR） ,but was identical to that of real-time turbidimetry and quantitative real-time PCR （qPCR）. The amplification time of the visual LAMP assay ranges from 20 to 40 minutes, much shorter than the 75 rain for qPCR and 3 hours for conventional PCR. Conclusion The visual LAMP assay could detect CoxieUa burnetti sensitively and specifically with less labor and time. It might be applicable for the routine surveil- lance of Coxiella burnetti in grassroots disease control and prevention institutes or in field test.