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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 establish rapid visualization of ring-mediated isothermal amplification (LAMP) targeting Beauveria bassiana. Methods The IS1111a gene of Beauveria bassiana was synthesized in vitro and the recombinant plasmid was constructed. Three sets of LAMP primers were designed by online primer design software, and the best primers were screened by real-time turbidimetry. At the same time, the optimal concentration of hydroxynaphthol blue was optimized. The visualized LAMP reaction system was established and its sensitivity and specificity were evaluated. Results The visualized LAMP reaction could detect the recombinant plasmid of Bayesia cynoglossa and the strain of Xinqiao accurately and did not cross-react with other rickettsia. The detection limit was 3.6 × 10-2 copies / reaction, Polymerase chain reaction (PCR) sensitivity increased by an order of magnitude, equivalent to real-time turbidimetric and real-time PCR detection sensitivity. Conclusion The established visual LAMP method can detect C. celosius infection sensitively and specifically, and is simple and quick to operate. It is suitable for the screening of the primary health epidemic prevention and epidemic situation.