恙虫病东方体目视LAMP现场化检测方法的建立

Establishment of a field visual approach for detecting Orientia tsutsugamushi in chigger mites by LAMP device

目的建立基于“染料-蜡丸”的目视LAMP检测恙螨中恙虫病东方体的方法,为恙虫病的防治提供新工具。方法选择Ot GroEL基因作为检测靶序列,在线设计并合成LAMP引物。构建r Ot-GroEL重组质粒作为阳性对照。建立基于SYBR Green I的“染料-蜡丸”目视LAMP,并联合便携式LAMP仪使其利于现场应用。结果新设计的引物针对Ot GroEL基因的237 bp靶区域,所构建的重组质粒r Ot-GroEL测序结果正确。在便携式LAMP仪上运用“染料-蜡丸”目视LAMP法,其检测下限为1×10^2拷贝质粒。结论成功建立了一种可与便携式LAMP仪联合使用的检测恙螨体内恙虫病东方体的“染料-蜡丸”目视LAMP法。

Objective To establish a field visual method for detecting the Orientia tsutsugamushi(Ot)in chigger mites using self-prepared dye-wax capsule kit on portable loop-mediated isothermal amplification(LAMP)device basis for developing a new field tool in control and prevention of scrub typhus.Methods The GroEL gene was selected as the target sequence,and the primer for LAMP detection was designed online.A recombinant plasmid r Ot-GroEL was constructed as the positive control.SYBR Green I based dye-wax capsule kit was used to establish a close-tube visual LAMP targeting GroEL.Field visual detection was simulated and verified using portable LAMP device.Results A set of LAMP primers specific to the Ot GroEL gene was chosen to amplify the 237 bp target region.Sequencing results showed that the recombinant r Ot-GroEL plasmid was successfully constructed.Detection limit of the plasmid was 1×10^2 copies using dye-wax capsule kit combined with portable LAMP device.Conclusion A close-tube visual LAMP method targeting Ot GroEL was established,which could be used for field visual detecting Ot in chigger mites with portable LAMP device.