蜱源淋巴细胞脉络丛脑膜炎病毒半巢式PCR检测方法的建立

Establishment of semi-nested PCR method for detection of tick-borne lymphocytic choriomeningitis virus

为建立快速、敏感的蜱源淋巴细胞脉络丛脑膜炎病毒(LCMV)检测方法,根据GenBank中已登录的蜱源LCMV NP基因序列设计半巢式PCR引物,通过优化反应条件建立半巢式PCR检测方法。结果显示,用该方法能扩增出248 bp的目的片段,与LCMV序列(MG554174)的同源性为100%。特异性试验结果显示,该方法对蜱传脑炎病毒、发热板血小板减少综合征病毒、阿龙山病毒的基因扩增无条带。敏感性试验结果显示,该方法最低可以检出0.45×10^(2)copies/μL的蜱源LCMV NP蛋白基因,是普通PCR的1 000倍。对总数为150只森林革蜱、草原革蜱和全沟硬蜱样品的检测发现,半巢式PCR、普通PCR的检出率分别是18%和0。蜱源LCMV半巢式PCR方法的建立为蜱源淋巴细胞脉络丛脑膜炎的防控提供了技术支持。

In order to establish a rapid and sensitive detection method for tick-borne lymphocytic choriomeningitis virus(LCMV),a set of semi-nested PCR primers was designed based on the conserved region of tick-borneμLCMV NP gene sequence published by GenB ank,and the optimal reaction conditions were optimized.The results showed that the target fragment of 248 bp could be amplified by this method,the homology withμLCMV sequence(MG554174) was 100%.The results of specificity test showed that there were no bands in the gene amplification of tick-borne encephalitis virus,severe fever with thrombocytopenia syndrome virus and Alongshan virus.Sensitivity test results showed that it could detect 0.45×10^(2) copies/μL of tick-derivedμLCMV NP gene,which was 1 000 times higher than that of conventional PCR.The detection rates of semi-nested PCR and conventional PCR were 18% and 0,respectively,for 150 Dermacentor nuttalli,Dermacentor silvarum and Ixodes persulcatus.The establishment of tick-derivedμLCMV semi-nested PCR provides technical support for the prevention and control of tick-derivedμLCM.