马泰勒虫和弩巴贝斯虫双重荧光定量PCR检测方法的建立  

Establishment a duplex real-time PCR method for detection of Theileria equi and Babesia caballi

作　　者：陈克伟 胡哲[1] 郭奎 刘荻萩[1] 戚亭[1] 郭巍[1] 杨光璞 杜承[1] 王晓钧[1] CHEN Ke-wei;HU Zhe;GUO Kui;LIU Di-qiu;QI Ting;GUO Wei;YANG Guang-pu;DU Cheng;WANG Xiao-jun(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第12期1282-1286,1292,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发专项(2017YFD0500404);黑龙江省自然科学基金面上项目(C2018071)。

摘　　要：马梨形虫病(EP)是由巴贝斯虫属的驽巴贝斯虫(B.caballi)和泰勒虫属的马泰勒虫(T.equi)感染引起的蜱传马属动物疾病。为建立EP两种病原的双重荧光定量PCR检测方法,本研究根据GenBank中T.equi和B.caballi 18S rRNA基因序列设计特异性引物和探针,经过反应体系和条件优化,建立了能够同时检测T.equi和B.caballi的双重荧光定量PCR检测方法。特异性试验结果显示该方法与马疱疹病毒Ⅰ型(EHV-1),马疱疹病毒Ⅳ型(EHV-4),马流产沙门氏菌(S.abortus equi),马链球菌(S.equi),马传染性贫血病毒(EIAV)均无交叉反应,特异性强。敏感性试验结果显示,该方法对T.equi和B.caballi的质粒标准品的检测下限均为100拷贝/μL,敏感性高;将T.equi和B.caballi的质粒标准品10倍倍比稀释为1×10^(8)拷贝/μL~1×10^(4)拷贝/μL共5个稀释度,进行组内、组间重复性试验,结果显示该方法的组内、组间变异系数均小于5%,重复性好。利用本实验建立的方法对2019年我国北方西部地区200份马属动物全血样品进行检测,结果显示T.equi的阳性率为47%(94/200),B.caballi的阳性率为2.5%(5/200),其中T.equi和B.caballi混合感染率为1%(2/200),该方法与套式PCR的符合率为97.5%,且该方法具有耗时较短、易操作等特点。本研究首次建立的EP双重荧光定量PCR检测方法对T.equi和B.caballi的鉴别检测、病原监测、流行病学调查等均具有重要的意义。Equine piroplasmosis(EP)is a tick-borne disease caused by apicomplexan protozoan parasites,Babesia caballi and Theileria equi.In order to establish a duplex real-time PCR detection method for EP,primers and probes were designed based on the conserved sequence of 18 S rRNA gene of T.equi and B.caballi.After optimizing the reaction conditions and reaction system,a duplex real-time PCR detection method for simultaneous detection of T.equi and B.caballi was successfully established.The results showed the methods had no cross-react with EHV-1,EHV-4,S.abortus equi,S.equi and EIAV.In the sensitivity test,the plasmid standards of T.equi and B.caballi were subjected to a 10-fold ratio dilution from 1×107 copies/μL to 1×101 copies/μL.The results showed that the method has the lowest detection limit of the plasmid standards of T.equi and B.caballi at 100 copies/μL.In the repeatability test,the plasmid standards of T.equi and B.caballi were diluted 10 times by 1×10^(8) copies/μL to 1×10^(4) copies/μL,and a total of 5 dilutions were carried out.Repeatability test were repeated 3 times for each dilution.The coefficient variation of intra-and inter-assay were less than 5%,indicating the established methods had high specificity,sensitivity and repeatability.The detection of 200 clinical samples collected from equids in 2019 showed that the positive rate of T.equi was 47%(94/200),and the positive rate of B.caballi was 2.5%(5/200),and the co-infection rate of T.equi and B.caballi was 1%(2/200).The coincidence rate of this method with the nest PCR was 97.5%.The established method could contribute to the accurate diagnosis,pathogenic surveillance and epidemiological investigation of T.equi and B.caballi infections in horses.

关 键 词：马梨形虫 马泰勒虫 驽巴贝斯虫 双重荧光定量PCR 

分 类 号：S855.9[农业科学—临床兽医学]