中山病病毒qRT-PCR与RT-PCR检测方法的建立及应用

Development and application of a real-time quantitative RT-PCR and RT-PCR method for detection of Chuzan disease virus

中山病病毒(CHUV)是一种严重危害犊牛神经系统的虫媒病毒,自2012年首次从我国云南师宗分离后,相继在广西与广东等地区也分离到该病毒。为建立CHUV的检测方法,本研究根据CHUV编码VP2蛋白的Seg-2基因序列设计引物和探针,通过条件优化建立了检测CHUV的荧光定量RT-PCR(qRT-PCR)和常规RT-PCR法。利用建立的qRT-PCR和RT-PCR分别检测CHUV、流行性出血病病毒、阿卡斑病毒、蓝舌病病毒和牛流行热病毒,结果显示除了均能检测CHUV以外,两种方法对其他病毒均无特异性扩增;利用两种方法对质粒标准品pLB-CHUV/V144/Seg-2(1×10^(0)拷贝/μL~1×10^(9)拷贝/μL)进行检测,结果显示两种方法的检测下限可分别达到10拷贝/μL(qRTPCR)与100拷贝/μL(RT-PCR);重复性试验结果显示,qRT-PCR的批内和批间变异系数均小于1%。利用这两种方法分别检测2019年5月~11月隔周采集3头感染CHUV哨兵牛的各25份共计75份的血液样品并计算二者的符合率;同时利用C-ELISA方法检测3头牛对应血清样品的CHUV抗体。结果显示,qRT-PCR检测到30份阳性样品,45份阴性样品;RT-PCR检测到22份阳性样品,53份阴性样品。两种方法的总符合率为89.33%;相比较于C-ELISA方法,qRT-PCR方法能在动物感染CHUV的早期(约早2周)检测出病毒核酸,更适用于检测病毒载量较低的血液样品,而RT-PCR的扩增产物可以通过测序了解待测病毒的遗传信息。本研究建立的两种检测方法可以互补使用,为CHUV的早期检测和流行病学研究等提供了快速、方便、可靠的技术支撑。

Chuzan disease virus(CHUV)is an arbovirus that seriously harms the nervous system of calves.Since the first isolation of CHUV from sentinel animals in Yunnan,China in 2012,the virus has also been isolated successively in Guangxi and Guangdong provinces.In order to establish a CHUV detection methods,we designed primers and probes based on the Seg-2 gene of CHUV for real-time quantitative RT-PCR(qRT-PCR)and RT-PCR methods.These two methods showed great specificity for CHUV detection,which have no cross reaction with epizootic haemorrhagic disease virus(EHDV),bluetongue virus(BTV),Akabane virus(AKAV)and bovine ephemeral fever virus(BEFV).The detection limit of viral nucleic acid was 10 copies/μL(qRTPCR)and 100 copies/μL(RT-PCR),respectively;the intra-and inter-group coefficients of variation were less than 1%.Meanwhile,the two methods were used to detect 75 blood samples of 3 cattle infected with CHUV,and the coincidence rate of the two methords was calculated.At the same time,C-ELISA was used to detect CHUV antibody in serum samples of 3 cattle.The results showed that 30 positive samples and 45 negative samples were detected by qRT-PCR;RT-PCR detected 22 positive samples and 53 negative samples.The total coincidence rate of the two methods was 89.33%;Compared with C-ELISA method,qRT-PCR method can detect virus nucleic acid in the early stage of animal infection with CHUV(about 2 weeks earlier),which is more suitable for detecting blood samples with low virus load.The two methods can be used complementarily,which provides rapid,convenient and reliable technical support for diagnosis,epidemiological investigation and epidemiological research of CHUV.