【作者】 张鹤；

【导师】 吴高峰； 闫喜军；

【作者基本信息】 沈阳农业大学 ， 动物疫病防控（专业学位）， 2024， 硕士

【摘要】 牛腹泻性病毒是养牛产业中最常见、对犊牛危害最大且致死率较高的疾病,引起幼牛腹泻的病毒较常见的主要有牛病毒性腹泻病毒(BVDV),牛冠状病毒(BCoV),牛轮状病毒(BRoV),这三种腹泻病毒在牛群中发病率较高。对此本实验对这三种病毒进行研究,为我国牛腹泻病毒病毒株的流行情况提供数据支持和理论依据,为疫病的早期诊断和快速筛查提供技术支撑。主要研究内容包括。1.牛腹泻病毒毒株分离鉴定将牛场收集到的临床样本进行PCR检测,选取疑似BVDV和BRoV的阳性样本,分别在MDBK细胞和MA-104细胞上面进行培养繁殖。使用病毒滴度测定、RT-PCR验证、致病性试验等方法进行鉴定,结果显示:(1)MDBK细胞出现聚集,脱落等特征性病变,间接免疫荧光显示BVDV1出现荧光。使用BVDV1引物序列扩增出现目的片段。进行动物毒力试验,试验牛出现持续性水样腹泻,精神沉郁,体温均出现升高现象,白细胞升高,并从白细胞中成功进行病毒分离,使用间接免疫荧光进行鉴定为阳性。遗传进化分析结果显示,该分离株与BVDV1-Y2病毒进化关系较为密切。成功分离到一株BVDV将其命名为BVDV-JSHWT分离株。(2)MA-104细胞出现细胞质病变,细胞聚集等特征性病变,使用BRoV引物序列扩增出现目的片段。基因测序分析,该分离株与RZV-Khorasan-N2K7和Tehran-N2K6两株病毒进化关系较为密切,同处于一独立分支。成功分离到一株BRoV将其命名为BRoV-SC-HWT分离株。2.多重荧光定量PCR方法建立最近几年分子技术的使用非常普遍,最近几年分子技术的使用非常普遍,尤其是荧光定量PCR方法,特别是多重荧光定量PCR方法方便,简洁,可以同时检测多种病原。本课题通过查找NCBI上BVDV、BRoV和BCoV的参考基因序列,分别选择5’UTR基因、VP6基因与N基因,设计引物探针,建立单重的RT-qPCR方法,在此基础上建立一种多重RT-qPCR方法。摸索探针引物浓度和比例,并进行特异性、敏感性和重复性及临床样本检测等试验进行验证。研究结果如下:使用建立三种病原单重的RT-qPCR方法和三重的RT-qPCR方法对牛病毒性腹泻,牛冠状病毒,牛轮状病毒,猪瘟病毒,牛传染性鼻气管炎病毒,牛副流感3型病毒,猫瘟病毒,犬瘟热病毒进行特异性检测,检测结果只有BVDV、BRV和BCoV扩增呈阳性,而其它病毒的无扩增曲线,具有高的特异性。将BVDV毒株,BCOV毒株,BROV毒株进行10~1-10~88个梯度的稀释后为模板使用建立的RT-qPCR进行灵敏性检测。检测结果表明本实验建立的BVDV最低检测限4.1 copies/μL(4.1×10~0copies/μL)BRoV 4.98 copies/μL(4.98×10~0copies/μL),BCoV 6.37 copies/μL(6.37×10~0copies/μL),所建立的多重RT-qPCR检测方法具有极高的敏感性。选取10-~5、10-~6、10-~73个不同浓度的样本进行重复性试验,3组数据变异系数范围小于1%,该方法稳定性成立。对已知的阳性样本进行检测,结果显示,本试验建立的RT-qPCR方法对阳性样本的检出率要高于商品化试剂盒,对样本的检测具有高度准确性。综上所述,本试验成功分离到BVDV-JSHWT分离株和BRoV-SC-HWT分离株,建立的三重RT-qPCR检测方法可用于BVDV,BRV和BCoV的快速检测。更多还原

【Abstract】 Bovine diarrheal virus is the most common disease in the cattle industry,which is the most harmful to calves and has a high fatality rate.The viruses that cause diarrhea in young cattle are mainly bovine viral diarrhea virus（BVDV）,bovine coronavirus（BCoV）and bovine rotavirus（BRoV）,which have a high incidence in cattle.In this study,these three viruses were studied to provide data support and theoretical basis for the epidemic situation of bovine diarrhea virus strains in China,and to provide technical support for early diagnosis and rapid screening of the disease.The main research contents include.1.Isolation and identification of bovine diarrhea virus strainThe clinical samples collected from the cattle farm were tested by PCR,and the positive samples suspected of BVDV and BRoV were selected for culture and propagation on MDBK cells and MA-104 cells,respectively.Virus titer determination,RT-PCR verification,pathogenicity test and other methods were used for identification,and the results showed:（1）MDBK cells showed characteristic lesions such as aggregation and shedding,and indirect immunofluorescence showed fluorescence of BVDV1.The target fragment was amplified using BVDV1 primer sequence.Animal virulence test was carried out.The experimental cattle showed persistent watery diarrhea,mental depression,elevated body temperature and elevated white blood cells.The virus was successfully isolated from the white blood cells and identified as positive by indirect immunofluorescence.Genetic analysis showed that this strain was closely related to the evolution of BVDV1-Y2 virus.A BVDV strain was successfully isolated and named as the BVDV-JSHWT isolate.（2）Characteristic lesions such as cytoplasmic lesions and cell aggregation were found in MA-104 cells,and target fragments were amplified with BRoV primer sequence.The gene sequencing analysis showed that this strain was closely related to RZV-Khorasan-N2K7 and Tehran N2K6 strains,which were in an independent branch.A BRoV strain was successfully isolated and was named the Bro V-SC-HWT isolate.2.Multiple fluorescence quantitative PCR method was establishedIn recent years,molecular technology has been widely used,especially the fluorescent quantitative PCR method,especially the multiple fluorescent quantitative PCR method is convenient and concise,and can detect multiple pathogens at the same time.In this study,by searching the reference gene sequences of BVDV,BRoV and BCoV on NCBI,selecting5’UTR gene,VP6 gene and N gene respectively,designing primer probes,establishing a single RT-qPCR method,and on this basis,establishing a multiple RT-qPCR method.Probe primer concentration and proportion were explored,and specificity,sensitivity,repeatability and clinical sample testing were performed for verification.The results are as follows:Specific detection of bovine viral diarrhea,bovine coronavirus,bovine rotavirus,swine fever virus,bovine infectious rhinotracheitis virus,bovine parainfluenza type 3 virus,cat distemper virus and canine distemper virus was performed by establishing a three-pathogen single RT-qPCR method and a triple RT-qPCR method.Only BVDV,BRV and BCoV amplification were positive,while other viruses showed no amplification curve and had high specificity.BVDV strains,BCOV strains and BROV strains were diluted with 8 gradients of10¹-10⁸,and then sensitivity was detected using the established RT-qPCR.The results showed that the minimum detection limit of BVDV established in this experiment was 4.1 copies/μL（4.1×10⁰copies/μL）and BRoV was 4.98 copies/μL（4.98×10⁰copies/μL）.BCoV was6.37 copies/μL（6.37×10⁰copies/μL）,and the established multiplex RT-qPCR assay was highly sensitive.Three samples with different concentrations of 10^-5,10^-6and 10^-7were selected for repeatability test,and the variation coefficient range of the three groups of data was less than 1%,indicating that the stability of the method was established.The known positive samples were tested,and the results showed that the RT-qPCR method established in this experiment had a higher detection rate of positive samples than commercial kits,and had high accuracy in the detection of samples.In summary,the BVDV-JSHWT and BRoV-SC-HWT isolates were successfully isolated in this experiment,and a triple RT-qPCR detection method was established.更多还原