牛腺病毒ELISA检测方法的建立及在咸阳地牛腺病毒血清流行病学调查中的应用

发布时间：2018-04-02 16:17

本文选题：牛腺病毒3型　切入点：棉鼠肺成纤维细胞　出处：《西北农林科技大学》2015年硕士论文

【摘要】：牛腺病毒(Bovine adenovirus,BAV),为腺病毒科(Adenoviridae)哺乳动物腺病毒属(Mastadenovirus)的成员。目前已报道牛腺病毒总共有10个血清型,BAV-1、-2、-3、-9和-10型牛腺病毒属于哺乳动物腺病毒属(Mastadenovirus),而BAV-4、-5、-6、-7和-8型属于腺胸腺病毒属(Atadenovirus)。BAVs感染呈世界范围内广泛流行,但是对BAVs在我国牛场的流行状况目前仍不清楚。鉴于不同牛腺病毒血清型间存在血清交叉反应,使用一种血清型BAV可同时对多个血清型BAVs进行检测,本研究以实验室成熟应用的3型牛腺病毒(BAV3)为检测抗原,建立检测牛腺病毒的间接ELISA方法,并以所建立间接ELISA对来自咸阳地区不同牛场2000多份血清进行牛腺病毒流行病学调查。取得的主要如下结果:1.全病毒抗原的制备及病毒滴度的测定通过0.1 MOI的BAV3接种棉鼠肺成纤维细胞(VIDO-DT1),对BAV3进行了大量扩增和病毒滴度测定。以测得TCID50值为106.47的BAV3为包被抗原原始浓度。2.间接ELISA检测方法的优化方阵滴定确定ELISA抗原最佳包被浓度为1:120,即106.47,血清稀释液的最佳使用浓度为l:80,封闭液用5%的脱脂奶粉在37°C下作用60 min,酶标二抗的最适浓度为1:1200,37°C作用1.5 h,底物作用15 min后,加1 mol/L的H2SO4终止反应。计算结果如下:30份阴性血清平均值(X)为0.1832,标准差(SD)为0.0657,确定阳性临界点(X+3SD)为0.3803,阴性临界点为(X+2SD)位0.3146。以该方法检测牛口蹄疫阳性血清、牛传染性鼻气管炎阳性血清、牛肺疫阳性血清、牛结核病阳性血清、牛流行热的标准阳性血清,以及BAV3的阳性,阴性血清。进行特异性试验,结果只有BAV3的阳性血清为阳性,其余均为阴性。并对此ELISA方法进行重复试验,批内重复变异系数在3.8511%~7.5916%之间,变异系数(CV)5%;批间重复变异系数在4.0124%~8.7293%之间,变异系数(CV)5%。证明此ELISA方法进行重复性良好。从上述各个方面对所建立的ELISA方法进行优化和改进,有效的提高了阳性值,并降低了阴性背景值,并验证了所建立的ELISA方法的稳定性。3.咸阳地区BAV3流行病学调查研究用建立的鉴别诊断间接ELISA方法对采集来自咸阳8个地区23个不同规模大小的牛场2000份牛血清进行了检测,结果显示:2000份牛样品中阳性样品数为933份,即整个样品阳性率平均值为49.23%,阳性群体中阳性率最低为31.96%,最高达91.74%。试验结果表明,可用于临床BAV血清抗体的检测。这为ELISA诊断试剂盒的研制开发奠定了基础,为建立规模化牛场的环境卫生及BAV感染的快速诊断方法提供了科学依据。
[Abstract]:Bovine adenovirusBAV, a member of the mammal adenovirus genus Adenoviridaeae, is a member of the family Adenoviridae.However, the prevalence of BAVs in cattle farms in China is still unclear.In view of the existence of serum cross-reaction among different bovine adenovirus serotypes, BAVs of multiple serotypes can be detected simultaneously by using one serotype of BAV. In this study, bovine adenovirus type 3 (BAV3), which is used in laboratory, was used as the detection antigen.An indirect ELISA method for detection of bovine adenovirus was established, and an epidemiological survey of bovine adenovirus was carried out on more than 2000 sera from different cattle farms in Xianyang area with indirect ELISA.The main result is as follows: 1.The preparation of whole virus antigen and the determination of virus titer were carried out by inoculation of VIDO-DT1 with 0. 1 MOI BAV3 of cotton mouse lung fibroblasts. A large number of BAV3 was amplified and virus titer was determined.The original concentration of encapsulated antigen was BAV3 with TCID50 value of 106.47.Optimization of indirect ELISA titration method: the optimum coating concentration of ELISA antigen is 1: 120, that is 106.47, the optimal concentration of serum dilution solution is 1: 80, and the optimal concentration of enzyme labeled second antibody is 60 min with 5% skimmed milk powder at 37 掳C, and the optimal concentration of serum dilution solution is 1: 80 with 5% skim milk powder at 37 掳C for 60 min.1: 1200 掳C for 1.5 h and substrate for 15 min.The H2SO4 termination reaction with 1 mol/L was added.The calculated results are as follows: the average value of 30 negative sera is 0.1832, the standard deviation is 0.0657, the positive critical point is 0.3803, and the negative critical point is 0.3146.The positive serum of bovine foot and mouth disease, the positive serum of bovine infectious rhinotracheitis, the positive serum of bovine pulmonary disease, the positive serum of bovine tuberculosis, the standard positive serum of bovine epidemic fever, and the positive and negative serum of BAV3 were detected by this method.The specificity test showed that only the positive serum of BAV3 was positive and the others were negative.The repeat test of ELISA showed that the coefficient of variation was between 3.8511% and 7.5916%, and the coefficient of variation was 4.0124% and 8.7293%, respectively, and the coefficient of variation was higher than that of CV5.The coefficient of variation was 7.5916%, and the coefficient of variation was between 4.0124% and 8.7293%, respectively.It is proved that this ELISA method is reproducible.From the above aspects, the established ELISA method is optimized and improved, which effectively increases the positive value, reduces the negative background value, and verifies the stability of the established ELISA method. 3.BAV3 Epidemiological investigation in Xianyang area 2000 bovine serum samples from 23 cattle farms of different sizes from 8 districts of Xianyang were detected by indirect ELISA method for differential diagnosis.The results showed that the positive rate of the whole sample was 49.23, the lowest was 31.96 in the positive population, and the highest was 91.74. The results showed that the number of positive samples was 933, the average positive rate of the whole sample was 49.23, and the positive rate was 31.96 in the positive population.The results show that it can be used to detect the serum antibody of BAV.It lays a foundation for the research and development of ELISA diagnostic kit and provides a scientific basis for the establishment of a rapid diagnostic method for environmental hygiene and BAV infection in large scale cattle farms.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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