牛副流感病毒3型Q-PCR及间接ELISA方法的建立

发布时间：2017-12-26 15:52

本文关键词：牛副流感病毒3型Q-PCR及间接ELISA方法的建立　出处：《黑龙江八一农垦大学》2017年硕士论文　论文类型：学位论文

更多相关文章： 牛副流感3型 Q-PCR 间接ELISA NP基因

【摘要】：牛副流感病毒3型(Bovine parainfluenza virus type 3,BPIV3)为单股负链RNA病毒,副黏病毒科、呼吸道病毒属成员,与牛疱疹病毒I型(Bovine herpes virus I,Bo HV-I)、牛呼吸道合胞体病毒(Bovine respiratory syncytial virus,BRSV)、牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)共同构成了牛呼吸道疾病综合征(Bovine respiratory disease complex,BRDC)的主要病毒性病原,在世界范围内每年都会给养牛业造成巨大的经济损失。流行病学调查表明,该病毒在我国已呈现广泛分布。因此,建立能够方便、准确的检测BPIV3的方法已迫在眉睫。病原学诊断方面,本研究根据Gen Bank上已公布BPIV3的P基因中保守区域设计特异性引物及Taq Man-MGB探针,并建立了Taq Man探针和SYBR Green I两种检测BPIV3的荧光定量PCR方法,并对两种方法的特异性、敏感性、重复性进行验证。结果表明,Taq Man探针法及SYBR染料法构建的标准曲线在103~107 copies/μL内均具有较好的线性关系,特异性实验中,两种方法检测BPIV3a及BPIV3c的结果为阳性,而对牛的其他呼吸道病毒无交叉反应。敏感性试验中,Taq Man Q-PCR对标准品的最小检出量为101 copies/μL,SYBR Green I Q-PCR对标准品的最小检出量为103 copies/μL。重复性试验中,Ct值变异系数均小于1.0%。血清学诊断方面,本研究选取在不同毒株中相对保守的NP蛋白,对NP蛋白进行抗原表位的预测及初步筛选,结合现有文献的报道,最终确定NP蛋白的N端8 aa~156 aa(NP-N)及C端368 aa~507 aa(NP-C)为优势抗原区,设计合成两对特异性引物并扩增。构建原核表达载体p ET-28a-NP-N-C,并在E.coli BL21(DE3)中诱导表达。以纯化的融合蛋白NP-N-C作为包被抗原,以BPIV3阴性、阳性血清为对照建立了检测BPIV3抗体间接ELISA诊断方法。通过实验对比,确立最佳反应条件,通过SPSS分析构建ROC曲线,确定临界值,计算该方法的特异性、敏感性,并对其重复性进行验证。最佳反应条件如下:抗原最佳包被浓度为4μg/m L,37℃,1 h后4℃过夜为抗原最佳包被条件,最佳血清稀释度为1:80。5%的脱脂乳为封闭液的最佳浓度,37℃条件下最佳封闭时间为1 h,血清的最佳作用条件为37℃1 h。二抗最佳稀释度选择1:5000。37℃条件下,二抗的最佳孵育时间为1 h。TMB底物最佳作用时间为15 min。该方法的临界值为0.267时,所建立方法的特异性为97.4%,敏感性为95.3%。重复性实验中,以同一批次及不同批次包被的酶标板检测已知血清,变异系数均小于10%,本研究所建立的两种Q-PCR检测方法具有检测3种不同基因型的BPIV3的潜力,为BPIV3的病原学早期诊断及定量分析提供了更快速、稳定、可靠的方法。本研究建立的检测BPIV3抗体的间接ELISA方法能够应用于BPIV3的血清流行病学调查及追溯性诊断。本研究所建立的两种Q-PCR病原学诊断方法和间接ELISA血清学诊断方法可以为BPIV3相关疾病的防治提供可靠指导。
[Abstract]:[Abstract]: bovine parainfluenza virus type 3 (Bovine parainfluenza virus type 3, BPIV3) is a single stranded RNA virus, Paramyxoviridae, respiratory virus is a member, and bovine herpes virus type I (Bovine herpes virus I, Bo HV-I), bovine respiratory syncytial virus (Bovine respiratory syncytial virus, BRSV), bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) constitute the bovine respiratory disease syndrome (Bovine respiratory disease complex, BRDC) the main pathogenic viruses, every year will cause huge economic losses to the cattle industry in the world. Epidemiological surveys show that the virus has been widely distributed in China. Therefore, it is imminent to establish a convenient and accurate method for detecting BPIV3. Etiologic diagnosis, according to the study published in Bank Gen P BPIV3 gene specific primers were designed in the conserved region of Taq and Man-MGB probe, and the establishment of fluorescent quantitative PCR for Taq Man and SYBR Green I two probe for detecting BPIV3, and verify the two methods the specificity, sensitivity and repetition of. The results showed that the standard curve constructed by Taq Man probe method and SYBR dye method had a good linear relationship in 103~107 copies/ L L. In the specific experiment, the results of BPIV3a and BPIV3c detected by the two methods were positive, but there was no cross reaction to other respiratory viruses in cattle. In the sensitivity test, the minimum detection amount of Taq Man Q-PCR for standard products is 101 copies/ mu L, and the minimum detection amount of SYBR Green I Q-PCR is 103 copies/ mu L. In the repeatability test, the coefficient of variation of Ct value is less than 1%. Serological diagnosis, were selected in this study is relatively conservative in different strains of NP protein, NP protein to predict epitopes and preliminary screening, combined with the existing literature, and ultimately determine the NP protein N terminal 8 aa~156 AA (NP-N) and C terminal 368 aa~507 AA (NP-C) for the advantages of antigenic regions the design and synthesis of two pairs of specific primers and amplified. The prokaryotic expression vector, P ET-28a-NP-N-C, was constructed and expressed in E.coli BL21 (DE3). The purified fusion protein NP-N-C was used as the envelope antigen, and the indirect ELISA detection method for the detection of BPIV3 antibody was established with BPIV3 negative and positive serum as the control. Through experimental comparison, we established the best reaction condition, constructed ROC curve by SPSS analysis, determined the critical value, calculated the specificity and sensibility of the method, and verified its reproducibility. The optimal reaction conditions were as follows: the optimal concentration of antigen was 4 g/m L, 37 C, 1 h and 4 C for the best envelope, and the best serum dilution was 1:80. 5% of the defatted milk was the best concentration of the closed liquid. The optimum closing time was 1 h at 37 C, and the optimum condition for the serum was 37 and 1 h. The optimum dilution of two was selected as 1:5000. Under 37 centigrade, the best incubation time of two resistance was 1 h. The optimum reaction time of TMB substrate was 15 min. When the critical value of the method is 0.267, the specificity of the method is 97.4% and the sensitivity is 95.3%. Repeat the experiment, in the same batch and different batches of the ELISA plate coated by the detection of known serum, the coefficient of variation was less than 10%, two kinds of Q-PCR detection methods established in this study can detect 3 different genotypes of BPIV3 potential method for BPIV3 diagnosis and etiological analysis provides a more quantitative early fast, stable and reliable. The indirect ELISA method for detecting BPIV3 antibodies in this study can be applied to the sero epidemiological investigation and traceability diagnosis of BPIV3. Two Q-PCR etiological diagnosis methods and indirect ELISA serological diagnosis methods established in this study can provide reliable guidance for the prevention and treatment of BPIV3 related diseases.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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