本文關(guān)鍵詞：牛副流感病毒3型Q-PCR及間接ELISA方法的建立　出處：《黑龍江八一農(nóng)墾大學》2017年碩士論文　論文類型：學位論文

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【摘要】：牛副流感病毒3型(Bovine parainfluenza virus type 3,BPIV3)為單股負鏈RNA病毒,副黏病毒科、呼吸道病毒屬成員,與牛皰疹病毒I型(Bovine herpes virus I,Bo HV-I)、牛呼吸道合胞體病毒(Bovine respiratory syncytial virus,BRSV)、牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV)共同構(gòu)成了牛呼吸道疾病綜合征(Bovine respiratory disease complex,BRDC)的主要病毒性病原,在世界范圍內(nèi)每年都會給養(yǎng)牛業(yè)造成巨大的經(jīng)濟損失。流行病學調(diào)查表明,該病毒在我國已呈現(xiàn)廣泛分布。因此,建立能夠方便、準確的檢測BPIV3的方法已迫在眉睫。病原學診斷方面,本研究根據(jù)Gen Bank上已公布BPIV3的P基因中保守區(qū)域設計特異性引物及Taq Man-MGB探針,并建立了Taq Man探針和SYBR Green I兩種檢測BPIV3的熒光定量PCR方法,并對兩種方法的特異性、敏感性、重復性進行驗證。結(jié)果表明,Taq Man探針法及SYBR染料法構(gòu)建的標準曲線在103~107 copies/μL內(nèi)均具有較好的線性關(guān)系,特異性實驗中,兩種方法檢測BPIV3a及BPIV3c的結(jié)果為陽性,而對牛的其他呼吸道病毒無交叉反應。敏感性試驗中,Taq Man Q-PCR對標準品的最小檢出量為101 copies/μL,SYBR Green I Q-PCR對標準品的最小檢出量為103 copies/μL。重復性試驗中,Ct值變異系數(shù)均小于1.0%。血清學診斷方面,本研究選取在不同毒株中相對保守的NP蛋白,對NP蛋白進行抗原表位的預測及初步篩選,結(jié)合現(xiàn)有文獻的報道,最終確定NP蛋白的N端8 aa~156 aa(NP-N)及C端368 aa~507 aa(NP-C)為優(yōu)勢抗原區(qū),設計合成兩對特異性引物并擴增。構(gòu)建原核表達載體p ET-28a-NP-N-C,并在E.coli BL21(DE3)中誘導表達。以純化的融合蛋白NP-N-C作為包被抗原,以BPIV3陰性、陽性血清為對照建立了檢測BPIV3抗體間接ELISA診斷方法。通過實驗對比,確立最佳反應條件,通過SPSS分析構(gòu)建ROC曲線,確定臨界值,計算該方法的特異性、敏感性,并對其重復性進行驗證。最佳反應條件如下:抗原最佳包被濃度為4μg/m L,37℃,1 h后4℃過夜為抗原最佳包被條件,最佳血清稀釋度為1:80。5%的脫脂乳為封閉液的最佳濃度,37℃條件下最佳封閉時間為1 h,血清的最佳作用條件為37℃1 h。二抗最佳稀釋度選擇1:5000。37℃條件下,二抗的最佳孵育時間為1 h。TMB底物最佳作用時間為15 min。該方法的臨界值為0.267時,所建立方法的特異性為97.4%,敏感性為95.3%。重復性實驗中,以同一批次及不同批次包被的酶標板檢測已知血清,變異系數(shù)均小于10%,本研究所建立的兩種Q-PCR檢測方法具有檢測3種不同基因型的BPIV3的潛力,為BPIV3的病原學早期診斷及定量分析提供了更快速、穩(wěn)定、可靠的方法。本研究建立的檢測BPIV3抗體的間接ELISA方法能夠應用于BPIV3的血清流行病學調(diào)查及追溯性診斷。本研究所建立的兩種Q-PCR病原學診斷方法和間接ELISA血清學診斷方法可以為BPIV3相關(guān)疾病的防治提供可靠指導。
[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.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65

【參考文獻】

相關(guān)期刊論文 前6條

1 楊建樂;趙貴民;侯佩莉;王洪梅;李杰;何洪彬;;牛副流感病毒3型抗體間接ELISA檢測方法的建立與初步應用[J];動物醫(yī)學進展;2016年11期

2 冉旭華;張\，

本文編號：1337850