本文選題：犬瘟熱病毒 + 抗原檢測(cè)�。� 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：犬瘟熱是由犬瘟熱病毒引起犬的一種高度接觸性、致死性傳染病。犬瘟熱病毒屬于副粘病毒科麻疹病毒屬,同屬成員還有麻疹病毒、牛瘟病毒、小反芻獸疫病毒和鯨豚麻疹病毒。感染犬瘟熱病毒犬臨床癥狀為精神沉郁、體溫升高、厭食、拉稀,后期出現(xiàn)神經(jīng)癥狀,表現(xiàn)為共濟(jì)失調(diào)、四肢癱瘓等。最終,口吐白沫、尖叫而死亡。犬瘟熱病毒易感動(dòng)物主要是幼犬和貂,成年動(dòng)物發(fā)病但不死亡。我國(guó)養(yǎng)犬業(yè)、皮毛經(jīng)濟(jì)動(dòng)物業(yè)及野生動(dòng)物安全受到了該病嚴(yán)重威脅�，F(xiàn)有的犬瘟熱病毒檢測(cè)方法主要包括血清中和試驗(yàn)、免疫熒光試驗(yàn)、ELISA、RT-PCR、病毒分離及包涵體檢查。但是這些方法都需要專業(yè)人員在特殊儀器下進(jìn)行操作,并且耗時(shí)幾小時(shí)到幾天不等。因此建立一種在臨床上操作簡(jiǎn)易、快速、可靠并且廉價(jià)的犬瘟熱病毒檢測(cè)方法非常重要。本研究使用了兩株可以與重組CDV-N蛋白和犬瘟熱病毒結(jié)合的抗CDV-N蛋白單克隆抗體,研發(fā)了一個(gè)可以檢測(cè)犬瘟熱病毒抗原的免疫層析試紙條。本研究的內(nèi)容及結(jié)果如下:將兩株抗CDV-N蛋白單克隆抗體1B7和2F11,分別腹腔注射Balb/c小鼠制備單抗腹水,純化并鑒定。以膠體金標(biāo)記純化的1B7單抗作為示蹤劑,將純化的單克隆抗體2F11和商品化的羊抗鼠IgG二抗分別作檢測(cè)線(T線)和質(zhì)控線(C線)噴點(diǎn)于硝酸纖維素膜上,建立了檢測(cè)動(dòng)物排出物和排泄物中犬瘟熱病毒抗原的膠體金免疫層析方法。判定標(biāo)準(zhǔn)為:①待檢的樣品中含有CDV,可與金標(biāo)單抗形成CDV-1B7復(fù)合物,經(jīng)層析膜流經(jīng)T線和C線時(shí),CDV-1B7復(fù)合物就會(huì)和T線上的2F11單抗以及C線上的羊抗鼠IgG二抗結(jié)合,形成兩條紅色的線,判定為陽(yáng)性結(jié)果;②若待檢血清中不含CDV,則只有質(zhì)控線出現(xiàn);③若質(zhì)控線未形成紅色線條時(shí),則該方法檢測(cè)無(wú)效。經(jīng)過(guò)探索該膠體金的最佳反應(yīng)條件,我們最終確定T線包被濃度為600μg/mL、C線濃度為800μg/mL、金標(biāo)單抗最佳標(biāo)記pH為8.6、濃度為14.26μg/mL。本研究中制備的膠體金免疫試紙條特異性良好,只在檢測(cè)犬瘟熱病毒時(shí)出現(xiàn)陽(yáng)性結(jié)果,對(duì)其他病毒如犬細(xì)小病毒(CPV)、犬冠狀病毒(CCV)、犬副流感病毒(CPIV)、偽狂犬病毒(PRV)、新城疫病毒(NDV)無(wú)交叉反應(yīng);在敏感性試驗(yàn)中,該方法檢測(cè)細(xì)胞毒最低濃度為102.5 TCID50/mL;對(duì)同一樣品不同批次重復(fù)試驗(yàn),該方法檢驗(yàn)均為同一結(jié)果,并且在4℃可穩(wěn)定儲(chǔ)存6個(gè)月不改變特性;與商品化CDV檢測(cè)試劑盒對(duì)比,兩者有良好的符合性。綜上所述,本試驗(yàn)建立了犬瘟熱病毒抗原膠體金免疫層析檢測(cè)方法,為犬瘟熱病毒檢測(cè)提供了一種快速、簡(jiǎn)易的方法。
[Abstract]:Canine distemper is a highly contagious and fatal disease caused by canine distemper virus. Canine distemper virus belongs to the measles virus genus of paramyxovirus family, and its members include measles virus, rinderpest virus, small ruminant zoonotic virus and whale dolphin measles virus. The clinical symptoms of canine distemper virus infection were depression, elevated body temperature, anorexia, dilation, and later neurological symptoms, such as ataxia, quadriplegia and so on. Finally, foaming at the mouth, screaming and dying. Canine distemper virus susceptible animals are mainly puppies and minks, adult animals develop but do not die. China's canine industry, fur and economic animal industry and wildlife safety has been seriously threatened by the disease. The existing detection methods of canine distemper virus mainly include serum neutralization test, immunofluorescence test ELISART-PCR, virus isolation and inclusion body examination. But these methods require professionals to operate under special instruments and take hours to days. Therefore, it is very important to establish a simple, rapid, reliable and inexpensive method for detection of canine distemper virus. In this study, two monoclonal antibodies against CDV-N protein and canine distemper virus were used to detect the antigen of canine distemper virus, and an immunochromatographic test strip was developed to detect the antigen of canine distemper virus. The contents and results of this study are as follows: two McAbs, 1B7 and 2F11, were injected intraperitoneally into Balb/c mice to prepare ascites, purified and identified. The purified 1B7 McAb labeled with colloidal gold was used as tracer. The purified monoclonal antibody (2F11) and the commercial goat anti mouse IgG (second antibody) were sprayed on the nitrocellulose membrane. A colloidal gold immunochromatographic method for the detection of canine distemper virus antigen in animal excreta and excreta was established. It was determined that the sample to be tested by the standard of: 1 contained CDV, which could form CDV-1B7 complex with gold labeled McAb. When the membrane was flowing through T and C lines, the CDV-1B7 complex would bind to 2F11 monoclonal antibody on T line and sheep anti-rat IgG second antibody on C line. If there is no CDV in the serum, only the quality control line appears if the red line is not formed, then the method is ineffective. After exploring the optimum reaction conditions of the colloidal gold, we finally determined that the concentration of T line coating was 600 渭 g / mL, the best pH of gold labeled McAb was 8.6, and the concentration was 14.26 渭 g / mL. The colloidal gold immunoassay strip prepared in this study has a good specificity and only positive results in the detection of canine distemper virus. There was no cross reaction for other viruses such as CPV, CCV, CPIVV, PRV, NDV, and so on, and no cross reaction was found for other viruses, such as canine parainfluenza virus (CPV), pseudorabies virus (PRV) and Newcastle disease virus (NDV). The lowest concentration of cytotoxicity detected by this method was 102.5 TCID 50% mL.The results of repeated tests for different batches of the same sample were the same, and it could be stably stored at 4 鈩，

本文編號(hào)：1811318