牛传染性鼻气管炎病毒荧光定量PCR方法建立及其灭活疫苗研究

发布时间：2018-05-18 04:28

本文选题：牛传染性鼻气管炎病毒 + 荧光定量PCR　；参考：《黑龙江八一农垦大学》2015年硕士论文

【摘要】：牛传染性鼻气管炎(Infectious bovine rhinotracheitis，IBR)是一种急性、热性、接触性传染病，由牛传染性鼻气管炎病毒(IBRV)引起。临床主要表现为高热、呼吸困难、上呼吸道炎症和母畜流产等。该病虽然致死率低，但对牛的育肥、产奶量和国际贸易有严重影响。建立灵敏、快速、可靠的检测方法，净化牛群，免疫接种疫苗，仍是预防和控制该病的重要措施。由于没有合适的实验动物模型，，传统疫苗免疫后仍缺乏有效的免疫效力评价方法。因此，本研究在建立牛传染性鼻气管炎病毒的TaqMan-MGB荧光定量PCR方法的基础上，对健康家兔及灭活苗免疫家兔的病毒侵染进行了初步研究，对于疫苗免疫效力的评价及有效防控本病具有重要的现实意义。本研究首先根据GenBank已公布的IBRV Bartha Nu/67株基因序列针对gB基因设计特异性引物和Taqman-MGB探针，建立了检测牛传染性鼻气管炎病毒的TaqMan-MGB荧光定量PCR方法。结果显示，标准曲线相关系数(R2)为0.998，103-108拷贝/μL内具有较好的线性关系。该方法灵敏度是常规PCR灵敏度的100倍，检测下限为1.49×101拷贝/μL。特异性良好，对牛呼吸道其他病毒无交叉反应。适用于临床疑似样品的快速定量检测。从17份疑似IBR症状的奶牛鼻拭子中，经分离培养，PCR、间接免疫荧光试验试验、微量血清中和实验与基因序列同源性分析鉴定，确定获得一株IBRV，命名为IBRV DQ/14株。用TCID50为10-7.33/100μL的分离株鼻腔接种家兔，用鼻拭子采集第1-14d的鼻腔粘液，每隔7d分离血清。无菌采集第3、5、7、11和49d的剖杀兔组织脏器，荧光定量PCR、病理组织学和免疫组化检测。结果表明，接毒后第2-7d家兔出现临床症状并排毒至第7d，7-14d产生大量IgG抗体并维持数周。病理组织学和免疫组化结果显示，肺脏出现炎性反应，鼻甲骨和肺脏上皮细胞均有病毒复制。研究表明该分离株对家兔有致病性。用BEI灭活IBRV DQ/14分离株，以Montanide ISA206佐剂乳化制备灭活疫苗。分别接种断奶仔兔和Balb/c小鼠，进行安全性检验。间隔21d后两次免疫日本大耳家兔，14d后进行攻毒实验。结果表明疫苗安全性良好。攻毒后，免疫组未出现临床症状，免疫组化结果表明该疫苗阻止了分离株对呼吸道粘膜的侵染，免疫保护率为100%。综上所述，本研究成功建立了可用于临床快速检测IBRV的TaqMan-MGB荧光定量PCR方法；证明了IBRV DQ/14分离株对日本大耳家兔有较强的致病性，制备的灭活疫苗安全，可有效阻止病毒对呼吸系统的侵染。为IBRV的防制提供了有效措施。
[Abstract]:Infectious bovine rhinotrachetitis (IBR) is an acute, feverish, contact infectious disease caused by bovine infectious rhinotracheitis virus (IBRV). Clinical manifestations include high fever, dyspnea, upper respiratory inflammation and female abortion. Although the fatality rate is low, it has a serious effect on cattle fattening, milk production and international trade. It is still an important measure to prevent and control the disease by establishing a sensitive, rapid and reliable detection method, purifying cattle and vaccinating them. Due to the absence of suitable animal models, the traditional vaccine still lacks an effective method to evaluate the immune efficacy after immunization. Therefore, the infection of bovine infectious rhinotracheitis virus in healthy rabbits and inactivated rabbits was studied on the basis of the method of TaqMan-MGB fluorescence quantitative PCR for bovine infectious rhinotracheitis virus. It has important practical significance for the evaluation of vaccine immune effectiveness and the effective prevention and control of this disease. In this study, based on the sequence of IBRV Bartha Nu/67 strain published by GenBank, specific primers and Taqman-MGB probes were designed to detect bovine infectious rhinotracheitis virus by TaqMan-MGB fluorescence quantitative PCR. The results show that the correlation coefficient of the standard curve is 0.998103-108 copies / 渭 L with a good linear relationship. The sensitivity of this method is 100 times higher than that of conventional PCR, and the detection limit is 1.49 脳 101copy / 渭 L. It has good specificity and has no cross reaction to other viruses in bovine respiratory tract. It is suitable for rapid quantitative detection of suspected clinical samples. An IBRV strain was obtained from 17 nasal swabs of cows suspected to have IBR symptoms by isolation and culture, indirect immunofluorescence assay, microserum neutralization test and homology analysis of gene sequence. Rabbits were inoculated with nasal cavity with TCID50 of 10-7.33 / 100 渭 L, nasal swab was used to collect nasal mucus from day 1 to 14, and serum was isolated every 7 days. Tissue organs, fluorescence quantitative PCR, histopathology and immunohistochemistry were collected from rabbits at day 3, 5, 7, 11 and 49, respectively. The results showed that the rabbits developed clinical symptoms at 2-7 days and produced a large number of IgG antibodies at 7-14 days after detoxification for several weeks. Histopathological and immunohistochemical results showed that inflammatory reaction was found in the lungs and virus replication was found in both nasal turbinate bone and lung epithelial cells. The results showed that the isolate had pathogenicity to rabbits. BEI was used to inactivate IBRV DQ/14 isolate and Montanide ISA206 adjuvant emulsified to prepare inactivated vaccine. Weaning rabbits and Balb/c mice were inoculated for safety test. Japanese rabbits were immunized twice for 14 days after 21 days. The results showed that the vaccine was safe. The results of immunohistochemistry showed that the vaccine prevented the isolated strain from infecting the respiratory mucosa, and the immune protection rate was 100%. In conclusion, this study successfully established the TaqMan-MGB fluorescence quantitative PCR method which can be used for rapid clinical detection of IBRV, and proved that the isolated strain of IBRV DQ/14 has strong pathogenicity to Japanese large ear rabbits, and the inactivated vaccine prepared is safe. It can effectively prevent the infection of respiratory system. It provides effective measures for the prevention and control of IBRV.
【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】：2015
【分类号】：S852.65

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