牛冠状病毒实时荧光定量PCR检测方法的建立及应用

发布时间：2018-05-12 22:38

本文选题：牛冠状病毒 + 实时荧光定量PCR　；参考：《山东师范大学》2015年硕士论文

【摘要】：牛冠状病毒（Bovine Coronavirus，BCoV）属于冠状病毒科，冠状病毒属2a亚群，，是引起新生犊牛腹泻、成年牛冬痢和呼吸道感染的重要病原。流行病学调查表明，牛冠状病毒在牛场中普遍存在，主要通过消化道和呼吸道传播，感染的牛长期带毒并在一定时期内持续排毒，易造成牛群的大范围感染。因此，对带毒牛的及时检出和牛场环境监测就显得尤为重要。目前国内外尚无针对BCoV感染治疗的特效药物，奶业发达国家应用灭活疫苗或减毒疫苗预防本病，而我国尚无相关商品化疫苗。因此，亟需建立该病毒快速敏感的检测方法，并对我国部分地区奶牛场的BCoV流行情况进行调查，及早发现BCoV感染的牛，掌握流行病学数据，以便及时采取更有效的针对性措施进行防控，以减少该病毒对养牛业造成的经济损失。本研究根据GenBank收录的牛冠状病毒N基因序列，设计合成了1对扩增N基因部分片段的特异性引物，以BCoV病毒的cDNA为模板，进行PCR扩增，PCR产物纯化回收后送华大基因测序，将测序正确后的N基因片段连接到pEASY-T3载体上，经酶切鉴定、测序验证获得了重组质粒pEASY-T3-N。以得到的重组质粒pEASY-T3-N为阳性标准品，进行一系列稀释，选取6个梯度稀释的标准品作为模板进行荧光定量PCR反应，建立标准曲线和回归方程。然后运用常规PCR和实时荧光定量PCR方法，对2013～2014年本实验室采集的5个规模化奶牛场的212份临床样本进行病原学检测。最后设计合成了1对用于扩增N基因全长的特异性引物，将PCR扩增后产物送测序鉴定，应用DNAstar生物学软件将测得的N基因全长序列与GenBank中的序列进行系统发生分析。序列测定表明，我们克隆获得的100bp大小的片段正确，并且成功获得了重组质粒pEASY-T3-N阳性标准品。在建立的牛冠状病毒SYBR Green Ⅰ实时荧光定量PCR检测方法中，一个反应体系中模板最低检测限为7.8copies/μL，比普通RT-PCR更加灵敏；熔解曲线只出现特异性的单个峰，而没有明显的引物二聚体或者非特异性扩增的峰出现，证明该方法具有良好的特异性。同时与牛轮状病毒（bovine rotavirus，BRV)、牛病毒性腹泻病毒(bovine viral diarrhea virus，BVDV)、牛传染性鼻气管炎病毒（infectious bovine rhinotracheitis virus，IBRV）、牛肠道病毒（bovine enterovirus，BEV）、牛流行热病毒（bovine ephemeral fevervirus，BEFV）和牛副流感病毒3型（bovine parainfluenza virus type3，BPIV-3）均无交叉反应；批内、批间重复变异系数均低于1.5%。因此，该方法敏感性高，特异性强，重复性好，为牛冠状病毒的临床诊断和流行病学调查提供了技术保障。通过对临床样本进行PCR检测发现：在107份鼻拭子样本中，BCoV感染阳性率为5.61%，同时检测到BCoV分别与BVDV、IBRV、BPIV3混合感染阳性率依次为4.60%、4.70%、5.60%；在105份粪便样本中，BCoV感染阳性率33.33%；同时检测到BCoV分别与BVDV、IBRV、BEV混合感染阳性率依次为18.10%、5.71%、29.52%。通过以上调查表明牛冠状病毒在牛场中是普遍存在的，且与其他病原混合感染严重，为牛病防疫带来了重大隐患。通过扩增阳性样本中的BCoV N基因全长序列，测序分析发现，测的的N基因全长序列与GenBank中收录的BCoV N基因差异不大，为疫苗研制提供指导。
[Abstract]:Bovine Coronavirus (BCoV) belongs to the coronavirus family and the coronavirus 2A subgroup. It is an important pathogen causing newborn calf diarrhea, adult bovine and respiratory infection. Epidemiological investigation shows that the bovine coronavirus is prevalent in cattle farms, mainly through the digestive tract and respiratory tract, and the infected cattle have long been poisoned and are in the field. Continuous detoxification in a certain period is easy to cause a large scale infection of cattle. Therefore, it is particularly important to detect and monitor cattle farm environment in time. At present, there are no special drugs for the treatment of BCoV infection at home and abroad. In developed dairy countries, inactivated vaccines or attenuated vaccines are used to prevent this disease, but there is no related commercialized vaccine in China. Therefore, it is urgent to establish a rapid and sensitive detection method of the virus, and to investigate the epidemic situation of BCoV in dairy farms in some areas of China, find BCoV infected cattle early and grasp epidemiological data so as to take more effective and effective measures to prevent and control the economic losses caused by the virus to the cattle industry.
In this study, the specific primers of 1 pairs of N gene fragments were designed and synthesized according to the N gene sequence of the bovine coronavirus virus included in GenBank. The BCoV virus cDNA was used as the template for PCR amplification. The PCR product was purified and recovered and returned to the large gene sequence. The correct sequence of the N gene was connected to the pEASY-T3 vector, and the sequencing was identified by enzyme digestion and sequencing. The recombinant plasmid pEASY-T3-N. was obtained to obtain the recombinant plasmid pEASY-T3-N as the positive standard, and a series of dilution was carried out. 6 gradient dilution standards were selected as the template for the fluorescence quantitative PCR reaction, and the standard curve and regression equation were established. Then the conventional PCR and real time fluorescence quantitative PCR method were used for the 2013~2014 year experiment. 212 clinical samples of 5 large dairy cattle farms collected by the room were tested for etiology. Finally, 1 pairs of specific primers used to amplify the full length of N gene were designed, and the products of PCR amplification were sequenced and identified. The total length of the measured N gene sequence and the sequence in GenBank were analyzed by DNAstar biological software.
Sequence determination showed that the fragment of the 100bp size obtained by the clone was correct and the recombinant plasmid pEASY-T3-N positive standard was successfully obtained. The minimum detection limit of the template in a reaction system was 7.8copies/ mu L in the established real-time quantitative PCR detection method of the bovine coronavirus SYBR Green I, which was more sensitive than the ordinary RT-PCR; the fusion was more sensitive than the common RT-PCR. The curve only showed a specific single peak, but no apparent primer two or non specific amplification peak appeared, which proved that the method had good specificity. It was also with bovine rotavirus (BRV), bovine viral diarrhea virus (bovine viral diarrhea virus, BVDV), bovine infectious nasotracheitis virus (infectious bovi). NE rhinotracheitis virus, IBRV), bovine enterovirus (bovine enterovirus, BEV), bovine epidemic fever virus (bovine ephemeral fevervirus, BEFV) and bovine parainfluenza virus type 3 (bovine) have no cross reaction; in batch, the coefficient of interbatch duplication variation is lower than that, and the method is highly sensitive and specific. Good reproducibility provides technical support for clinical diagnosis and epidemiological investigation of bovine coronavirus.
PCR detection of clinical samples showed that in 107 samples of nasal swabs, the positive rate of BCoV infection was 5.61%, and the positive rate of mixed infection of BCoV and BVDV, IBRV, BPIV3 was 4.60%, 4.70%, 5.60%, and the positive rate of BCoV infection was 33.33% in 105 fecal samples, and BCoV was mixed with BVDV, IBRV, BEV, respectively. The positive rate of the infection was 18.10%, 5.71%. 29.52%. through the above investigation showed that the bovine coronavirus was common in the cattle field, and the mixed infection with other pathogens was serious, which brought a major hidden danger to the epidemic prevention of bovine disease. The full length sequence of the BCoV N gene in the positive samples and the sequence analysis found that the total length of the measured N gene and GenBank There is little difference in BCoV N gene, which provides guidance for vaccine development.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.653

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