BDV的核酸检测方法和西部地区BDV感染的分子流行病学研究

发布时间：2018-11-17 14:26

【摘要】： 背景当中国的SARS、马来西亚的尼巴病毒(NiV)、美国的西尼罗河病毒(WNV)以及全球的禽流感病毒所引起的一场场席卷全球的各种病毒性疾病爆发流行时,人们在极度恐慌的同时也进行了反思,为什么不能预测这些传染病的爆发流行?为什么对疾病的流行缺乏有效的监控措施和疫情预报?现有的防治策略和措施为什么收效甚微?这些都促使我们对新发病毒性疾病的防治工作和公共卫生体系的建设有了新的认识。及早的对可能成为的新发感染性疾病致病原的病毒给予关注,尽早的研究发现这些病毒的起源、传播、与临床表现和预后相关的宿主因素、诊断方法和治疗措施,为防止这些病毒的大流行打下基础,已成为目前迫切需要解决的重大公共卫生问题。目的本研究旨在关注中枢神经新发感染性病毒Borna病病毒(Borna disease virus,BDV),拟对BDV一步法实时RT-PCR检测方法进行一些有益的探索,并和前期开发建立的BDV荧光定量套式逆转录酶聚合酶链反应(FQ-nRT-PCR)检测方法进行比较,优选出快速、准确、敏感、特异的检测BDV的方法,为大规模流行病学监测提供一种简便、可靠的检测手段。同时,应用所建立的检测方法,对采集自中国新疆伊犁地区的和重庆三峡库区的马、猪外周血、脑组织样本分别进行BDV检测,以获得我国西部部分地区的BDV的病毒流行病学资料,为BDV感染性疾病的可能爆发流行提供预警,同时也为某些神经精神疾病的病因学诊断及其防治提供新的思路和依据。方法 1根据GenBank提供的基因序列,针对BDV基因的保守区域(BDV p24基因),设计并合成特异性引物和荧光标记TaqMan探针。从含有目的基因的质粒出发,利用PCR方法扩增获得目的基因,克隆至pBS-T载体进行体外转录,将得到的RNA纯化并使用RiboGreen方法进行定量后作为阳性标准品,初步建立BDV的一步法实时RT-PCR方法,对其敏感性和特异性进行探讨;同时与前期本课题组建立检测BDV的FQ-nRT-PCR方法进行比较分析。 2自2007年6月起,采集自中国西部部分地区动物标本共480份(新疆伊犁地区天然牧场上放养马的外周血和脑组织标本120份、重庆及三峡库区乡村饲养的猪外周血标本360份),使用密度梯度离心法分离外周血淋巴细胞,应用Trizol法提取细胞总RNA。用优选的BDV检测方法,对样本进行BDV检测。对阳性样本进行PCR产物序列鉴定、序列分析等研究,在可能的情况下进行病毒分离培养,并提交流行病学调查报告。结果 1对所设计的BDV引物和探针序列,登录美国国立卫生研究院网站(http://www.ncbi.nlm.nih.gov/BLAST/)进行Blast检索,所设计的引物和探针可以与已公布的BDV病毒株序列匹配。 2本研究建立BDV一步法实时RT-PCR方法的最低检出限即敏感性为1.7×102copies/μl,标准曲线的定量范围分别为1.7×102～1.7×106copies/μl,相关系数R2分别为0.9998,不与其他病毒发生非特异反应。该方法具有较好的灵敏性和特异性,在BDV流行病学研究中可以选择使用。 3对采集自中国新疆伊犁地区的和重庆及三峡库区的马、猪外周血、脑组织样本分别进行BDV检测,新疆伊犁地区有3例马外周血和脑组织中同时检测到博尔纳病毒,阳性率为2.5%(3/120)。扩增产物序列与其他国外马源BDV分离株同源性大于93%,与标准株He/80同源性达到98%以上。重庆及三峡库区的BDV p24基因片段阳性率在360只猪的PBMC BDV-p24基因的阳性检出率为4.44%。序列分析结果表明,重庆及三峡库区猪感染的BDV核苷酸序列与马He/80同源性为100%,并且编码的氨基酸序列也相同。结论 1本研究构建了BDV p24基因检测用的RNA及DNA标准品,并初步建立了BDV的一步法实时RT-PCR检测方法,与本课题组前期建立的FQ-nRT-PCR检测方法相比,FQ-nRT-PCR方法,具有更好的灵敏性和特异性,并且有稳定性和重复性好等特点,适于流行病学研究和BDV相关性疾病的诊断。 2初步流行病学研究提示我国西部新疆伊犁地区马群中存在BDV的自然感染。该地区BDV流行株与标准株He/80存在高度的同源性。 3初步流行病学研究提示我国重庆及三峡库区猪中存在BDV的自然感染。该地区感染的BDVP24核苷酸序列与He/80株具有高度同源性。
[Abstract]:Background When China's SARS, Malaysia's Nipavirus (NiV), the West Nile virus (WNV) in the United States, and the global bird flu virus have spread across the world's various viral diseases, people are in extreme panic and in the meantime It's a reflection. Why can't you predict these infectious diseases? Outbreaks? Why is a lack of effective monitoring of the prevalence of the disease? The epidemic forecast? The existing strategies and measures for prevention and control Little effect? These have led to our work on the prevention and control of new viral diseases and the construction of the public health system. New awareness. Early detection of the virus that may be the cause of the new infectious disease, as early as possible, found the origin, spread, host factors, diagnostic methods, and treatment measures related to the clinical and prognosis of these viruses, in order to prevent the large prevalence of these viruses The foundation has become the most important public key to be solved at present health The purpose of this study is to focus on the new Borna disease virus (BDV) of the central nervous system. The R-test method is used for some beneficial exploration, and compared with the detection method of the BDV fluorescence quantitative nested reverse transcriptase polymerase chain reaction (FQ-nRT-PCR), which is established in the early stage, and the method for detecting the BDV is preferably rapid, accurate, sensitive and specific, and is a large-scale epidemiological monitoring. The invention provides a simple and reliable detection method, The viral epidemiological data of the BDV in the region provide early warning for the possible outbreak of the BDV infectious disease, and also for the etiology of certain neuropsychiatric disorders break and The method 1 according to the gene sequence provided by GenBank, aiming at the conserved region (BDV p24 gene) of the BDV gene, The method comprises the following steps of: designing and synthesizing a specific primer and a fluorescence labeled TaqMan probe; starting from a plasmid containing a target gene, amplifying the obtained target gene by using a PCR method, cloning the obtained target gene into a pBS-T vector to perform in-vitro transcription, purifying the obtained RNA and using the RiboGreen method for quantitative detection as a positive marker, The sensitivity and specificity of the one-step real-time RT-PCR method of BDV were discussed in this paper. the FQ-nT-PCR method for the detection of BDV was compared and analyzed. From June 2007, 480 (peripheral blood and brain tissue markers of the horse in the natural pasture of Xinjiang Ili) were collected from a total of 480 animal specimens from the western part of China. 360 copies of peripheral blood samples from 120, Chongqing and the Three Gorges Reservoir, and the density gradient was used. Separation of peripheral blood lymphocytes by heart method and extraction with Trizol method Total RNA of the cells. The samples were tested for BDV using the preferred BDV test method. The positive samples were identified by PCR product sequence identification, sequence analysis, and the like. research, In the case of possible virus isolation and culture, and to submit an epidemiological investigation report. As a result, 1 pair of the designed BDV primers and probe sequences were registered in the National Institutes of Health website (

http:// www. ncbi. nlm. nih. gov/ blast/ ), and the designed primers and probes can be matched with the published BDV strains. The minimum detection limit of the one-step real-time RT-PCR method for BDV is 1. 7-102copies/. mu. l, and the quantitative range of the standard curve is 1. 7-102-1, respectively. 7-106copies/. mu.l, and the correlation coefficient R2 is 0.9998, and is not related to it the method has good sensitivity and specificity, and can be selected and used in the BDV epidemiological study. The results showed that the positive rate was 2.5% (3/ 120) in the peripheral blood and the brain tissue of 3 horses in the Yili area of Xinjiang, and the positive rate was 2.5% (3/ 120). and the homology of the amplified product sequence to other foreign horse-source BDV isolates is more than 93 percent, and the homology of the amplified product sequence to the standard strain He/ 80 is more than 98 percent. The positive rate of BDV p24 gene was 4.44% in the PBDV-p24 gene of 360 pigs. The results of the sequence analysis showed that the positive rate of BDV p24 gene was 4.44%., weight Conclusion 1 This study constructed the RNA and DNA standard for detection of BDV p24 gene, and set up a one-step real-time RT-P of BDV. Compared with the FQ-nRT-PCR detection method established in the earlier stage of the research group, the method for detecting the CR-nRT-PCR method with better sensitivity and specificity and good stability and repeatability and the like, and is suitable for the epidemiological study and the diagnosis of the BDV-related diseases. 2. The preliminary epidemiological study indicates that there is BDV in the horse population in the western Xinjiang Yili area. Natural infection. The BDV epidemic in the region has a high degree of homology with the standard strain He/ 80.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R181.3

【引证文献】