【摘要】： 背景 當(dāng)中國(guó)的SARS、馬來(lái)西亞的尼巴病毒(NiV)、美國(guó)的西尼羅河病毒(WNV)以及全球的禽流感病毒所引起的一場(chǎng)場(chǎng)席卷全球的各種病毒性疾病爆發(fā)流行時(shí),人們?cè)跇O度恐慌的同時(shí)也進(jìn)行了反思,為什么不能預(yù)測(cè)這些傳染病的爆發(fā)流行?為什么對(duì)疾病的流行缺乏有效的監(jiān)控措施和疫情預(yù)報(bào)?現(xiàn)有的防治策略和措施為什么收效甚微?這些都促使我們對(duì)新發(fā)病毒性疾病的防治工作和公共衛(wèi)生體系的建設(shè)有了新的認(rèn)識(shí)。及早的對(duì)可能成為的新發(fā)感染性疾病致病原的病毒給予關(guān)注,盡早的研究發(fā)現(xiàn)這些病毒的起源、傳播、與臨床表現(xiàn)和預(yù)后相關(guān)的宿主因素、診斷方法和治療措施,為防止這些病毒的大流行打下基礎(chǔ),已成為目前迫切需要解決的重大公共衛(wèi)生問(wèn)題。 目的 本研究旨在關(guān)注中樞神經(jīng)新發(fā)感染性病毒Borna病病毒(Borna disease virus,BDV),擬對(duì)BDV一步法實(shí)時(shí)RT-PCR檢測(cè)方法進(jìn)行一些有益的探索,并和前期開(kāi)發(fā)建立的BDV熒光定量套式逆轉(zhuǎn)錄酶聚合酶鏈反應(yīng)(FQ-nRT-PCR)檢測(cè)方法進(jìn)行比較,優(yōu)選出快速、準(zhǔn)確、敏感、特異的檢測(cè)BDV的方法,為大規(guī)模流行病學(xué)監(jiān)測(cè)提供一種簡(jiǎn)便、可靠的檢測(cè)手段。同時(shí),應(yīng)用所建立的檢測(cè)方法,對(duì)采集自中國(guó)新疆伊犁地區(qū)的和重慶三峽庫(kù)區(qū)的馬、豬外周血、腦組織樣本分別進(jìn)行BDV檢測(cè),以獲得我國(guó)西部部分地區(qū)的BDV的病毒流行病學(xué)資料,為BDV感染性疾病的可能爆發(fā)流行提供預(yù)警,同時(shí)也為某些神經(jīng)精神疾病的病因?qū)W診斷及其防治提供新的思路和依據(jù)。 方法 1根據(jù)GenBank提供的基因序列,針對(duì)BDV基因的保守區(qū)域(BDV p24基因),設(shè)計(jì)并合成特異性引物和熒光標(biāo)記TaqMan探針。從含有目的基因的質(zhì)粒出發(fā),利用PCR方法擴(kuò)增獲得目的基因,克隆至pBS-T載體進(jìn)行體外轉(zhuǎn)錄,將得到的RNA純化并使用RiboGreen方法進(jìn)行定量后作為陽(yáng)性標(biāo)準(zhǔn)品,初步建立BDV的一步法實(shí)時(shí)RT-PCR方法,對(duì)其敏感性和特異性進(jìn)行探討;同時(shí)與前期本課題組建立檢測(cè)BDV的FQ-nRT-PCR方法進(jìn)行比較分析。 2自2007年6月起,采集自中國(guó)西部部分地區(qū)動(dòng)物標(biāo)本共480份(新疆伊犁地區(qū)天然牧場(chǎng)上放養(yǎng)馬的外周血和腦組織標(biāo)本120份、重慶及三峽庫(kù)區(qū)鄉(xiāng)村飼養(yǎng)的豬外周血標(biāo)本360份),使用密度梯度離心法分離外周血淋巴細(xì)胞,應(yīng)用Trizol法提取細(xì)胞總RNA。用優(yōu)選的BDV檢測(cè)方法,對(duì)樣本進(jìn)行BDV檢測(cè)。對(duì)陽(yáng)性樣本進(jìn)行PCR產(chǎn)物序列鑒定、序列分析等研究,在可能的情況下進(jìn)行病毒分離培養(yǎng),并提交流行病學(xué)調(diào)查報(bào)告。 結(jié)果 1對(duì)所設(shè)計(jì)的BDV引物和探針序列,登錄美國(guó)國(guó)立衛(wèi)生研究院網(wǎng)站(http://www.ncbi.nlm.nih.gov/BLAST/)進(jìn)行Blast檢索,所設(shè)計(jì)的引物和探針可以與已公布的BDV病毒株序列匹配。 2本研究建立BDV一步法實(shí)時(shí)RT-PCR方法的最低檢出限即敏感性為1.7×102copies/μl,標(biāo)準(zhǔn)曲線的定量范圍分別為1.7×102～1.7×106copies/μl,相關(guān)系數(shù)R2分別為0.9998,不與其他病毒發(fā)生非特異反應(yīng)。該方法具有較好的靈敏性和特異性,在BDV流行病學(xué)研究中可以選擇使用。 3對(duì)采集自中國(guó)新疆伊犁地區(qū)的和重慶及三峽庫(kù)區(qū)的馬、豬外周血、腦組織樣本分別進(jìn)行BDV檢測(cè),新疆伊犁地區(qū)有3例馬外周血和腦組織中同時(shí)檢測(cè)到博爾納病毒,陽(yáng)性率為2.5%(3/120)。擴(kuò)增產(chǎn)物序列與其他國(guó)外馬源BDV分離株同源性大于93%,與標(biāo)準(zhǔn)株He/80同源性達(dá)到98%以上。重慶及三峽庫(kù)區(qū)的BDV p24基因片段陽(yáng)性率在360只豬的PBMC BDV-p24基因的陽(yáng)性檢出率為4.44%。序列分析結(jié)果表明,重慶及三峽庫(kù)區(qū)豬感染的BDV核苷酸序列與馬He/80同源性為100%,并且編碼的氨基酸序列也相同。 結(jié)論 1本研究構(gòu)建了BDV p24基因檢測(cè)用的RNA及DNA標(biāo)準(zhǔn)品,并初步建立了BDV的一步法實(shí)時(shí)RT-PCR檢測(cè)方法,與本課題組前期建立的FQ-nRT-PCR檢測(cè)方法相比,FQ-nRT-PCR方法,具有更好的靈敏性和特異性,并且有穩(wěn)定性和重復(fù)性好等特點(diǎn),適于流行病學(xué)研究和BDV相關(guān)性疾病的診斷。 2初步流行病學(xué)研究提示我國(guó)西部新疆伊犁地區(qū)馬群中存在BDV的自然感染。該地區(qū)BDV流行株與標(biāo)準(zhǔn)株He/80存在高度的同源性。 3初步流行病學(xué)研究提示我國(guó)重慶及三峽庫(kù)區(qū)豬中存在BDV的自然感染。該地區(qū)感染的BDVP24核苷酸序列與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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R181.3

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 張英英;中國(guó)新疆和重慶地區(qū)多物種博爾納病病毒的檢測(cè)及種系發(fā)生分析[D];重慶醫(yī)科大學(xué);2010年

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本文編號(hào)：2338113