本文選題：牛冠狀病毒 + 實(shí)時(shí)熒光定量PCR　； 參考：《山東師范大學(xué)》2015年碩士論文

【摘要】：牛冠狀病毒（Bovine Coronavirus，BCoV）屬于冠狀病毒科，冠狀病毒屬2a亞群，，是引起新生犢牛腹瀉、成年牛冬痢和呼吸道感染的重要病原。流行病學(xué)調(diào)查表明，牛冠狀病毒在牛場中普遍存在，主要通過消化道和呼吸道傳播，感染的牛長期帶毒并在一定時(shí)期內(nèi)持續(xù)排毒，易造成牛群的大范圍感染。因此，對(duì)帶毒牛的及時(shí)檢出和牛場環(huán)境監(jiān)測就顯得尤為重要。目前國內(nèi)外尚無針對(duì)BCoV感染治療的特效藥物，奶業(yè)發(fā)達(dá)國家應(yīng)用滅活疫苗或減毒疫苗預(yù)防本病，而我國尚無相關(guān)商品化疫苗。因此，亟需建立該病毒快速敏感的檢測方法，并對(duì)我國部分地區(qū)奶牛場的BCoV流行情況進(jìn)行調(diào)查，及早發(fā)現(xiàn)BCoV感染的牛，掌握流行病學(xué)數(shù)據(jù)，以便及時(shí)采取更有效的針對(duì)性措施進(jìn)行防控，以減少該病毒對(duì)養(yǎng)牛業(yè)造成的經(jīng)濟(jì)損失。 本研究根據(jù)GenBank收錄的牛冠狀病毒N基因序列，設(shè)計(jì)合成了1對(duì)擴(kuò)增N基因部分片段的特異性引物，以BCoV病毒的cDNA為模板，進(jìn)行PCR擴(kuò)增，PCR產(chǎn)物純化回收后送華大基因測序，將測序正確后的N基因片段連接到pEASY-T3載體上，經(jīng)酶切鑒定、測序驗(yàn)證獲得了重組質(zhì)粒pEASY-T3-N。以得到的重組質(zhì)粒pEASY-T3-N為陽性標(biāo)準(zhǔn)品，進(jìn)行一系列稀釋，選取6個(gè)梯度稀釋的標(biāo)準(zhǔn)品作為模板進(jìn)行熒光定量PCR反應(yīng)，建立標(biāo)準(zhǔn)曲線和回歸方程。然后運(yùn)用常規(guī)PCR和實(shí)時(shí)熒光定量PCR方法，對(duì)2013～2014年本實(shí)驗(yàn)室采集的5個(gè)規(guī)模化奶牛場的212份臨床樣本進(jìn)行病原學(xué)檢測。最后設(shè)計(jì)合成了1對(duì)用于擴(kuò)增N基因全長的特異性引物，將PCR擴(kuò)增后產(chǎn)物送測序鑒定，應(yīng)用DNAstar生物學(xué)軟件將測得的N基因全長序列與GenBank中的序列進(jìn)行系統(tǒng)發(fā)生分析。 序列測定表明，我們克隆獲得的100bp大小的片段正確，并且成功獲得了重組質(zhì)粒pEASY-T3-N陽性標(biāo)準(zhǔn)品。在建立的牛冠狀病毒SYBR Green Ⅰ實(shí)時(shí)熒光定量PCR檢測方法中，一個(gè)反應(yīng)體系中模板最低檢測限為7.8copies/μL，比普通RT-PCR更加靈敏；熔解曲線只出現(xiàn)特異性的單個(gè)峰，而沒有明顯的引物二聚體或者非特異性擴(kuò)增的峰出現(xiàn)，證明該方法具有良好的特異性。同時(shí)與牛輪狀病毒（bovine rotavirus，BRV)、牛病毒性腹瀉病毒(bovine viral diarrhea virus，BVDV)、牛傳染性鼻氣管炎病毒（infectious bovine rhinotracheitis virus，IBRV）、牛腸道病毒（bovine enterovirus，BEV）、牛流行熱病毒（bovine ephemeral fevervirus，BEFV）和牛副流感病毒3型（bovine parainfluenza virus type3，BPIV-3）均無交叉反應(yīng)；批內(nèi)、批間重復(fù)變異系數(shù)均低于1.5%。因此，該方法敏感性高，特異性強(qiáng)，重復(fù)性好，為牛冠狀病毒的臨床診斷和流行病學(xué)調(diào)查提供了技術(shù)保障。 通過對(duì)臨床樣本進(jìn)行PCR檢測發(fā)現(xiàn)：在107份鼻拭子樣本中，BCoV感染陽性率為5.61%，同時(shí)檢測到BCoV分別與BVDV、IBRV、BPIV3混合感染陽性率依次為4.60%、4.70%、5.60%；在105份糞便樣本中，BCoV感染陽性率33.33%；同時(shí)檢測到BCoV分別與BVDV、IBRV、BEV混合感染陽性率依次為18.10%、5.71%、29.52%。通過以上調(diào)查表明牛冠狀病毒在牛場中是普遍存在的，且與其他病原混合感染嚴(yán)重，為牛病防疫帶來了重大隱患。通過擴(kuò)增陽性樣本中的BCoV N基因全長序列，測序分析發(fā)現(xiàn)，測的的N基因全長序列與GenBank中收錄的BCoV N基因差異不大，為疫苗研制提供指導(dǎo)。
[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.

【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.653

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張禮德;王葳;;牛冠狀病毒的培養(yǎng)增殖及含毒滴度檢測新進(jìn)展[J];國外獸醫(yī)學(xué).畜禽疾病;1992年01期

2 傅彩霞;靳興軍;鄭瑞峰;郭峰;韓磊;李佳;吳惠明;王玉田;張躍;杜鵑;;北京地區(qū)規(guī)�；膛鋈N病毒性腹瀉病的血清學(xué)調(diào)查[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2012年05期

3 韓廷義;汪生貴;;牛冠狀病毒RT-LAMP檢測方法的建立[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2013年05期

4 姚火春,杜念興,徐為燕,陸承平;牛冠狀病毒的血清流行病學(xué)調(diào)查[J];南京農(nóng)業(yè)大學(xué)學(xué)報(bào);1990年02期

5 徐文忠,陸承平,杜念興,徐為燕,陳溥言;牛冠狀病毒單克隆抗體的研究[J];南京農(nóng)業(yè)大學(xué)學(xué)報(bào);1992年01期

6 陸承平;牛冠狀病毒的檢出及分布[J];動(dòng)物檢疫;1986年03期

7 柳強(qiáng);侯喜林;車車;劉雙翼;劉合義;;牛冠狀病毒和輪狀病毒的雙重RT-PCR檢測方法的建立及應(yīng)用[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2009年09期

8 楊盛華，常國權(quán)，鄒嘯環(huán)，王新平，張仁誠，劉暢;從猝死癥黃牛分離出冠狀病毒[J];中國獸醫(yī)學(xué)報(bào);1995年02期

9 劉合義;孫留霞;王進(jìn)軼;劉雙翼;王紅;余麗蕓;樸范澤;侯喜林;;牛冠狀病毒RT-PCR檢測方法的建立[J];中國獸醫(yī)雜志;2009年02期

10 陳煥春，金梅林，何國慶，蘇雄，陳毓川;單克隆抗體ELISA檢測犢牛糞便中冠狀病毒抗原[J];中國獸醫(yī)雜志;1995年01期

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