本文選題：豬δ冠狀病毒(PDCoV) + RT-PCR��； 參考：《江西農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬δ冠狀病毒(Porcine Deltacoronavirus,PDCoV)是近年來新發(fā)現(xiàn)的可引起豬只,特別是新生仔豬腹瀉的冠狀病毒,其引發(fā)的腹瀉具有較高的發(fā)病率和死亡率,是造成新生仔豬死亡的重要原因之一。豬Delta冠狀病毒,屬于冠狀病毒科(Coronaviridae)冠狀病毒屬(Coronavirus)。PDCoV在2012年報道后,陸續(xù)在美國、中國等出現(xiàn)。目前對于PDCoV的致病和復(fù)制機(jī)理尚不清楚,僅了解其引起仔豬發(fā)病特征與豬流行性腹瀉病毒相似。鑒于PDCoV在豬群中的廣泛流行性及潛在威脅,本試驗(yàn)建立了針對PDCoV核酸的RT-PCR(Reverse Transcriptional Polymerase Chain Reaction)和反轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增(Reverse Transcriptional Loop-Mediated Isothermal Amplification,RT-LAMP)的檢測方法及針對PDCoV抗體的間接ELISA(Enzyme Linked Immunosorbent Assay)的檢測方法。1.PDCoV RT-PCR方法的建立及其流行病學(xué)調(diào)查比對GenBank中PDCoV全基因序列,根據(jù)其保守區(qū)域設(shè)計(jì)一對特異性擴(kuò)增N基因片段的引物,利用RT-PCR方法擴(kuò)增獲得329 bp的片段,與預(yù)期目的片段大小一致,擴(kuò)增產(chǎn)物經(jīng)克隆后測序,將獲得的序列與GenBank數(shù)據(jù)庫序列進(jìn)行比對,比對結(jié)果表明試驗(yàn)所獲得的序列與數(shù)據(jù)庫中PDCoV序列的同源性高達(dá)99.1%,證明所擴(kuò)增的序列屬于PDCoV;對建立的RT-PCR方法的特異性、敏感性、穩(wěn)定性鑒定,結(jié)果表明該方法的特異性好、敏感性高、穩(wěn)定性強(qiáng);應(yīng)用建立的RT-PCR方法檢測了656份2012—2017年采集自江西省各地區(qū)的腹瀉母豬糞便及腹瀉仔豬糞便/腸道樣本,結(jié)果顯示腹瀉樣本中PDCoV的檢出率為30.79%(202/656),母豬糞便中PDCoV的檢出率(27.78%)稍低于仔豬糞便及腸道樣品PDCoV的檢出率(30.97%),最早可從2012年的樣品中檢測出PDCoV。2.PDCoV RT-LAMP檢測方法的建立根據(jù)比對結(jié)果,選取PDCoV N基因全長為目標(biāo)序列,設(shè)計(jì)2套RT-LAMP引物。以PDCoV陽性病料提取的總RNA為模板,建立了PDCoV RT-LAMP的檢測方法。對反應(yīng)條件優(yōu)化后,結(jié)果表明該反應(yīng)的最佳條件是63℃恒溫水浴70min,且該方法靈敏度高,特異性好。應(yīng)用RT-LAMP、RT-PCR和套式RT-PCR對192份臨床樣品進(jìn)行檢測,結(jié)果表明RT-LAMP的靈敏度為RT-PCR的100倍,與套式RT-PCR相當(dāng)。3.PDCoV N基因的原核表達(dá)以PDCoV陽性病料提取的總RNA為模板,擴(kuò)增PDCoV N基因的全基因序列,擴(kuò)增片段經(jīng)雙酶切后與表達(dá)載體pColdⅠ連接,構(gòu)建重組表達(dá)質(zhì)粒pCold-PDCoV-N,將重組質(zhì)粒轉(zhuǎn)入BL21(DE3)感受態(tài)細(xì)胞,OD600nm值為04-0.6時,冰浴30min后加入終濃度為0.8mM IPTG,15℃誘導(dǎo)表達(dá)21 h,破碎菌體得到大小約為41kDa的重組蛋白,且蛋白主要以可溶性的形式存在。使用PDCoV陽性血清作為一抗進(jìn)行Western Blot試驗(yàn),結(jié)果表明其免疫原性良好。4.PDCoV間接ELISA檢測方法的建立以純化的PDCoV N重組蛋白為包被抗原,采用棋盤滴定確定最佳抗原包被濃度和抗體,對酶標(biāo)二抗稀釋倍數(shù)、不同封閉液、封閉時間、一抗作用時間、二抗作用時間和顯色時間進(jìn)行優(yōu)化建立PDCoV間接ELISA檢測方法,結(jié)果表明抗原包被量為5μg、3%犢牛血清封閉2 h,檢測血清稀釋100倍,反應(yīng)45 min,酶標(biāo)二抗稀釋4000倍,反應(yīng)為1h為最優(yōu)條件。建立的ELISA方法陰陽性臨界值為0.316,與豬流行性腹瀉病毒等6種豬常見病原陽性血清均無交叉反應(yīng),具有良好的特異性。批內(nèi)和批間重復(fù)性試驗(yàn)的變異系數(shù)均小于10%,重復(fù)性和穩(wěn)定性均較好。應(yīng)用建立的間接ELISA方法,對收集自江西各地區(qū)282份豬血清進(jìn)行檢測,陽性率為12.8%(36/282),表明PDCoV在我省豬群中普遍存在。本實(shí)驗(yàn)建立的PDCoV抗體捕獲間接ELISA為臨床上豬群PDCoV感染監(jiān)測和流行病學(xué)調(diào)查提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Swine delta coronavirus (Porcine Deltacoronavirus, PDCoV) is a new coronavirus, which has been discovered in recent years, which can cause piglets, especially newborn piglet diarrhea. The diarrhea caused by the pig is one of the most important reasons for the death of newborn piglets. The porcine Delta coronavirus belongs to the coronavirus (Coronaviridae) coronavirus. After reported in 2012, virus genus (Coronavirus).PDCoV appeared in the United States, China and so on. The pathogenesis and replication mechanism of PDCoV are still unclear. It is only known that the pathogenic characteristics of the piglets are similar to those of the porcine epidemic diarrhea virus. In view of the widespread prevalence and potential threat of PDCoV in the pigs, this experiment established the PDCoV nucleic acid. The detection method of RT-PCR (Reverse Transcriptional Polymerase Chain Reaction) and reverse recording loop mediated isothermal amplification (Reverse Transcriptional Loop-Mediated Isothermal Amplification, RT-LAMP) and the establishment of the method for detecting indirect antibodies against the antibodies. The epidemiological survey was compared to the PDCoV whole gene sequence in GenBank, and the primers of a pair of specific amplified N gene fragments were designed according to the conservative region. The fragment of 329 BP was amplified by the RT-PCR method. The sequence of the amplified product was compared with the sequence of the GenBank database and the sequence of the amplified product was compared with the sequence of the GenBank database. The results showed that the sequence obtained by the experiment was 99.1% homologous to the PDCoV sequence in the database, which proved that the amplified sequence was PDCoV, and the specificity, sensitivity and stability of the RT-PCR method established. The results showed that the method had good specificity, high sensitivity and strong stability, and 656 2012 - 20 samples were detected by the application of the established RT-PCR method. 17 years from the diarrhoea sow feces and diarrhoea piglet feces / intestinal samples collected from various regions of Jiangxi Province, the results showed that the detection rate of PDCoV in diarrhea samples was 30.79% (202/656), and the detection rate of PDCoV in sow feces (27.78%) was slightly lower than that of piglet feces and intestinal samples (30.97%), and the earliest can be detected from the samples in 2012. The 2.PDCoV RT-LAMP detection method was established according to the comparison results. The total length of the PDCoV N gene was selected as the target sequence and 2 sets of RT-LAMP primers were designed. The detection method of PDCoV RT-LAMP was established with the total RNA of the PDCoV positive disease material as the template. The optimum conditions for the reaction showed that the best condition of the reaction was at 63 C at constant temperature water bath 70min, and the formula was used. The sensitivity of the method was high and the specificity was good. 192 clinical samples were detected with RT-LAMP, RT-PCR and nested RT-PCR. The results showed that the sensitivity of RT-LAMP was 100 times of RT-PCR. The prokaryotic expression of the.3.PDCoV N gene with the set of RT-PCR was the template for the total RNA of the PDCoV positive disease material, and the whole gene sequence of the PDCoV N gene was amplified and the amplified fragment was amplified. After double enzyme digestion, the recombinant plasmid pCold-PDCoV-N was constructed with the expression vector pCold I, and the recombinant plasmid was transferred into the BL21 (DE3) receptive cell. When the OD600nm value was 04-0.6, the final concentration was 0.8mM IPTG after the ice bath 30min, and the 21 h was expressed at 15 degrees C, and the broken strain obtained the recombinant protein about the 41kDa size, and the protein was mainly in the form of soluble form. PDCoV positive serum was used as an anti Western Blot test. The results showed that the positive immunogenicity of.4.PDCoV indirect ELISA detection method was established with the purified PDCoV N recombinant protein as the envelope antigen, the best antigen concentration and antibody were determined by the chessboard titration, the dilution multiple of enzyme standard two, the closed liquid, and the closure were closed. Time, anti action time, two anti action time and color time were optimized to establish PDCoV indirect ELISA detection method. The results showed that the antigen envelope was 5 mu g, 3% calf serum closed 2 h, serum dilution 100 times, reaction 45 min, enzyme standard two diluted 4000 times, and the reaction was the best condition. The critical value of ELISA method was 0.. The critical value of yin and Yang was 0.. 316, there was no cross reaction in the positive sera of 6 pigs, such as porcine epidemic diarrhea virus, which had good specificity. The coefficient of variation in both batch and interbatch repeatability tests was less than 10%, and the repeatability and stability were better. The indirect ELISA method was used to detect 282 pig serum collected from various regions of Jiangxi. The positive rate was 1. 2.8% (36/282) indicates that PDCoV is common in the pig population of our province. The indirect ELISA captured by PDCoV antibody in this experiment provides an experimental basis for the monitoring and epidemiological investigation of swine PDCoV infection in clinical pigs.
【學(xué)位授予單位】：江西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 劉玲玲;曹貝貝;張利衛(wèi);韓麗;韋學(xué)雷;蘭培英;胡慧;王亞賓;;新發(fā)PDCoV和TGEV雙重RT-PCR檢測方法的建立及初步應(yīng)用[J];農(nóng)業(yè)生物技術(shù)學(xué)報;2016年12期

2 張帆帆;宋德平;郭楠楠;葉昱;周信榮;李安琪;張敏;彭棋;陳燕君;黃冬艷;唐玉新;;以原核表達(dá)的豬δ冠狀病毒N蛋白為包被抗原的間接ELISA方法的建立[J];中國預(yù)防獸醫(yī)學(xué)報;2016年10期

3 逄鳳嬌;張柏猛;何孔旺;俞正玉;徐向偉;郭容利;范寶超;溫立斌;李彬;姜平;;豬δ冠狀病毒M基因RT-PCR檢測方法的建立及應(yīng)用[J];畜牧與獸醫(yī);2016年09期

4 任玉鵬;張斌;湯承;曹恭貌;岳華;;同時檢測PEDV和TGEV及PDCoV的多重RT-PCR方法的建立及初步應(yīng)用[J];中國獸醫(yī)科學(xué);2016年06期

5 張帆帆;宋德平;周信榮;黃冬艷;李安琪;彭棋;陳燕君;吳瓊;何后軍;唐玉新;;新現(xiàn)豬Delta冠狀病毒RT-PCR檢測方法的建立及其應(yīng)用[J];中國農(nóng)業(yè)科學(xué);2016年07期

6 陳建飛;王瀟博;焦賀勛;時洪艷;張鑫;劉建波;石達(dá);馮力;;國內(nèi)首株豬德爾塔冠狀病毒(Porcine deltacoronavirus)的分離鑒定[J];中國預(yù)防獸醫(yī)學(xué)報;2016年03期

7 Yu Chen;Deyin Guo;;Molecular mechanisms of coronavirus RNA capping and methylation[J];Virologica Sinica;2016年01期

，

本文編號：2011292