【摘要】：【目的】豬Delta冠狀病毒(Porcine Deltacoronavirus,PDCoV)是近年來(lái)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。試驗(yàn)擬建立新現(xiàn)PDCoV RT-PCR檢測(cè)方法,并調(diào)查當(dāng)前江西腹瀉豬群中PDCoV的感染情況�！痉椒ā客ㄟ^(guò)對(duì)Gen Bank數(shù)據(jù)庫(kù)中PDCoV全基因組序列的比對(duì)分析,找出保守序列,用Primer 3.0在線(xiàn)軟件設(shè)計(jì)1對(duì)擴(kuò)增PDCoV核衣殼(N)蛋白基因片段的特異性引物,基于該引物建立PDCoV RT-PCR檢測(cè)方法;用建立的方法檢測(cè)腹瀉豬糞便及腸道樣品,挑選陽(yáng)性擴(kuò)增產(chǎn)物進(jìn)行克隆、測(cè)序;用本試驗(yàn)獲得的PDCoV序列與其他國(guó)家或地區(qū)的PDCoV序列以及豬流行性腹瀉病毒(PEDV)、豬傳染性胃腸炎病毒(TGEV)共同構(gòu)建進(jìn)化樹(shù),分析PDCoV各毒株及其與PEDV和TGEV之間的進(jìn)化關(guān)系;以PEDV、TGEV、豬庫(kù)布病毒(PKoV)、豬星狀病毒(PAstV)、豬繁殖與呼吸綜合征病毒(PRRSV)及豬瘟病毒(CSFV)RNA為模板驗(yàn)證引物的特異性;構(gòu)建PDCoV核衣殼蛋白基因片段重組質(zhì)粒,并以系列稀釋的重組質(zhì)粒為模板確定所建立的PDCoV RT-PCR方法的敏感性;應(yīng)用建立的RT-PCR方法調(diào)查249份2012—2015年江西省豬群腹瀉樣品中PDCoV的存在情況,隨機(jī)抽取一定數(shù)量的RT-PCR陽(yáng)性擴(kuò)增產(chǎn)物進(jìn)行克隆、測(cè)序進(jìn)一步驗(yàn)證反應(yīng)的特異性�！窘Y(jié)果】1以腹瀉豬糞便樣品RNA為模板,應(yīng)用所設(shè)計(jì)的特異性引物擴(kuò)增出了329 bp的單一條帶,與預(yù)期目的片段大小一致,擴(kuò)增產(chǎn)物經(jīng)克隆后測(cè)序,將獲得的序列與NCBI Gen Bank數(shù)據(jù)庫(kù)序列進(jìn)行比對(duì),比對(duì)結(jié)果表明試驗(yàn)所獲得的序列與數(shù)據(jù)庫(kù)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;2將8條本試驗(yàn)獲得的PDCoV N基因片段序列與18株其他國(guó)家或地區(qū)的PDCoV毒株以及PEDV、TGEV的相應(yīng)N基因片段序列構(gòu)建進(jìn)化樹(shù),結(jié)果表明26條PDCo V序列屬于同一個(gè)分支,而PEDV和TGEV則分別屬于不同的分支。試驗(yàn)獲得的PDCoV序列與韓國(guó)PDCoV毒株KNU14-04親緣關(guān)系最近,同源性高達(dá)99.1%;與兩株香港毒株HKU 15-44和HKU 15-155親緣關(guān)系相對(duì)較遠(yuǎn);3本試驗(yàn)建立的RT-PCR方法特異性強(qiáng),僅能擴(kuò)增出PDCoV,而對(duì)PEDV、TGEV、PKoV、PAstV、PRRSV及CSFV核酸無(wú)交叉擴(kuò)增現(xiàn)象;4所建立的RT-PCR方法靈敏度高,將含擴(kuò)增片段的重組質(zhì)粒以1.0×106拷貝/μL為起始濃度依次10倍稀釋至1.0×101拷貝/μL作為模板驗(yàn)證方法的靈敏度,結(jié)果表明所建立的方法對(duì)PDCoV最低可檢出量為1.0×103拷貝/μL;5應(yīng)用建立的RT-PCR方法檢測(cè)了249份2012—2015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31.33%(78/249),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(31.92%),最早可從2012年的樣品中檢測(cè)出PDCoV�！窘Y(jié)論】試驗(yàn)成功建立了檢測(cè)新現(xiàn)PDCoV的RT-PCR方法;應(yīng)用所建立的RT-PCR方法檢測(cè)了249份2012—2015年江西地區(qū)豬群臨床腹瀉樣品中PDCoV存在情況,結(jié)果表明PDCoV是一種普遍存在于腹瀉豬群中的病毒;研究建立的RT-PCR方法對(duì)豬群PDCoV的臨床診斷和流行病學(xué)調(diào)查等具有應(yīng)用價(jià)值。
[Abstract]:[Objective] the swine Delta coronavirus (Porcine Deltacoronavirus, PDCoV) is a newly discovered coronavirus which can cause pigs, especially the newborn piglet diarrhea. The diarrhea caused by the swine has a high incidence and mortality, which is one of the important causes of the death of newborn piglets. The experiment is to establish a new method of detection of PDCoV RT-PCR. And investigate the infection of PDCoV in the current Jiangxi diarrhea pigs. [Methods] through the comparison and analysis of the whole genome sequence of PDCoV in the Gen Bank database, the conservative sequence was found, and the specific Primer gene fragment of the amplified PDCoV Nucleocapsid (N) protein gene fragment was designed by Primer 3 online software, and the PDCoV RT-PCR detection method was established based on the primer. The established method was used to detect the feces and intestinal samples of diarrhea pigs and select positive amplification products for cloning and sequencing. The PDCoV sequence obtained in this test and the PDCoV sequence of other countries or regions, and porcine epidemic diarrhea virus (PEDV) and porcine infectious gastroenteritis virus (TGEV) were used together to construct the evolutionary tree, and the PDCoV strains and their PEDV and TG were analyzed. The evolutionary relationship between EV; PEDV, TGEV, PKoV, PAstV, porcine reproductive and respiratory syndrome virus (PRRSV) and swine fever virus (CSFV) RNA as a template to verify the specificity of the primers; construct the recombinant plasmid of the PDCoV nucleocapsid protein gene fragment and determine the PDCoV RT by a series of diluted recombinant plasmids. The sensitivity of the -PCR method was used to investigate the existence of PDCoV in 249 samples of swine diarrhea in Jiangxi province from 2012 to 2015, and a certain number of RT-PCR positive amplification products were randomly selected to clone them, and the specificity of the reaction was further verified. [results] 1, the RNA of fecal samples from diarrhea pigs was used as a template, and the application was designed. Specific primers amplified a single band of 329 BP, consistent with the expected target fragment size. The amplified products were cloned and sequenced. The sequences obtained were compared with the NCBI Gen Bank database sequence. The comparison results showed that the sequence obtained by the test was 99.1% homology to the PDCoV sequence in the database, proving that the amplified sequence was P. DCoV; 2 the sequence of PDCoV N gene fragments obtained from 8 experiments and the PDCoV strains of 18 other countries or regions and the sequence of corresponding N gene fragments of PEDV and TGEV were constructed. The results showed that 26 PDCo V sequences belonged to the same branch, while PEDV and TGEV were in different branches. The phylogenetic relationship of strain KNU14-04 is up to 99.1% recently; the relationship with two strains of Hongkong strain HKU 15-44 and HKU 15-155 is relatively distant; 3 the RT-PCR method established in this experiment is specific and can only amplify PDCoV, while PEDV, TGEV, PKoV, PAstV, PRRSV and CSFV nucleic acids are not intersected, and 4 established RT-PCR methods are highly sensitive and will be expanded. The amplified recombinant plasmid was diluted to 1 x 106 copies / L as the initial concentration to 1 x 101 copies / mu L as a template verification method. The results showed that the minimum detectable amount of the proposed method was 1 x 103 copies / mu L, and 5 used RT-PCR method was used to detect 249 copies of Jiangxi province from Jiangxi province from 2012 to 2015. The results showed that the detection rate of PDCoV in diarrhoea samples was 31.33% (78/249), and the detection rate of PDCoV in sow feces (27.78%) was slightly lower than that of piglet feces and intestinal samples (31.92%), and the earliest possible test of PDCoV. [Conclusion] from samples in 2012 was successfully established. The RT-PCR method of new PDCoV was detected, and the RT-PCR method was used to detect the presence of PDCoV in 249 samples of clinical diarrhea samples from Jiangxi region from 2012 to 2015. The results showed that PDCoV was a virus commonly found in the diarrhea pigs, and the established RT-PCR method was used for the clinical diagnosis and epidemiological investigation of pig group PDCoV. It is of applied value.
【作者單位】： 江西農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金面上項(xiàng)目(31372457) 江西省科技廳落地計(jì)劃項(xiàng)目(KJLD13029)
【分類(lèi)號(hào)】：S852.651

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