本文選題：豬嵴病毒 + RT-PCR��； 參考：《揚(yáng)州大學(xué)》2015年碩士論文

【摘要】：豬嵴病毒(Porcine Kobuvirus, PKVs)是小RNA病毒科(Picornaviridae)嵴病毒屬(kobuvirus)的成員,于2008年首次發(fā)現(xiàn)。在世界多個國家的豬群中均發(fā)現(xiàn)了該病毒,越來越多的證據(jù)表明該病毒與豬腹瀉疫情有關(guān)。豬腹瀉病影響和危害我國養(yǎng)豬生產(chǎn)和養(yǎng)豬業(yè)發(fā)展,自2010年以來,我國大部分省份爆發(fā)了以水樣腹瀉、脫水、嘔吐為特征的哺乳仔豬病毒性腹瀉疫情,發(fā)病率和死亡率高,給養(yǎng)豬業(yè)帶來巨大損失,這其中病毒性腹瀉疫情引起養(yǎng)豬業(yè)的普遍關(guān)注。在腹瀉豬群中檢測到了豬嵴病毒,并且豬嵴病毒可能會導(dǎo)致仔豬腹瀉。豬嵴病毒目前尚未實(shí)現(xiàn)體外分離,對該病毒的研究大多是對其分子流行病學(xué)的調(diào)查研究。在PKVs的基因結(jié)構(gòu)中,3D基因豬嵴病毒最保守的基因,其編碼的蛋白在病毒增殖的動物機(jī)體內(nèi)和感染的細(xì)胞培養(yǎng)液中發(fā)現(xiàn)的一種非結(jié)構(gòu)性的病毒特異性蛋白,被稱為病毒感染相關(guān)蛋白。VP1基因是嵴病毒基因組中最具可變性的區(qū)域,編碼的蛋白是衣殼蛋白中暴露最多的免疫顯性蛋白,同小RNA病毒科其他病毒一樣VP1基因編碼的蛋白可引起宿主機(jī)體產(chǎn)生免疫反應(yīng),是嵴病毒的抗原位點(diǎn),是微RNA病毒分屬的重要依據(jù)。本研究建立了針對PKVs 3D基因和VP1基因的RT-PCR檢測方法,對3D基因和VP1基因進(jìn)行基因特征分析并對江蘇地區(qū)豬嵴病毒進(jìn)行全基因組測序分析,旨在通過對江蘇地區(qū)豬群中豬嵴病毒的分布流行情況進(jìn)行分子流行病學(xué)調(diào)查,了解該病毒在豬群中的感染狀況,豐富豬嵴病毒的流行病學(xué)數(shù)據(jù),為防治豬腹瀉疫情提供一定的參考。1豬嵴病毒3D基因RT-PCR檢測方法的建立根據(jù)豬嵴病毒(Porcine kobuvirus, PKVs) 3D基因保守序列設(shè)計(jì)一對特異性引物,建立了檢測豬棉拭樣本中的豬嵴病毒的RT-PCR方法,該方法能特異性的檢測豬嵴病毒,而對其它病原如豬流行性腹瀉病毒(Porcine epidemic diarrhea virus, PEDV)、豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus,TGEV)、豬A群輪狀病毒(Group A porcine rotavirus, GARV)、豬瘟病毒(Classical swine fever virus)、豬呼吸與繁殖障礙綜合征病毒(Porcine Re productive and Respiratory Syndrome virus, PRRSV)、豬圓環(huán)病毒(porcine circovirus, PCV)、豬大腸桿菌則不能檢出,用該方法能檢測的濃度為0.1263pg,靈敏度高,可用于對江蘇地區(qū)豬群中豬嵴病毒的檢測。2豬腹瀉病原混合感染檢測及豬嵴病毒3D基因和VP1基因特征分析用建立的豬嵴病毒3D基因RT-PCR檢測方法對2012年8月至2013年12月從江蘇13個地區(qū)46個豬場采集的396份豬腹瀉肛拭樣本進(jìn)行檢測。結(jié)果顯示豬場陽性率和病料陽性率分別為80.4%和45.7%,表明豬嵴病毒廣泛存在于江蘇地區(qū)豬群中。隨機(jī)選取19份病料進(jìn)行豬嵴病毒3D基因分析。19個豬嵴病毒3D基因的核苷酸和氨基酸相似性分別為88.0%～100%和69.4%～100%,而與其它嵴病毒的核苷酸和氨基酸相似性分別為69.6%～78.8%和27.8%～56.9%。進(jìn)化樹分析顯示不同地區(qū)的豬嵴病毒3D基因處于同一分支,說明江蘇地區(qū)豬嵴病毒沒有一定的地理限制。185份病料經(jīng)檢測發(fā)現(xiàn)含有2種及兩種以上其它病原,其中有68份PKVs病料檢測陽性的樣本有31份檢測到其它病原,說明在江蘇地區(qū)豬嵴病毒與其它病原發(fā)生混合感染。使用設(shè)計(jì)的針對豬嵴病毒VP1基因的特異性引物,對2014年8月至2015年2月從江蘇地區(qū)采集的91份腹瀉仔豬棉拭樣本和126份臨床健康仔豬棉拭樣本進(jìn)行檢測,腹瀉仔豬豬嵴病毒感染率64.8%(59/91)；臨床健康仔豬感染豬嵴病毒感染率為19.8%(25/126)。隨機(jī)選取28個VP1基因測序分析顯示,28個VP1基因的氨基酸同源性為80.3%～100%,核苷酸同源性為79.6%-100%。進(jìn)化樹分析顯示,28個VP1基因分為四個群,其中腹瀉豬感染的豬嵴病毒VP1基因分布在四個不同的群；所有臨床健康豬群感染的豬嵴病毒VP1基因分布在同一個群并顯示較近的基因同源性。結(jié)果表明江蘇地區(qū)腹瀉豬群中流行的豬嵴病毒表現(xiàn)一定的基因多樣性,而臨床健康豬群中則不然。這種差異可能與豬嵴病毒的潛在致病性有關(guān)。3豬嵴病毒全基因組的測序與基因遺傳進(jìn)化分析根據(jù)GenBank公布的PKVs全基因組序列,設(shè)計(jì)12對針對PKVs全基因組的特異性引物,對從江蘇地區(qū)檢測的2株P(guān)KVs進(jìn)行全基因測序分析。JS-01-CHN檢測自腹瀉仔豬,除去poly(A)基因組全長為8214nt,開放閱讀框(ORF)基因全長為7470nt,5'-UTR長度為576nt,與參考毒株比較發(fā)現(xiàn)JS-01-CHN的VP1基因編碼的氨基酸第237位插入一個氨基酸,在3'-UTR基因的108位插入一個胸腺嘧啶。JS-02a-CHN來自臨床健康仔豬,除去poly (A)基因組全長為8121nt,在其2B編碼區(qū)缺失30個氨基酸,這是首次發(fā)現(xiàn)感染健康PKVs的2B區(qū)的氨基酸缺失,這可能是PKVs進(jìn)化過程中變異的結(jié)果。序列分析發(fā)現(xiàn),兩株P(guān)KVs全基因序列與參考序列編碼區(qū)氨基酸和核苷酸同源性分別為94.3%-96.4%和87.8%-89.3%,而與其它嵴病毒則為56.2%-65.1%和59.0%-66.1%。5'-UTR和3'-UTR與其它PKVs核苷酸同源性為95.1%-97.0%和89.1%-97.0%。進(jìn)化樹分析顯示JS-02a-CHN和JS-01-CHN處于同一分支并和PKVs較近。對PKVs全基因組進(jìn)行重組分析發(fā)現(xiàn),JS-01-CHN存在潛在的重組事件,這可能是全基因組不同基因片段拼接導(dǎo)致的結(jié)果,為了解PKVs的重組特點(diǎn),需要對更多的全基因序列進(jìn)行測序分析。
[Abstract]:Porcine Kobuvirus (PKVs) is a member of the small RNA virus family (Picornaviridae), a member of the cristovirus (kobuvirus) genus (kobuvirus). It was first discovered in 2008. The virus was found in pigs in many countries of the world. More and more evidence shows that the virus is related to the epidemic of porcine diarrhea. Since 2010, most provinces of our country have outbreak of viral diarrhea of suckling piglets characterized by water sample diarrhea, dehydration and vomiting. The incidence and mortality rate are high, which bring huge losses to the pig industry. In this case, the virus diarrhea epidemic causes the swine industry to pay attention to the swine industry. Porcine crista virus may cause diarrhea in piglets. The swine crista virus has not yet been isolated in vitro. Most of the studies on the virus are investigated for its molecular epidemiology. In the gene structure of PKVs, the most conservative gene of the 3D gene porcine crista virus is encoded in the animal body and infected cell culture fluid of the virus. A non structural virus specific protein known as the virus infection related protein.VP1 gene is the most mutable region in the crista virus genome, the encoded protein is the most exposed immune dominant protein in the capsid protein, and the VP1 gene encoded by the protein of the other viruses of the small RNA family can cause the host organism to produce The immunoreaction, an antigen site of crest virus, is an important basis for the classification of RNA virus. In this study, a RT-PCR detection method for the PKVs 3D gene and VP1 gene was established. The genomic characteristics of the 3D and VP1 genes were analyzed and the whole genome sequencing of the swine crista virus in Jiangsu area was analyzed. The aim is to pass the pig cristae in the pigs in the Jiangsu region. The epidemic situation of the virus distribution is investigated by molecular epidemiology, to understand the infection status of the virus in the pigs, to enrich the epidemiological data of porcine crista virus, and to provide a reference for the prevention and control of porcine diarrhea in the epidemic situation of porcine crest virus (.1) 3D gene RT-PCR detection method based on the conservative sequence of the Porcine kobuvirus (PKVs) 3D gene. A pair of specific primers was designed to establish a RT-PCR method for detecting porcine crest virus in pig's cotton swab samples. This method can specifically detect porcine crista virus, while other pathogens such as Porcine epidemic diarrhea virus (PEDV), swine infectious gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), and A group of pigs Group A porcine rotavirus (GARV), swine fever virus (Classical swine fever virus), porcine respiratory and reproductive disorder syndrome virus (Porcine Re productive), porcine Escherichia coli, can not be detected. The method can detect the concentration of 0.126 3pg, with high sensitivity, it can be used to detect the swine crista virus in the pigs in Jiangsu area and to detect the mixed infection of.2 porcine diarrhea pathogen and the 3D gene and VP1 gene of porcine crista virus (crest virus). The 3D gene of swine crista virus (crest virus 3D) gene RT-PCR was used to collect 396 porcine anal swabs from 46 pig farms from August 2012 to December 2013. The results showed that the positive rate and positive rate of pig farm were 80.4% and 45.7% respectively, indicating that the swine crista virus was widely found in the pigs in Jiangsu area. The nucleotide and amino acid similarity of the 3D gene of the.19 crest virus 3D gene was divided into 88% to 100% and 69.4% to 100% in the random selection of the swine crista virus 3D gene. The nucleotide and amino acid similarity of crest virus was 69.6% to 78.8% and 27.8% ~ 56.9%. phylogenetic tree analysis showed that the 3D gene of swine crista virus in different regions was in the same branch. It showed that the swine crista virus in Jiangsu region had no geographical limitation and found that there were 2 species and more than two other pathogens, including 68 PKVs. 31 samples of the positive samples were detected, indicating that the swine crista virus was mixed with other pathogens in Jiangsu. Using specific primers designed for the porcine crest virus VP1 gene, 91 cotton swabs and 126 healthy piglets were collected from the Jiangsu region from August 2014 to February 2015. The infection rate of swine cristae virus was 64.8% (59/91), and the infection rate of swine crista virus infection was 19.8% (25/126) in healthy piglets. The sequence analysis of 28 VP1 genes showed that the amino acid homology of 28 VP1 genes was 80.3% to 100%. The nucleotide homology was 79.6%-100%. evolution tree analysis, 28 VP1 genes were divided into two genes. Four groups, among which the swine crista virus VP1 gene infected by diarrhea pigs was distributed in four different groups, and the VP1 gene of swine crista virus infected by all clinical healthy pigs was distributed in the same group and showed the homologous gene. The results showed that the swine cristae virus in the diarrhea pigs in Jiangsu region showed a certain genetic diversity, and the clinical health was healthy. This difference may be related to the potential pathogenicity of porcine crista virus, which is related to the sequencing of the whole genome of.3 swine crista virus and gene genetic evolution analysis based on the PKVs genome sequence published by GenBank, and designed 12 pairs of specific primers for the whole genome of PKVs, and complete the whole gene sequencing of 2 strains of PKVs detected from Jiangsu region. The total length of the poly (A) genome was 8214nt, the full length of the open reading frame (ORF) gene was 7470nt, the 5'-UTR length was 576nt, and the 237th bits of the JS-01-CHN's VP1 gene encoded amino acids were inserted into one amino acid, and a thymine.JS-02a-CHN from the 3'-UTR gene was inserted in the 108 bit of the 3'-UTR gene to insert a thymine.JS-02a-CHN from.JS-01-CHN. In clinical healthy piglets, the total length of the poly (A) genome is 8121nt, and 30 amino acids are missing in its 2B coding region. This is the first discovery of the amino acid deletion in the 2B region infected with healthy PKVs. This may be the result of the variation in the evolutionary process of PKVs. The sequence analysis found that the sequence of the two PKVs whole bases was homologous with the amino acids and nucleotides in the reference sequence coding region. The homology of 56.2%-65.1%, 59.0%-66.1%.5'-UTR and 3'-UTR with other PKVs nucleotides for 95.1%-97.0% and 89.1%-97.0%. evolution tree analysis showed that JS-02a-CHN and JS-01-CHN were in the same branch and were similar to PKVs. The recombinant analysis of the whole genome of PKVs was found to be 94.3%-96.4% and 87.8%-89.3%. There is a potential recombination event, which may be the result of the splicing of different genome segments in the whole genome. In order to solve the recombinant characteristics of PKVs, more whole gene sequences need to be sequenced.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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