遼寧省仔豬病毒性腹瀉病的病原調(diào)查及PEDV的遺傳變異分析
發(fā)布時(shí)間：2018-11-05 17:16
【摘要】：引起仔豬傳染性腹瀉病的病原主要有豬流行性腹瀉病毒(PEDV)、豬傳染性胃腸炎病毒(TGEV)和豬輪狀病毒(RV),這三種病原的傳染途徑和發(fā)病過程相類似,具有發(fā)病急、傳播速度快,哺乳仔豬感染率和死亡率都較高的特點(diǎn),臨床診斷和控制難度較大。近年來遼寧省哺乳仔豬傳染性病毒性腹瀉病發(fā)生較為嚴(yán)重,給養(yǎng)豬業(yè)造成了巨大的經(jīng)濟(jì)損失。為查明遼寧省仔豬病毒性腹瀉病主要病原的種類,明確主要流行病原的分布情況,從而制定有針對(duì)性的防控措施,開展了遼寧省仔豬病毒性腹瀉的病原調(diào)查及PEDV的遺傳變異分析。1.仔豬病毒性腹瀉的病原調(diào)查采用RT-PCR方法對(duì)2013年4月份至次年3月份遼寧省9個(gè)市采集的335份腹瀉病料進(jìn)行PEDV、TGEV、RV的檢測(cè),根據(jù)采集病料的時(shí)間和地點(diǎn)進(jìn)行統(tǒng)計(jì)分析。結(jié)果顯示陽性的病毒主要為PEDV和TGEV,其陽性率分別為38.2%和15.2%,二者在9個(gè)市均有分布。說明遼寧省仔豬病毒性腹瀉的主要病原是PEDV,在遼寧省感染分布廣泛,污染程度較高,并且全年持續(xù)存在。2.豬流行性腹瀉LN-J毒株的分離鑒定臨床調(diào)查發(fā)現(xiàn)已注射PEDV疫苗的豬場(chǎng)仍有本病發(fā)生,為查明遼寧地區(qū)PEDV流行毒株的來源及變異情況。采集遼寧某地區(qū)疑似PED腹瀉仔豬的腸內(nèi)容物,應(yīng)用Vero細(xì)胞進(jìn)行傳代培養(yǎng),分離到一株毒株,TCID50為10-4.30/0.1ml,通過電鏡觀察該病毒為130nm左右的球形顆粒;通過RT-PCR鑒定,該病毒為RNA病毒,而且核酸片段大小與所設(shè)計(jì)的PEDV引物相同,經(jīng)過核酸序列分析證明為PEDV,暫命名為PEDV LN-J毒株。3.豬流行性腹瀉LN-J毒株S基因的遺傳變異分析由于PEDV S基因編碼的表面纖突糖蛋白具有抗原表位,S基因的變異可能引起該病毒抗原性和毒力的變化,因此,選擇S基因進(jìn)行遺傳變異分析。設(shè)計(jì)了2對(duì)擴(kuò)增S全基因的引物S1和S2,進(jìn)行RT-PCR擴(kuò)增,成功獲得了和目的片段大小相符的條帶,并構(gòu)建了pUM-S1和pUM-S2重組質(zhì)粒,測(cè)序后通過生物軟件進(jìn)行分析、拼接,得到4161bp的全基因,利用DNAStar分析發(fā)現(xiàn),PEDV LN-J毒株的S基因和其他許多地方毒株均有較高的同源性,構(gòu)建系統(tǒng)進(jìn)化樹,發(fā)現(xiàn)該毒株與HB-2011-1、BJ-2001-2和CHGD-01親緣關(guān)系較近。說明遼寧地區(qū)流行的PEDV屬于輸入性傳播,但存在變異趨勢(shì)。
[Abstract]:The main pathogens causing infectious diarrhea in piglets are porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine rotavirus (RV),). The infection rate and mortality of suckling piglets are high, and the clinical diagnosis and control are difficult. In recent years, the infectious viral diarrhea of suckling piglets in Liaoning Province is more serious, which has caused huge economic losses to the pig industry. In order to find out the main pathogens of piglets viral diarrhea in Liaoning Province, and to determine the distribution of the main pathogens of the disease, and to formulate targeted prevention and control measures, The pathogen of viral diarrhea of piglets and the analysis of genetic variation of PEDV in Liaoning province were carried out. 1. RT-PCR method was used to detect 335 diarrhea samples collected from 9 cities in Liaoning Province from April 2013 to March of next year. The results were analyzed statistically according to the time and place of collection. The results showed that the positive rates of PEDV and TGEV, were 38.2% and 15.2% respectively. The results showed that the main pathogen of viral diarrhea of piglets in Liaoning Province was PEDV, which was widely distributed in Liaoning Province, and had a high degree of pollution. Isolation and Identification of Porcine epidemic diarrhea LN-J strain Clinical investigation showed that the disease still occurred in swine farm which had been injected with PEDV vaccine. In order to find out the source and variation of PEDV epidemic strain in Liaoning area. The intestinal contents of suspected PED diarrhea piglets in a certain area of Liaoning province were collected and subcultured with Vero cells. A virus strain with TCID50 of 10-4.30% 0.1 ml was isolated. The virus was observed to be a spherical particle about 130nm by electron microscope. The virus was identified as RNA virus by RT-PCR, and the nucleic acid fragment size was the same as that of the designed PEDV primer. The nucleic acid sequence analysis showed that the virus was tentatively named PEDV, as PEDV LN-J strain. Genetic variation Analysis of S Gene of Porcine epidemic diarrhea virus strain S the variation of S gene may cause the change of antigenicity and virulence of PEDV S gene because the surface fibrin encoded by PEDV S gene has antigen epitopes. S gene was selected for genetic variation analysis. Two pairs of primers S1 and S2 were designed to amplify the S gene, and the RT-PCR amplification was carried out, and the bands corresponding to the size of the target fragment were successfully obtained. The recombinant plasmids of pUM-S1 and pUM-S2 were constructed, and the recombinant plasmids were sequenced and analyzed by biological software. The whole gene of 4161bp was obtained. By DNAStar analysis, the S gene of PEDV LN-J strain was found to have high homology with many other local strains. A phylogenetic tree was constructed, and it was found that the S gene of PEDV LN-J strain was similar to that of HB-2011-1, strain. The relationship between BJ-2001-2 and CHGD-01 was close. The results showed that the prevalence of PEDV in Liaoning province belonged to imported transmission, but there was a trend of variation.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28
本文編號(hào)：2312751
[Abstract]:The main pathogens causing infectious diarrhea in piglets are porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine rotavirus (RV),). The infection rate and mortality of suckling piglets are high, and the clinical diagnosis and control are difficult. In recent years, the infectious viral diarrhea of suckling piglets in Liaoning Province is more serious, which has caused huge economic losses to the pig industry. In order to find out the main pathogens of piglets viral diarrhea in Liaoning Province, and to determine the distribution of the main pathogens of the disease, and to formulate targeted prevention and control measures, The pathogen of viral diarrhea of piglets and the analysis of genetic variation of PEDV in Liaoning province were carried out. 1. RT-PCR method was used to detect 335 diarrhea samples collected from 9 cities in Liaoning Province from April 2013 to March of next year. The results were analyzed statistically according to the time and place of collection. The results showed that the positive rates of PEDV and TGEV, were 38.2% and 15.2% respectively. The results showed that the main pathogen of viral diarrhea of piglets in Liaoning Province was PEDV, which was widely distributed in Liaoning Province, and had a high degree of pollution. Isolation and Identification of Porcine epidemic diarrhea LN-J strain Clinical investigation showed that the disease still occurred in swine farm which had been injected with PEDV vaccine. In order to find out the source and variation of PEDV epidemic strain in Liaoning area. The intestinal contents of suspected PED diarrhea piglets in a certain area of Liaoning province were collected and subcultured with Vero cells. A virus strain with TCID50 of 10-4.30% 0.1 ml was isolated. The virus was observed to be a spherical particle about 130nm by electron microscope. The virus was identified as RNA virus by RT-PCR, and the nucleic acid fragment size was the same as that of the designed PEDV primer. The nucleic acid sequence analysis showed that the virus was tentatively named PEDV, as PEDV LN-J strain. Genetic variation Analysis of S Gene of Porcine epidemic diarrhea virus strain S the variation of S gene may cause the change of antigenicity and virulence of PEDV S gene because the surface fibrin encoded by PEDV S gene has antigen epitopes. S gene was selected for genetic variation analysis. Two pairs of primers S1 and S2 were designed to amplify the S gene, and the RT-PCR amplification was carried out, and the bands corresponding to the size of the target fragment were successfully obtained. The recombinant plasmids of pUM-S1 and pUM-S2 were constructed, and the recombinant plasmids were sequenced and analyzed by biological software. The whole gene of 4161bp was obtained. By DNAStar analysis, the S gene of PEDV LN-J strain was found to have high homology with many other local strains. A phylogenetic tree was constructed, and it was found that the S gene of PEDV LN-J strain was similar to that of HB-2011-1, strain. The relationship between BJ-2001-2 and CHGD-01 was close. The results showed that the prevalence of PEDV in Liaoning province belonged to imported transmission, but there was a trend of variation.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28
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相關(guān)期刊論文 前1條
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相關(guān)碩士學(xué)位論文 前1條
1 陳慧娟;豬流行性腹瀉病毒河南株生物學(xué)特性研究及FQ-PCR檢測(cè)方法的建立[D];河南農(nóng)業(yè)大學(xué);2014年
，本文編號(hào)：2312751
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