本文選題：豬流行性腹瀉 + RT-PCR��； 參考：《東北農(nóng)業(yè)大學》2015年碩士論文

【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)引起的一種嚴重的病毒性傳染病。該病毒屬于尼多病毒目,冠狀病毒科,冠狀病毒屬成員,可以引起新生仔豬嚴重的嘔吐、腹瀉、脫水為主要特征的高度接觸性腸道傳染病,各個年齡階段的豬群都會發(fā)病,哺乳仔豬病死率高達100%。自2010年冬季以來,新生哺乳仔豬的腹瀉病開始在全國各地規(guī)�；i場暴發(fā),本次流行的腹瀉病具有發(fā)病日齡早、死亡率高的新特點。為此本研究在2013-2014年黑龍江省6個地級市的10座規(guī)模化豬場采取新生哺乳仔豬臨床表現(xiàn)為腹瀉癥狀的糞便與小腸進行實驗室RT-PCR病原檢測分析,結(jié)果表明黑龍江省地區(qū)規(guī)�；i場新生仔豬PEDV RT-PCR檢出率為56%,PEDV+TGEV RT-PCR檢出率為6%,PRV RT-PCR檢出率為10%,TGEV RT-PCR檢出率為10%,表明引起黑龍江省規(guī)模化豬場新生仔豬腹瀉的主要病原是PEDV,同時也存在與其他腹瀉病毒的混合感染,由于PEDV-M基因比較穩(wěn)定,是研究PEDV遺傳變異的優(yōu)先選擇基因,因此針對陽性病料擴增出6條PEDV-M基因序列進行遺傳進化分析,這6條PEDV-M基因序列之間的核苷酸同源性為98.2%~99%,與疫苗株CV777的核苷酸同源性為97.6%~98.2%。利用MEGA 6.0進行遺傳進化樹的構(gòu)建,擴增出的6條PEDV-M基因的遺傳進化樹分為二個大系,大部分遺傳距離較近,處于同一分支。其中LJJHD-M-13和LJJQ-M-14與泰國KU06RB08、泰國KU07RB08的親緣關(guān)系較近,LJJC-M-14、LJJJ-M-14、LJJH-M-13、LJMM-M-14這四株毒株與韓國M1595、韓國CPF259的親緣關(guān)系較近,同時擴增出的6條PEDV-M基因不與CV777毒株為一系,與其親緣性較遠。說明這些地區(qū)的PEDV流行毒株已經(jīng)發(fā)生變異,形成獨特的流行進化分支,因此新的疫苗毒株和免疫方法的研究對預防和控制該病具有重要的意義。本研究進一步建立了PEDV的RT-LAMP檢測方法。根據(jù)GenBank中發(fā)表的PEDV基因序列,用DNAstar軟件進行分析對比后,針對PEDV-N基因進行引物設(shè)計。利用Primer Explorer5.0軟件設(shè)計引物,經(jīng)過嚴格篩選,最終確定一組引物,包括外引物F3、B3,內(nèi)引物FIP、BIP。用此引物對病毒RNA進行RT-LAMP反應,并對實驗反應條件進行了優(yōu)化。結(jié)果表明,RT-LAMP檢測方法的最佳反應條件為:反應溫度為61℃,反應時間為50min,Mg2+2.5μL,dNTPs1.5μL,外引物和內(nèi)引物最佳濃度比為1:4,即外引物(F3:B3=1:1)為1μL,內(nèi)引物(FIP:BIP=1:1)為4μL。經(jīng)過靈敏性、特異性、穩(wěn)定性試驗,結(jié)果表明,RT-LAMP的敏感性是RT-PCR的100倍,特異性強,且穩(wěn)定性好。用本實驗建立的RT-LAMP方法對125份樣品提取的RNA進行檢測后,與RT-PCR方法檢測結(jié)果對比,二者符合率為100%,該檢測方法,具有操作簡便、敏感性高、特異性強、不需要昂貴的儀器設(shè)備,非常適合基層的檢疫部門對豬流行性腹瀉的監(jiān)測與防控,在現(xiàn)實中具有廣闊的應用價值。
[Abstract]:Porcine epidemic diarrhea (PED) is a serious viral infectious disease caused by porcine epidemic diarrhea virus (PEDV). The virus is a member of the family Nidoviridae, the family Coronavirus, and can cause severe vomiting, diarrhea, dehydration in newborn piglets, a highly contagious intestinal infection characterized by disease in pigs of all ages. The mortality of suckling piglets is as high as 100. Since the winter of 2010, the diarrhea disease of newborn suckling piglets began to break out in large scale pig farms all over the country. This epidemic diarrhea disease has a new characteristic of early onset and high mortality. In this study, the feces and small intestine of newborn breast-feeding piglets with clinical symptoms of diarrhea were used in 10 large-scale pig farms in 6 prefectural cities of Heilongjiang Province from 2013 to 2014 to detect the pathogeny of the disease by RT-PCR. The results showed that the detection rate of PEDV RT-PCR was 56 and the detection rate of PEDV TGEV RT-PCR was 6. The detection rate of PRV RT-PCR was 10. The detection rate of TGEV RT-PCR was 10. It indicated that the main pathogen causing diarrhea of newborn piglets in Heilongjiang Province was PEDVV, which also has mixed infections with other diarrhoeal viruses, Because PEDV-M gene is relatively stable, it is the preferred gene to study the genetic variation of PEDV, so six PEDV-M gene sequences were amplified by genetic evolution analysis. The nucleotide homology of these six PEDV-M genes was 98.299 and 97.6- 98.2 with CV777. MEGA6.0 was used to construct the genetic evolution tree. The six PEDV-M gene genetic evolution trees were divided into two lines, most of which were close to each other and were in the same branch. Among them, LJJHD-M-13 and LJJQ-M-14 are closely related to KU06RB08 and KU07RB08 of Thailand, and LJJC-M-14 LJJJ-14 LJJH-M-13 and LJMM-14 of LJJHD-M-13 and LJJQ-M-14 to Korean M1595 and CPF259 respectively. It is concluded that PEDV strains in these areas have mutated and formed a unique branch of epidemic evolution. Therefore, the study of new vaccine strains and immune methods is of great significance for the prevention and control of the disease. In this study, a new method for the detection of PEDV by RT-LAMP was established. According to the PEDV gene sequence published in GenBank, the primer design for PEDV-N gene was carried out by DNAstar software. Primer explorer5.0 software was used to design primers. After strict screening, a group of primers, including external primer F3B3 and internal primer FIPBIPs, were determined. This primer was used to react with RT-LAMP on viral RNA, and the reaction conditions were optimized. The results showed that the optimum reaction conditions were as follows: reaction temperature 61 鈩，

本文編號：2111520