黑龙江省新生仔猪病毒性腹泻RT-PCR检测及LAMP检测方法的建立

发布时间：2018-07-10 01:39

  本文选题：猪流行性腹泻 + RT-PCR　； 参考：《东北农业大学》2015年硕士论文

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的一种严重的病毒性传染病。该病毒属于尼多病毒目,冠状病毒科,冠状病毒属成员,可以引起新生仔猪严重的呕吐、腹泻、脱水为主要特征的高度接触性肠道传染病,各个年龄阶段的猪群都会发病,哺乳仔猪病死率高达100%。自2010年冬季以来,新生哺乳仔猪的腹泻病开始在全国各地规模化猪场暴发,本次流行的腹泻病具有发病日龄早、死亡率高的新特点。为此本研究在2013-2014年黑龙江省6个地级市的10座规模化猪场采取新生哺乳仔猪临床表现为腹泻症状的粪便与小肠进行实验室RT-PCR病原检测分析,结果表明黑龙江省地区规模化猪场新生仔猪PEDV RT-PCR检出率为56%,PEDV+TGEV RT-PCR检出率为6%,PRV RT-PCR检出率为10%,TGEV RT-PCR检出率为10%,表明引起黑龙江省规模化猪场新生仔猪腹泻的主要病原是PEDV,同时也存在与其他腹泻病毒的混合感染,由于PEDV-M基因比较稳定,是研究PEDV遗传变异的优先选择基因,因此针对阳性病料扩增出6条PEDV-M基因序列进行遗传进化分析,这6条PEDV-M基因序列之间的核苷酸同源性为98.2%~99%,与疫苗株CV777的核苷酸同源性为97.6%~98.2%。利用MEGA 6.0进行遗传进化树的构建,扩增出的6条PEDV-M基因的遗传进化树分为二个大系,大部分遗传距离较近,处于同一分支。其中LJJHD-M-13和LJJQ-M-14与泰国KU06RB08、泰国KU07RB08的亲缘关系较近,LJJC-M-14、LJJJ-M-14、LJJH-M-13、LJMM-M-14这四株毒株与韩国M1595、韩国CPF259的亲缘关系较近,同时扩增出的6条PEDV-M基因不与CV777毒株为一系,与其亲缘性较远。说明这些地区的PEDV流行毒株已经发生变异,形成独特的流行进化分支,因此新的疫苗毒株和免疫方法的研究对预防和控制该病具有重要的意义。本研究进一步建立了PEDV的RT-LAMP检测方法。根据GenBank中发表的PEDV基因序列,用DNAstar软件进行分析对比后,针对PEDV-N基因进行引物设计。利用Primer Explorer5.0软件设计引物,经过严格筛选,最终确定一组引物,包括外引物F3、B3,内引物FIP、BIP。用此引物对病毒RNA进行RT-LAMP反应,并对实验反应条件进行了优化。结果表明,RT-LAMP检测方法的最佳反应条件为:反应温度为61℃,反应时间为50min,Mg2+2.5μL,dNTPs1.5μL,外引物和内引物最佳浓度比为1:4,即外引物(F3:B3=1:1)为1μL,内引物(FIP:BIP=1:1)为4μL。经过灵敏性、特异性、稳定性试验,结果表明,RT-LAMP的敏感性是RT-PCR的100倍,特异性强,且稳定性好。用本实验建立的RT-LAMP方法对125份样品提取的RNA进行检测后,与RT-PCR方法检测结果对比,二者符合率为100%,该检测方法,具有操作简便、敏感性高、特异性强、不需要昂贵的仪器设备,非常适合基层的检疫部门对猪流行性腹泻的监测与防控,在现实中具有广阔的应用价值。
[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，

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