猪细环病毒2型间接ELISA和LAMP检测方法的建立
发布时间：2019-06-07 15:44
【摘要】：输血传播病毒(Transfusion transmitted virus,TTV)又称细环病毒(Torque teno virus)。猪细环病毒即猪源TTV病毒(Torque teno sus virus,TTSu V),属于细环病毒科(Anelloviridae),壬型细环病毒属(Iotatorquevirus)和Kappatorquevirus病毒属。TTSu V由美国学者于1999年首次从猪群中发现,之后世界范围内陆续有病例的报道。目前对于TTSuV的直接致病性和复制机理尚不清楚,只了解其与某些猪病发生可能存在某种协同作用。鉴于TTSu V在猪群中广泛流行性及潜在威胁,本试验针对TTSu V2建立了其相应的ELISA和LAMP检测方法。1.TTSu V2 ORF1基因序列分析比对GenBank中TTSu V2 ORF1基因序列,设计一对特异性引物,利用PCR方法扩增获得两条TTSuV2 ORF1序列。测序结果显示两条序列均包含了整个TTSuV2ORF1区域,与TTV2Hn93株(JQ664305.1)同源性分别为97,2%和97.7%。运用MEGA6.0和DNAstar将获得的两条序列与GenBank中TTSuV2 ORF1基因序列进行遗传进化和同源性分析。从遗传进化关系来看,TTSu V2可分为四个分支,各分支间同源性在53.6~82.2%之间,我国范围内存在四个分支,表明我国可能呈现TTSu V2多亚群的流行。2.TTSu V2 ORF1截短基因原核的表达在TTSu V2 ORF1基因序列分析基础上,选择两端截短的ORF1基因作为靶序列进行PCR扩增,将扩增片段与表达载体pcoldⅠ连接,构建了重组表达质粒pcold-TTSu V2-ORF1,转入BL21(DE3)感受态细胞后经0.5mM IPTG诱导24 h后表达了大小约为34 KD的带有His标签的重组蛋白,蛋白形式主要呈可溶性。Western Blot试验显示纯化的蛋白反应原性良好,可作为下一步ELISA试验抗原基础。3.TTSu V2间接ELISA检测方法的建立以纯化的重组蛋白作为包被抗原,分别对血清稀释度及反应时间、封闭液类型及封闭时间、酶标二抗(HRP标记的兔抗猪IgG)稀释度及反应时间等梯度进行优化建立了TTSu V2间接ELISA检测方法。其中封闭条件为3%犊牛血清封闭2 h,血清稀释度为1:400,反应为45 min,酶标二抗稀释度为1:4000,反应为1 h。对阴性血清反应结果通过统计学计算得出阴阳性血清临界值为0.182。特异性试验说明该方法特异性强。批内批间重复性试验表明该方法重复性良好。运用该方法对采自江西3家规模化猪场的84份血清样品进行检测,阳性检出率分别为72%(13/18)、57.5%(19/33)、36.3%(12/33)。本试验建立的间接ELISA方法可运用于临床上猪血清中TTSuV2抗体的检测。4.TTSu V2环介导等温扩增(LAMP)检测方法的建立本试验选取TTSu V2 UTR和ORF1前端区域为靶序列,利用生物软件网站设计了两对LAMP引物。对反应参数和反应条件摸索后,建立了TTSu V2 LAMP检测方法。TTSu V2 LAMP方法最佳反应条件为64℃恒温90 min,最低病毒检出限为100 copies/?l,特异性良好,与相关猪源病毒不存在交叉反应。该LAMP检测方法具有强特异性,高灵敏度等优势,有望成为快速检测TTSu V2的主要手段之一。
[Abstract]:Transfusion transmitted virus (Transfusion transmitted virus,TTV) also known as microcyclic virus (Torque teno virus). Porcine TTV virus (Torque teno sus virus,TTSu V),) belongs to (Iotatorquevirus) and Kappatorquevirus viruses of (Anelloviridae), nontype microcircovirus family. TPTSu V was first discovered in pigs by American scholars in 1999. the TPTSu V was first discovered in pigs by American scholars in 1999. the TPTSu V was first discovered in pigs by American scholars in 1999. Since then, there have been reports of cases around the world. At present, the direct pathogenicity and replication mechanism of TTSuV are not clear, only that it may have some synergistic effect with the occurrence of some pig diseases. In view of the widespread epidemic and potential threat of TTSu V in pig populations, the corresponding ELISA and LAMP detection methods for TTSu V 2 were established in this experiment. 1. TTSu V 2 ORF1 gene sequence was compared with TTSu V 2 ORF1 gene sequence in GenBank, and a pair of specific primers were designed. Two TTSuV2 ORF1 sequences were amplified by PCR. The sequencing results showed that the two sequences contained the whole TTSuV2ORF1 region, and the homology with TTV2Hn93 strain (JQ664305.1) was 97%, 2% and 97.7%, respectively. MEGA6.0 and DNAstar were used to analyze the genetic evolution and homology of the two sequences with TTSuV2 ORF1 gene sequences in GenBank. According to the genetic evolution relationship, TTSu V 2 can be divided into four branches, the homology among them is 53.6 鈮，
本文编号：2494906
[Abstract]:Transfusion transmitted virus (Transfusion transmitted virus,TTV) also known as microcyclic virus (Torque teno virus). Porcine TTV virus (Torque teno sus virus,TTSu V),) belongs to (Iotatorquevirus) and Kappatorquevirus viruses of (Anelloviridae), nontype microcircovirus family. TPTSu V was first discovered in pigs by American scholars in 1999. the TPTSu V was first discovered in pigs by American scholars in 1999. the TPTSu V was first discovered in pigs by American scholars in 1999. Since then, there have been reports of cases around the world. At present, the direct pathogenicity and replication mechanism of TTSuV are not clear, only that it may have some synergistic effect with the occurrence of some pig diseases. In view of the widespread epidemic and potential threat of TTSu V in pig populations, the corresponding ELISA and LAMP detection methods for TTSu V 2 were established in this experiment. 1. TTSu V 2 ORF1 gene sequence was compared with TTSu V 2 ORF1 gene sequence in GenBank, and a pair of specific primers were designed. Two TTSuV2 ORF1 sequences were amplified by PCR. The sequencing results showed that the two sequences contained the whole TTSuV2ORF1 region, and the homology with TTV2Hn93 strain (JQ664305.1) was 97%, 2% and 97.7%, respectively. MEGA6.0 and DNAstar were used to analyze the genetic evolution and homology of the two sequences with TTSuV2 ORF1 gene sequences in GenBank. According to the genetic evolution relationship, TTSu V 2 can be divided into four branches, the homology among them is 53.6 鈮，
本文编号：2494906
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