水貂阿留申病毒TaqMan-MGB荧光定量PCR检测方法的建立与应用

发布时间：2018-01-11 23:15

本文关键词：水貂阿留申病毒TaqMan-MGB荧光定量PCR检测方法的建立与应用　出处：《中国预防兽医学报》2016年11期 　论文类型：期刊论文

【摘要】：为建立一种快速、敏感检测水貂阿留申病毒(AMDV)的方法,本研究根据AMDV的VP2基因保守区设计特异性引物和MGB探针,并优化检测反应条件,建立了AMDV的TaqMan荧光定量PCR检测方法。结果显示,以重组质粒为标准品建立的标准曲线在1.0×10~2拷贝/μL~1.0×10~8拷贝/μL内具有良好的线性关系,相关系数(R~2)为0.998。该方法仅对AMDV的靶基因扩增呈阳性,而对猪细小病毒、犬细小病毒、水貂肠炎细小病毒等相关病毒检测结果均为阴性,特异性良好。该方法对AMDV检测的灵敏度为10拷贝/μL,为普通PCR灵敏度的100倍;组内和组间重复性试验表明该方法的组内和组间变异系数均小于2%,具有良好的重复性。应用建立方法和普通PCR方法分别对40份疑似临床组织病料样品进行检测,该方法检出率比普通PCR高约7.5%。本研究结果表明建立的AMDV TaqMan-MGB荧光定量PCR方法灵敏、快速、特异性强、重复性好,适用于对AMDV的快速定量检测。
[Abstract]:In order to establish a rapid and sensitive method for detection of Aleutian virus (AMDV) in mink, specific primers and MGB probes were designed according to the conserved region of VP2 gene of AMDV. The detection reaction conditions were optimized and the TaqMan quantitative PCR detection method for AMDV was established. The standard curve established by using recombinant plasmid as standard material has a good linear relationship in the range of 1.0 脳 10 ~ (2) copy / 渭 L ~ (-1) ~ (-1) 脳 10 ~ (8) copy / 渭 L. The correlation coefficient was 0.998.The method was only positive for target gene amplification of AMDV, but positive for porcine parvovirus and canine parvovirus. The detection results of mink enteritis parvovirus and other related viruses were negative and specific. The sensitivity of this method to AMDV detection was 10 copies / 渭 L, which was 100 times of that of ordinary PCR. The intra-group and inter-group reproducibility tests showed that the coefficient of variation within and between groups of the method was less than 2%. With good reproducibility, 40 samples of suspected clinical tissue samples were detected by the established method and the general PCR method. The detection rate of this method is about 7.5% higher than that of ordinary PCR. The results of this study show that the established AMDV TaqMan-MGB fluorescent quantitative PCR method is sensitive, rapid, specific and reproducible. It is suitable for rapid quantitative detection of AMDV.
【作者单位】：吉林农业大学;辽宁出入境检验检疫局;辽宁省水产技术推广总站;四川出入境检验检疫局;
【基金】：国家质检总局科研项目(2013IK036) 十二五“国家科技支撑计划项目”(2013BAD12B01)
【分类号】：S852.65
【正文快照】： 水貂阿留申病(Aleutian mink disease,AMD)是由水貂阿留申病病毒(AMDV)引起的一种主要侵害水貂免疫细胞,导致自身免疫系统紊乱,同时并发自身免疫的慢性进行性传染病。该病以渐进性消瘦、高免疫蛋白血症、神经症状、肾脏肿大等为主要特征[1]。1946年,美国学者Hartsough首次观察

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