牛结节性皮肤病病毒TaqMan-MGB荧光PCR检测方法的建立

发布时间：2018-07-28 20:55

【摘要】：为建立快速、特异鉴别检测牛结节性皮肤病病毒(LSDV)的方法,本研究参照GenBank中发表的LSDV全基因组序列,设计并合成一对特异性引物和探针,经优化条件,建立了可鉴别LSDV的TaqMan-MGB荧光定量PCR检测方法。结果显示,该方法可特异性检测LSDV,而与山羊痘病毒、绵羊痘病毒、牛痘病毒和猪痘病毒均无交叉反应;标准曲线在各浓度范围内呈现良好的线性关系,该方法建立的标准曲线方程为y=-3.141x+32.10,线性相关系数为0.999,扩增效率达到108%,最低检出下限为1.48 copies/L;组内、组间重复性试验的变异系数均小于2%。采用此方法对52份模拟临床样品和112份临床样本的检测结果显示:32份模拟阳性样品检出LSDV核酸阳性,20份模拟阴性样品未检出LSDV;而用OIE推荐的普通PCR方法仅检出28份模拟阳性样品的LSDV核酸阳性,24份模拟样品的LSDV核酸阴性(其中20份为模拟阴性样品,4份为模拟阳性样品);两种方法均未从112份临床样品中检出LSDV核酸阳性。上述结果表明,荧光PCR的敏感性明显优于普通PCR,能够快速准确地检测LSDV,适用于临床样品的检测,为外来疫病防控提供了技术支撑。
[Abstract]:In order to establish a rapid and specific method for the identification and detection of bovine nodular dermatosis virus (LSDV), a pair of specific primers and probes were designed and synthesized according to the LSDV genome sequence published in GenBank. A fluorescence quantitative PCR method for the identification of LSDV by TaqMan-MGB was established. The results showed that the method could specifically detect LSDV, but had no cross reaction with goat pox virus, sheep pox virus, cowpox virus and porcine pox virus, and the standard curve showed a good linear relationship in different concentration range. The standard curve equation established by this method is yap-3.141x 32.10, the linear correlation coefficient is 0.999 9, the amplification efficiency reaches 108, the lowest detection limit is 1.48 copies-L, and the coefficient of variation of the repeatability test between groups is less than 2%. By using this method, 52 simulated clinical samples and 112 clinical samples were tested. The results showed that LSDV nucleic acid positive samples were detected in 32 LSDV positive samples and 20 analogue negative samples were not detected, but only 28 cases were detected by the ordinary PCR method recommended by OIE. LSDV nucleic acid was negative in 24 LSDV nucleic acid positive samples (of which 20 were simulated negative samples, 4 were simulated positive samples), and LSDV nucleic acid was not detected in 112 clinical samples by both methods. The results show that the sensitivity of fluorescent PCR is obviously better than that of conventional PCR, and it can be used to detect the clinical samples quickly and accurately, which provides technical support for the prevention and control of foreign epidemic diseases.
【作者单位】：重庆出入境检验检疫局检验检疫技术中心/国家牛病重点实验室/重庆市进境陆生动物疫病防控研究工程中心;重庆大学生物工程学院;西南大学荣昌校区;中国检验检疫科学研究院;
【基金】：国家质检公益性项目(201310093) 重庆市应用开发项目(CSTC2015yykf C8001) 国家十三五科研项目(2016YFD0500907-X)
【分类号】：S852.653

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