猪丹毒杆菌和溶血性曼氏杆菌二重TaqMan荧光定量PCR检测方法的建立

发布时间：2018-03-27 15:19

本文选题：猪丹毒杆菌　切入点：溶血性曼氏杆菌　出处：《中国兽医科学》2017年08期

【摘要】：为建立一种快速、准确检测猪丹毒杆菌和溶血性曼氏杆菌二重TaqMan荧光定量PCR方法,本研究根据猪丹毒杆菌和溶血性曼氏杆菌16S rRNA基因保守序列设计种特异性引物及探针,并优化反应条件,建立了检测猪丹毒杆菌和溶血性曼氏杆菌的二重TaqMan荧光定量PCR检测方法。结果表明,所建方法特异性良好,与其他25种常见细菌均无交叉反应。猪丹毒杆菌检测在2.07×108~2.07×103copies/L范围内具有良好的线性关系,最低可以检测到2.07×102copies/L的标准品阳性质粒,批内和批间变异系数均小于3%;溶血性曼氏杆菌检测在1.07×108~1.07×103copies/L范围内具有良好的线性关系,最低可以检测到1.07×102copies/L的标准品阳性质粒,批内和批间变异系数均小于3%。应用建立的二重荧光定量方法和普通荧光定量方法对47份临床样品进行检测,符合率为100%。本研究结果表明,所建立的方法可用于猪丹毒杆菌和溶血性曼氏杆菌感染的高通量快速诊断以及猪丹毒杆菌和溶血性曼氏杆菌的快速鉴定及定量分析。
[Abstract]:In order to establish a rapid and accurate method for the detection of dual TaqMan quantitative PCR of erysipelas porcine and M. haemolyticus, specific primers and probes were designed according to the conserved sequence of 16s rRNA gene of erysipelas and M.haemolyticus. A double TaqMan fluorescence quantitative PCR method for the detection of erysipelas and M.haemolyticus was established under optimized reaction conditions. The results showed that the method was of good specificity. There was no cross reaction with other 25 common bacteria. The detection of erysipelas had a good linear relationship in the range of 2.07 脳 10 ~ (8) ~ 2.07 脳 103copies/L, and a positive plasmid of 2.07 脳 102copies/L could be detected at least. The coefficient of variation within and between batches was less than 3, and the detection of M.haemolyticus had a good linear relationship in the range of 1.07 脳 10 ~ (8) ~ 1.07 脳 103copies/L, and the lowest positive plasmid of 1.07 脳 102copies/L could be detected in the range of 1.07 脳 10 ~ (8) ~ 1.07 脳 103copies/L. The coefficient of variation within and between batches was less than 3. The coincidence rate of 47 clinical samples was 100 by using the established double fluorescence quantitative method and the general fluorescence quantitative method. The established method can be used for high throughput rapid diagnosis of erysipelas and M.haemolyticus infection, and for rapid identification and quantitative analysis of erysipelas and M.haemolyticus.
【作者单位】：云南省畜牧兽医科学院;云南省陆良县动物疾病预防控制中心;
【基金】：云南省现代农业生猪产业技术体系建设项目(云财教[2013]160号) 云南省现代农业奶牛产业技术体系建设(云财农[2009]171号)
【分类号】：S852.61

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