驽巴贝斯虫TaqMan-MGB荧光定量PCR检测方法的建立
发布时间：2019-03-08 10:20
【摘要】：为建立快速检测驽巴贝斯虫Bc-48基因的方法,本研究根据Bc-48保守基因序列设计合成特异性引物和MGB荧光探针,以构建的重组质粒作为阳性标准品,经条件优化后建立了检测Bc-48基因的TaqMan-MGB荧光定量PCR方法。结果显示,该方法能够特异性检测驽巴贝斯虫,而对其它病原检测均为阴性;该方法最小检出量为5.17×10~1拷贝/μL的标准质粒,敏感性是常规PCR的100倍;组内与组间重复性试验的变异系数均小于1%,重复性好;采用所建立的方法和常规PCR分别对90份临床样品进行检测,结果显示本研究建立的驽巴贝斯虫TaqMan-MGB荧光定量PCR检测方法特异性强、敏感性高、重复性好,可以用于驽巴贝斯虫病临床样品诊断、流行病学调查。本研究为蜱传马梨形虫病的防控提供新的方法。
[Abstract]:In order to establish a rapid method for detecting the Bc-48 gene of Babesia caballi, specific primers and MGB fluorescence probes were designed and synthesized according to the conserved gene sequence of Bc-48. The recombinant plasmid was used as the positive standard. A TaqMan-MGB fluorescence quantitative PCR method for the detection of Bc-48 gene was established after optimization of the conditions. The results showed that the method could detect Babesia caballi specifically, but negative for other pathogens, and the sensitivity of the standard plasmid with a minimum detection quantity of 5.17 脳 10 渭 1 copy / 渭 L was 100 times higher than that of conventional PCR. The coefficient of variation of intra-group and inter-group repeatability test was less than 1%, and the repeatability was good. The established method and routine PCR were used to detect 90 clinical samples respectively. The results showed that the method established in this study was highly specific, sensitive and reproducible for the detection of Babesia caballi TaqMan-MGB. It can be used for the diagnosis and epidemiological investigation of Babes caballi disease. This study provides a new method for the prevention and control of tick-borne pear disease.
【作者单位】： 新疆农业大学动物医学学院;伊犁出入境检验检疫局综合技术服务中心;
【基金】：国家自然基金NSFC-新疆联合基金重点项目(U1403283)
【分类号】：S852.7
本文编号：2436707
[Abstract]:In order to establish a rapid method for detecting the Bc-48 gene of Babesia caballi, specific primers and MGB fluorescence probes were designed and synthesized according to the conserved gene sequence of Bc-48. The recombinant plasmid was used as the positive standard. A TaqMan-MGB fluorescence quantitative PCR method for the detection of Bc-48 gene was established after optimization of the conditions. The results showed that the method could detect Babesia caballi specifically, but negative for other pathogens, and the sensitivity of the standard plasmid with a minimum detection quantity of 5.17 脳 10 渭 1 copy / 渭 L was 100 times higher than that of conventional PCR. The coefficient of variation of intra-group and inter-group repeatability test was less than 1%, and the repeatability was good. The established method and routine PCR were used to detect 90 clinical samples respectively. The results showed that the method established in this study was highly specific, sensitive and reproducible for the detection of Babesia caballi TaqMan-MGB. It can be used for the diagnosis and epidemiological investigation of Babes caballi disease. This study provides a new method for the prevention and control of tick-borne pear disease.
【作者单位】： 新疆农业大学动物医学学院;伊犁出入境检验检疫局综合技术服务中心;
【基金】：国家自然基金NSFC-新疆联合基金重点项目(U1403283)
【分类号】：S852.7
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