家蚕微孢子虫荧光定量PCR检测方法及诊断试剂盒  

Fluorescent Quantitative PCR Detection of Nosema bombycis and the Dignostic Kit

作　　者：何永强 吴姗 鲁兴萌[2] 张弘[3] 邱海洪[2] 帅江冰 王素华 张晓峰 徐国群 李光才[2] 董强 

机构地区：[1]浙江出入境检验检疫局技术中心,杭州310012 [2]浙江大学动物科学学院,杭州310029 [3]浙江中医药大学第一附属医院,杭州310006

出　　处：《蚕业科学》2011年第2期260-265,共6页ACTA SERICOLOGICA SINICA

基　　金：国家质量监督检验检疫总局科研项目(No.2007IK017);浙江省重大科技专项(No.2010C12005-2);浙江出入境检验检疫局科技项目(No.ZK200610)

摘　　要：基于为蚕种生产与流通贸易提供快速、灵敏、准确检测家蚕微孢子虫的方法,以家蚕微孢子虫小亚单位核糖体RNA基因66 bp片段作为靶标,设计特异性引物和TaqMan探针,用构建的重组质粒制备标准品进行荧光定量PCR扩增,通过优化PCR反应体系,测定线性范围,评价方法的特异性、灵敏度和重复性,成功构建了家蚕微孢子虫的荧光定量PCR检测方法,并研制出诊断试剂盒。该方法的检测线性范围为1×108～1×102拷贝/mL的7个线性梯度,检测家蚕微孢子虫的敏感度达1×102拷贝/mL,特异性高,3次重复性检测应用试验的批内变异系数为3.6%～7.2%,批间变异系数为5.2%～7.8%。结果表明,建立的家蚕微孢子虫荧光定量PCR检测方法具有准确、灵敏、快速、特异性强的特点。In order to provide a rapid,sensitive and accurate method for detecting Nosema bombycis（Nb） in silkworm eggs production,circulation and trading,a 66 bp segment of the small subunit ribosomal RNA gene of N.bombycis was used as the target molecule for designing specific primers and TaqMan probe.The constructed recombinant plasmid was used to prepare standard agent for fluorescent quantitative PCR amplification.Through optimization of PCR reaction system,linear range of the detection method was determined,and specificity,sensitivity and repeatability of the method were evaluated.Finally,a fluorescent quantitative PCR detection method was established successfully and the corresponding diagnostic kit was developed.Linear range of this detection method was from 1×108 to 1×102 copies/mL,covering 7 linear gradients within this range.The detectable sensitivity of this method reached 1×102 copies/mL,showing high specificity in detection.Three repeated detection trials showed that the variation coefficients within batches and between batches were 3.6% ~7.2% and 5.2% ~7.8%,respectively.These results indicated that the established fluorescent quantitative PCR method for Nosema bombycis detection is accurate,sensitive,easy to operate and highly specific.

关 键 词：家蚕微孢子虫 荧光定量PCR 诊断试剂盒 

分 类 号：S884.21[农业科学—特种经济动物饲养]