| 家蚕微孢子虫荧光定量PCR检测方法及诊断试剂盒 |
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| 引用本文： | 何永强,吴姗,鲁兴萌,张弘,邱海洪,帅江冰,王素华,张晓峰,徐国群,李光才,董强.家蚕微孢子虫荧光定量PCR检测方法及诊断试剂盒[J].蚕业科学,2011,37(2):260-265. |
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| 作者姓名： | 何永强 吴姗 鲁兴萌 张弘 邱海洪 帅江冰 王素华 张晓峰 徐国群 李光才 董强 |
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| 作者单位： | 浙江出入境检验检疫局技术中心,杭州,310012;浙江大学动物科学学院,杭州,310029;浙江中医药大学第一附属医院,杭州,310006 |
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| 基金项目： | 国家质量监督检验检疫总局科研项目，浙江省重大科技专项，浙江出入境检验检疫局科技项目 |
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| 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。
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| 关 键 词： | 家蚕微孢子虫 荧光定量PCR 诊断试剂盒 |
Fluorescent Quantitative PCR Detection of Nosema bombycis and the Dignostic Kit |
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| HE Yong-Qiang,WU Shan,LU Xing-Meng,ZHANG Hong,QIU Hai-Hong,SHUAI Jiang-Bing,WANG Su-Hua,ZHANG Xiao-Feng,XU Guo-Qun,LI Guang-Cai,DONG Qiang.Fluorescent Quantitative PCR Detection of Nosema bombycis and the Dignostic Kit[J].Acta Sericologica Sinica,2011,37(2):260-265. |
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| Authors: | HE Yong-Qiang WU Shan LU Xing-Meng ZHANG Hong QIU Hai-Hong SHUAI Jiang-Bing WANG Su-Hua ZHANG Xiao-Feng XU Guo-Qun LI Guang-Cai DONG Qiang |
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| Institution: | HE Yong-Qiang1 WU Shan1 LU Xing-Meng2 ZHANG Hong3 QIU Hai-Hong2 SHUAI Jiang-Bing1 WANG Su-Hua1 ZHANG Xiao-Feng1 XU Guo-Qun1 LI Guang-Cai2 DONG Qiang1(1Technical Center of Zhejing Entry-Exit Inspection and Quarantine Bureau,Hangzhou 310012,China,2College of Animal Science,Zhejiang University,Hangzhou 310029,3The First Affiliated Hospital,Zhejiang University of Traditional Chinese Medicine,Hangzhou 310006,China) |
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| Abstract: | 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 ... |
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| Keywords: | Nosema bombycis Fluorescent quantitative PCR Diagnostic kit |
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