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当前位置: 首页 > 文章 > 荷斯坦牛脊椎畸形综合征TaqMan荧光PCR检测方法的建立与应用 华南农业大学学报 2015,36 (5) 26-30
Position: Home > Articles > Establishment and application of TaqMan real-time PCR to detect the complex vertebral malformation in Holstein cattle Journal of South China Agricultural University 2015,36 (5) 26-30

荷斯坦牛脊椎畸形综合征TaqMan荧光PCR检测方法的建立与应用

作  者：
刘志玲;吴晓薇;康伟;朱道中;刘中勇;陈增荣;郭霄峰
单  位：
广东出入境检验检疫局检验检疫技术中心;广州出入境检验检疫局;华南农业大学兽医学院
关键词：
荷斯坦牛;脊椎畸形综合征;TaqMan荧光PCR
摘  要：
【目的】建立快速筛查牛脊椎畸形综合征(Complex vertebral malformation,CVM)基因携带者的方法,在牛群中逐渐淘汰CVM基因携带者以减少养牛业损失.【方法】根据Gen Bank已发表的CVM基因的SLC35A3基因序列,设计合成了2对特异性引物和2条TaqMan探针,建立了牛脊椎畸形综合征Ta Man探针检测方法.对方法的特异性和敏感性进行分析后,应用该方法对临床样本进行检测,并与测序方法进行比较.【结果和结论】该方法能有效区分突变型、野生型和杂合型个体.各种基因型的检测灵敏度为:突变型质粒拷贝数1×10~3μL~(-1),杂合型质粒拷贝数1×10~4μL~(-1),野生型质粒拷贝数5×10~3μL~(-1).从292份荷斯坦牛全血中检出6份CVM基因携带者,与测序方法结果一致.本研究建立的方法简便快捷、准确率高,适合大样本筛选和口岸现场快速筛查.
译  名：
Establishment and application of TaqMan real-time PCR to detect the complex vertebral malformation in Holstein cattle
作  者：
LIU Zhiling;WU Xiaowei;KANG Wei;ZHU Daozhong;LIU Zhongyong;CHEN Zengrong;GUO Xiaofeng;Inspection and Quarantine Technology Center,Guangdong Entry-Exit Inspection and Quarantine Bureau;Guangdong Key Laboratory of Import and Export Technical Measures of Animal,Plant and Food;Guangzhou Entry-Exit Inspection and Quarantine;College of Veterinary Medicine,South China Agricultural University;
关键词：
Holstein cattle;;complex vertebral malformation;;TaqMan real-time PCR
摘  要：
【Objective】The objective of this study was to establish a rapid assay to detect complex vertebral malformation( CVM) gene carrier so as to gradually eliminate CVM gene carriers for a lower loss in the cattle-feeding industry. 【Method】In this study,a pair of primer and two Taq Man probes were designed according to the sequence of SLC35A3 gene from GenBank. A Taq Man real-time PCR assay for CVM gene was developed. DNA samples from clinical Holstein cattles were analysed by the assay,and the results were used for comparison with sequencing method.【Result and conclusion】The assay could effectively differentiate mutant type,wild type and hybrid type of CVM gene. The sensitivity of three types was 1 × 103,5 × 10~3 and 1 × 104μL~(-1),respectively. Six out of 292 tested Holstein cattles were identified to be CVM gene carriers. The results were consistent with the sequencing method. The Taq Man realtime PCR is a rapid and reliable method for extensive screening of CVM gene at the port of entry.

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