中国农业科学院农业信息研究所 中国农学会
Agricultural Information Institute of CAAS China Association of Agricultural Science Societies
En
Ch
中国农业科学院农业信息研究所 中国农学会 主办

AI检索

订阅

返回顶部
当前位置: 首页 > 文章 > 牛分枝杆菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立 南方农业学报 2012,43 (10) 1584-1589
Position: Home > Articles > Development of SYBR GREEN Ⅰreal-time PCR method for detecting Mycobacterium bovis Journal of Southern Agriculture 2012,43 (10) 1584-1589

牛分枝杆菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立

作  者：
谢志勤;谢芝勋;刘加波;庞耀珊;邓显文;谢丽基;彭宜;范晴;牟群;莫文胜
单  位：
桂林市动物疫病预防诊断中心;广西兽医研究所/广西畜禽疫苗新技术重点实验室;永福县动物疫病预防诊断中心
关键词：
牛结核病;牛分枝杆菌;SYBR GreenⅠ实时荧光定量PCR;检测
摘  要：
【目的】建立一种检测牛分枝杆菌的SYBR GreenⅠ实时荧光定量PCR,为防控牛结核病提供技术支持。【方法】根据牛分枝杆菌特异性基因序列(No.MBU87961,gi9954088)设计合成一对扩增牛分枝杆菌的特异性引物,从引物设计、各成分配比、样品采集及样品DNA提取等方面进行优化,并对建立的方法进行标准曲线、溶解曲线分析及特异性、敏感性、重复性、临床样品检验试验。【结果】牛分枝杆菌SYBR Green Ⅰ实时荧光定量PCR的检测模板范围为1.0×109～1.0×10拷贝/μL时,其标准曲线呈良好的线性关系,溶解曲线表现为单一波峰,Tm值为86.48～86.65℃。SYBR GreenⅠ实时荧光定量PCR的牛型分枝杆菌扩增结果为阳性,其他参试菌株均为阴性;可检测出10拷贝/μL的牛分枝杆菌DNA,敏感性是常规PCR的100倍;Ct值变异系数小于5%,重复性好;对50份PPD检测为阳性的牛鼻黏液、牛奶和淋巴结进行检测,其结果与常规PCR检测结果一致。【结论】建立的SYBR Green Ⅰ实时荧光定量PCR具有快速、便捷、准确等优点,适合于牛分枝杆菌的鉴别诊断。
译  名：
Development of SYBR GREEN Ⅰreal-time PCR method for detecting Mycobacterium bovis
作  者：
XIE Zhi-qin1, XIE Zhi-xun1, LIU Jia-bo1, PANG Yao-shan1, DENG Xian-wen1, XIE Li-ji1, PENG Yi1, FAN Qing 1, MOU Qun2, MO Wen-sheng 3 (1 Guangxi Veterinary Research Institute / Guangxi Key Laboratory of Animal Vaccine and New Technology, Nanning 530001,China; 2 Guilin Animal Disease Prevention and Diagnosis Center, Guilin, Guangxi 541001, China; 3 Yongfu Animal Disease Prevention and Diagnosis Center, Yongfu, Guangxi 541800, China)
关键词：
bovine tuberculosis; Mycobacterium bovis; SYBR GREEN Ⅰ real-time PCR; detection
摘  要：
[Objective]The present experiment was conducted to develop the SYBR GREEN Ⅰreal-time PCR method for detecting Mycobacterium bovis (M. bovis) in order to provide scientific references for its prevention and control. [Method]A pair of specific primers was designed and synthesized from species-specific sequence(No. MBU87961,gi9954088) of M. bovis to amplify M. bovis. The designed primer, each component ratio, sample collection, and DNA extration, were optimized in the experiment. The standard curve and melting curve of the established method were analyzed and its specifity, sensitivity, repeatability, and clinial sampes detecion were carried out. [Result]The results indicated that the melting curve presented good linear relation and single crest when the detection template of SYBR GREEN Ⅰreal-time PCR ranged from 1.0×109 to 1.0×10 copies/μL during Tm 86.48-86.65℃. All the M. bovis strains tested positive in this condition-specific analysis; however, the other strains tested negative. The established method could detect 10 copies/μL of M. bovis DNA, and its sensitivity was 100 times compared to routine PCR. The coefficient of variation of Ct value for this method was less than 5%, which showed better repeatability. The 50 clinical samples, including nasal swaps, milk, and lymph node preserved in the freezer, were tested by this real-time PCR, and the results showed no differences from the results obtained by routine PCR method. [Conclusion]The established SYBR GREEN Ⅰ real-time PCR method was fast, simple, accurate, and can be advantageously used for the detection of M. bovis.

相似文章

导出引用
计量
文章访问数: 10
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊

友情链接Friendly link
农业信息研究所Agricultural information institute 农业智能知识服务平台Agricultural intelligent knowledge service platform 农业学术服务平台Agricultural Knowledge Service System 中国知网CNKI 维普网VIP 万方WANFANG DATA
联系我们Contact us

电话Tell：010-82109652-808

邮箱E-mail：agrijournal@caas.cn

服务Service
关于我们About us 意见建议Opinions and suggest
版权说明Copyright
单位：中国农业科学院农业信息研究所 中国农学会 © 2017中国农业科学院农业信息研究所 ICP备案号：京ICP备09089781号-46