南方农业学报2012，Vol.43Issue(10)：1584-1589,6.DOI:10.3969/j.issn.2095-1191.2012.10.1584
牛分枝杆菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立
Development of SYBR GREEN Ⅰ real-time PCR method for detecting Mycobacterium bovis
摘要
Abstract
The present experiment was conducted to develop the SYBR GREEN I real-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 ex-tration, 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 I realtime PCR ranged from l.0×109 to 1.0x10 copies/μL during Tm 86.48-86.65℃. All the M. hovis 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.关键词
牛结核病/牛分枝杆菌/SYBR GreenⅠ实时荧光定量PCR/检测Key words
bovine tuberculosis/ Mycobacterium bovis/ SYBR GREEN I real-time PCR/ detection分类
农业科技引用本文复制引用
谢志勤,莫文胜,谢芝勋,刘加波,庞耀珊,邓显文,谢丽基,彭宜,范晴,牟群..牛分枝杆菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立[J].南方农业学报,2012,43(10):1584-1589,6.基金项目
新世纪百千万人才工程国家级人选专项基金项目(945200603) （945200603）
公益性行业(农业)科研专项项目(201103008) （农业）
广西特聘专家专项经费资助项目(2011B020) （2011B020）
广西科技攻关项目(桂科转0626001-5-1) （桂科转0626001-5-1）
广西水产畜牧局科研计划项目(桂渔牧科08283-20,桂渔牧科09254-18) （桂渔牧科08283-20,桂渔牧科09254-18）
