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

牛分枝杆菌SYBRGreen I实时荧光定量PC删方法的建立

作  者：
谢志勤[1];谢芝勋[1];刘加波[1];庞耀珊[1];邓显文[1];谢丽基[1];彭宜[1];范晴[1];牟群[2];莫文胜[3]
单  位：
广西兽医研究所
关键词：
牛结核病;牛分枝杆菌;SYBR;Green;I实时荧光定量PCR;检测;bovine tuberculosis; Mycobactedum bovis; SYBR GREEN I real-time PCR; detection
摘  要：
[目的]建立一种检测牛分枝杆菌的SYBRGreenI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SYBRGreenIX时荧光定量PCR的检测模板范围为1.O*10^9-1.0*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℃.SYBRGreenI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/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SYBRGreenI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 I real-time PCR method for  detecting Mycobacterium boris
摘  要：
[Objective]The present experiment was conducted to develop the SYBR GREEN I real-time PCR method for detecting Mycobacterium boris (M. boris) 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. boris to amplify M. boris. 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 real- time PCR ranged from 1.0*10^9 to 1.0*10 copies/μL during Tm 86.48-86.65℃. All the M. boris strains tested positive in this condition-specific analysis; however, the other strains tested negative. The established method could detect 10 copies/ txL of M. boris 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. boris.

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