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 Ⅰreal-time PCR method for detecting Mycobacterium bovis

【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。

[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.