伯氏疏螺旋体TaqMan-MGB探针荧光定量PCR方法的建立及对新疆地区蜱的检测

Establishment of a TaqMan-MGB Probe Real-time PCR Assay for Detection of Borrelia Burgdorferi in Ticks in Xinjiang

为建立一种TaqMan-MGB探针实时荧光定量PCR方法,来快速、特异地检测伯氏疏螺旋体,根据GenBank发表的伯氏疏螺旋体16SrRNA序列,应用分子生物学软件进行比对,选取保守序列设计特异性引物及TaqMan-MGB探针,并优化荧光定量PCR反应体系及条件,分别对大肠杆菌、沙门氏菌及钩端螺旋体进行扩增;构建伯氏疏螺旋体标准株16SrRNA基因片段阳性质粒,10倍系列稀释后进行real-timePCR扩增,测定该方法的敏感性。结果可见:仅伯氏疏螺旋体出现阳性扩增,而大肠杆菌、沙门氏菌及钩端螺旋体扩增均为阴性;该方法对阳性质粒的检测限约为100copies/μL。应用该方法对新疆地区的100份蜱DNA样本进行检测,发现7份为伯氏疏螺旋体阳性。结果表明,本研究建立的TaqMan-MGB探针荧光定量PCR方法具有较高的特异性及良好的敏感性,为今后伯氏疏螺旋体的快速诊断及流行病学调查提供了技术支撑。

In order to establish a TaqMan-MGB probe real-time PCR assay to rapidly and specifically detect Borrelia burgdorferi,the specific primers and TaqMan-MGB probe were designed according to the conservative sequence selected from 16SrRNA sequence of Borrelia burgdorferi published in GenBank,after optimization of reaction system and conditions,the real-time PCR was established. Then Salmonella,Escherichia coli and Leptospira were amplified by the established assay respectively;besides,the positive plasmids of 16SrRNA gene fragment of Borrelia burgdorferi standard strain were constructed,after 10 times dilution,the plasmids were amplified by the real-time PCR to measure its sensitivity. The results showed that the amplification of Borrelia burgdorferi was positive,but those of Salmonella,Escherichia coli and Leptospira were negative;the detection limit for positive plasmids was about 100 copies/μL. Then 100 DNA samples collected from ticks in Xinjiang were tested by the assay,and seven samples were detected to be positive. Therefore,it was concluded that the assay established in this study was with high specificity and sensitivity,which could provide technical supports for rapid diagnosis and epidemiological investigation of Borrelia burgdorferi in the future.