重组酶介导等温核酸扩增技术检测伯氏疏螺旋体方法的建立

Establishment of Recombinase Aided Amplification for Detection of Borrelia burgdorferi

本试验采用重组酶介导等温核酸扩增(RAA)技术建立伯氏疏螺旋体核酸的检测方法。根据伯氏疏螺旋体16S rRNA基因保守序列设计并合成特异性RAA引物,采用重组酶介导扩增技术电泳法,以10倍系列稀释1 ng/μL伯氏疏螺旋体B31基因组DNA为模板进行RAA,测定该方法的敏感性。通过对大肠杆菌、沙门菌、钩端螺旋体基因组DNA扩增验证该方法的特异性。结果显示,该方法对伯氏疏螺旋体标准株的B31基因组DNA检测限约为100 fg/μL,对大肠杆菌、沙门菌、钩端螺旋体均无扩增,具有良好的特异性。RAA具有特异性高、检测快速、灵敏等优点,本研究建立的伯氏疏螺旋体RAA检测方法,为伯氏疏螺旋体临床样本的快速诊断提供依据。

In this study,recomb inase aided amplification(RAA)was established for the detection of Borrelia burgdorferi(B.burgdorferi).The specific RAA primers were designed and synthesized according to the conserved sequence of 16S rRNA gene of B.burgdorferi.The recombinant enzyme-mediated amplification technique was used to electrophoresis,RAA was performed using 10-fold serial dilution of 1 ng/mL B31 genomic DNA of B.burgdorferias the template,and the sensitivity of the method was determined.The specificity of the method was verified by amplification of genomic DNA of E.coli,Salmonella and Leptospira.The results showed that the detection limit of B31 genome DNA of B.burgdorferi standard strain was about 100 fg/μL.It had no amplification to E.coli,Salmonella and Leptospira,which had good specificity.RAA has the advantages of high specificity,rapid detection and sensitivity.This study established RAA detection method for the clinical samples of B.burgdorferi.