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目的基于莱姆病螺旋体recA基因,建立一种检测鼠中莱姆病螺旋体的real-time PCR方法。方法通过GenBank分析比较莱姆病螺旋体recA基因,选择其保守序列设计MGB探针及引物并进行方法学评估。并应用建立的real-time PCR方法和nested PCR方法对收集的123份鼠标本进行检测分析。结果本研究建立的real-time PCR方法仅对莱姆病螺旋体检测阳性,其最小检出浓度为101copies/μL。标准曲线各浓度点Ct值批内、批间平均变异系数(CV)分别为1.56%和2.30%。123份鼠标本中,real-time PCR检测59例阳性,nested PCR检测43例阳性。结论新建立的real-time PCR方法具有快速、敏感和特异的优点,可用于鼠标本中莱姆病螺旋体的检测。
Objective To establish a real-time PCR method for the detection of Borrelia burgdorferi in mice based on the recA gene of Borrelia burgdorferi. Methods GenBank analysis was used to compare the recA gene of Borrelia burgdorferi. MGB probes and primers were selected based on their conserved sequences and evaluated by methodology. The collected 123 mouse samples were detected by real-time PCR and nested PCR. Results The real-time PCR method established in this study was positive only for the detection of Borrelia burgdorferi and its minimum detection concentration was 101 copies / μL. The Ct values of each concentration point in the standard curve were 1.56% and 2.30%, respectively, within and between batches. Of the 123 mice, 59 were positive by real-time PCR and 43 by nested PCR. Conclusion The newly established real-time PCR method has the advantages of fast, sensitive and specific, and can be used for the detection of Lyme disease in mice.