一种莱姆病螺旋体real-time PCR方法的建立及其在鼠标本检测中的应用评价

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10¹ copies/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。

Evaluation of a new real-time PCR assay for detection of Borrelia burgdorferi in rodents

We established and evaluated a real-time PCR assay for detection of Borrelia burgdorferi (B. burgdorferi) in rodents. MGB probe and specific primers were designed based on the conserved sequences of recA gene of B. burgdorferi and methodology evaluation was performed. The 123 rodent tissue samples were detected by the established real-time PCR and nested PCR methods. Results showed real-time PCR assay based on recA gene was successfully established. Specificity evaluation showed specific amplification was only achieved from genomic DNA of B. burgdorferi. The lowest detection limit of the new assay was about 10 copies of recA gene from B. burgdorferi genomic DNA. The average intra-and-inter coefficient of variations (CV) of the Ct value were 1.56% and 2.30% respectively. In 123 rodent samples, 59 samples were detected positive by real-time PCR, compared to 43 positives by nested PCR. The new real-time PCR assay is suitable for detecting Borrelia burgdorferi in rodents.