实时荧光定量TaqMan-MGB探针法检测杆菌样巴尔通体

Detection of Bartonella bacilliformis by real-time PCR with TaqMan-MGB probe

摘要:

【目的】应用TaqMan探针实时荧光定量PCR技术建立特异性强、敏感性高和稳定性好的快速杆菌样巴尔通体检测方法。【方法】应用生物信息学方法查找杆菌样巴尔通体特有基因，从中筛选出一段特有的基因序列为模板设计探针和引物。通过比较Ct值和荧光强度确定扩增反应的最佳退火温度、探针和引物浓度；将扩增产物连接到pEASY-T载体上制备标准品，绘制标准曲线，分析扩增效率和线性关系；评估方法的特异性、敏感性及重复性。【结果】优化后退火温度为60 °C，探针和引物浓度均为200 nmol/L，反应体系20 μL。特异性实验显示只有杆菌样巴尔通体扩增出荧光信号，其他种属细菌均未见荧光信号；标准曲线线性关系良好(R2=1)，扩增效率E=98.18%；最低检出限为每个PCR反应3个拷贝；组内和组间的变异系数CV值分别为0.21%–0.42%和0.29%–0.59%，在允许范围内。【结论】研究建立的实时荧光定量TaqMan-MGB探针法特异性强、灵敏度高、稳定性好，可快速检测鉴定杆菌样巴尔通体，为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。

Abstract:

[Objective] Bartonella bacilliformis is a fastidious haemotropic gram-negative bacterium, an etiological agent of a life-threatening illness, termed Carrion’s disease or bartonellosis. We developed a rapid, highly sensitive and specific assay, based on real-time fluorescence quantitative PCR for identifying B. bacilliformis. [Methods] A primer pair and probe set for B. bacilliformis were designed based on a species-specific gene of B. bacilliformis, based on bioinformatics method by scanning and comparing of all bacteria on GenBank data. The annealing temperature and the final concentration of the TaqMan probe and primers were optimized. The amplification product of the target gene was cloned into pEASY-T vector for preparing the standard and making a standard curve. The standard curve was made using 10×dilution series of the reference plasmid and the efficiency and linearity of PCR amplification were analyzed. Specificity, sensitivity and reproducibility of the PCR system were assessed. [Results] The optimized annealing temperature is 60 °C and the probe and primer concentration are 200 nmol/L for 20 μL reaction system. The TaqMan probe-based fluorescence quantitative PCR did not show cross reactivity with other Bartonella species, non-Bartonella bacteria and the other animals. The correlation coefficient R2 and E-value of the standard curve were 1.0 and 98.18%, respectively. [Conclusion] The real-time PCR with TaqMan-MGB probe assay is highly specific and sensitive for the detection of B. bacilliformis.