TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

Real-time PCR-based detection of Bartonella vinsonii sub sp. Berkhoffii by TaqMan minor groove binder probe

摘要:

摘要:【目的】应用TaqMan-MGB探针技术，建立具有种水平特异性、高敏感性的荧光定量PCR方法，用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplified region，SCAR)技术基础上，依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物，分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线，评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV 值分别为0.12%－0.70%和0.14%－0.55%，在允许范围内;标准曲线线性关系良好(R2=1)，扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种，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:

Abstract:［Objective］Bartonella vinsonii subsp．berkhoffii is a fastidious haemotropic Gram-negative bacterium that has been identified as an emerging causative agent for zoonotic diseases of human and dogs．This study aimed to develop a TaqMan-MGB probe based，highly sensitive and species-specific fluorescence quantitative PCR assay for rapid detection of B．vinsonii subsp． berkhoffii．［Methods］The species-specific primersand probe set for B．vinsonii subsp．berkhoffii were designed．The annealing temperature，final concentration of the TaqMan-MGB probe and primers were optimized．Specificity，sensitivity and reproducibility of the PCR system were assessed．The standard curve was made using 10 × dilution series of the plasmid standard to analyze stability and PCR efficiency．［Results］ The real-time PCR with TaqMan-MGB assay was highly specific and sensitive for the detection of B．vinsonii subsp．berkhoffii．TaqMan-MGB probe-based fluorescence quantitative PCR did not show cross reactivity with the other Bartonella species，non-Bartonella bacteria and dogs and human．The detection limit of the TaqMan-MGB assay for the detection of B．vinsoniisub sp．berkhoffii was 11 copiesof the plasmid DNA per PCR reaction．The coefficient of variation CV% from the intra- and intergroupwas in the range of 0.12%－0.70% and 0.14%－0.55% which were acceptable．The correlation coefficient and E-value of the standard curve were 1.0 and 104.7%，which reflected a very good linearity and high efficiency．［Conclusion］ The TaqMan MGB-based probe fluorescence quantitative PCR assay was a reliable，species-specific，sensitive and useful tool for rapid detection of B.vinsonii subsp． berkhoffii.