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

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.