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 x 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. vinsonii subsp. berkhoffii was 11 copies of the plasmid DNA per PCR reaction. The coefficient of variation CV% from the intra- and inter-group was 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.