Molecular detection of Mycoplasma haemomuris subspecies using dnaK-targeted real-time PCR with SYBR Green I and melting curve analysis

Hemoplasmas cause severe infections in mammals, but these pathogens are difficult to detect and identify at the species and subspecies level because of the need for time-consuming sequence based methods. Here, we used real-time PCR with SYBR Green I targeting of the dnaK gene followed by standard melting curve analysis to achieve rapid detection and differentiation of the Mycoplasma haemomuris subspecies 'Candidatus Mycoplasma haemomuris subsp. musculi' and 'Candidatus M. haemomuris subsp. ratti'. The melting temperatures of the PCR products, 84.63 +/- 0.14 degrees C for 'Candidatus M. haemomuris subsp. musculi', and 80.72 +/- 0.16 degrees C for 'Candidatus M. haemomuris subsp. ratti', provided clear differentiation between them. Murine hemoplasma DNA samples, which were used as references, were confirmed for species by an analysis of 16S rRNA sequences. The protocol described herein provides a new rapid detection and identification method suitable for use with two recognized subspecies of M. haemomuris. (C) 2016 Elsevier Ltd. All rights reserved.