Differential detection of hemotropic Mycoplasma species in cattle by melting curve analysis of PCR products.

We developed a real-time PCR procedure followed by melting curve analysis using the green fluorescence dye SYBR Green I for rapid detection and differentiation of hemplasmas in cattle. Analysis of the melting temperature (Tm) of the PCR products allowed for differentiation of the 2 bovine hemoplasmas, Mycoplasma wenyonii and a provisional species, ;Candidatus Mycoplasma haemobos' (a synonym of ;Candidatus M. haemobovis'). The Tm (mean +/- S.E.) of the PCR products from the bovine hemoplasmas were 86.98 +/- 0.12 degrees C for M. wenyonii and 82.04 +/- 0.27 degrees C and 86.98 +/- 0.12 degrees C, respectively; [corrected] Candidatus M. haemobos' in the melting experiments. The protocol described in the present study can decrease the time to results by simultaneous detection and differentiation of the two hemoplasmas in cattle. By using this protocol, we examined hemoplasma prevalence in 109 cattle in Miyagi Prefecture and found that 67 (61.5%) were infected with M. wenyonii, 25 (22.9%) were infected with ;Candidatus M. haemobos' and 14 (12.8%) were infected with both.