Use of high-resolution melting curve analysis to identify Mycoplasma species commonly isolated from ruminant, avian, and canine samples.

A real-time polymerase chain reaction assay coupled with high resolution melting curve analysis (PCR-HRM) was developed for identifying and distinguishing Mycoplasma species commonly isolated from ruminant, avian, and canine samples. The real-time PCR used 1 set of universal primers specific for the spacer region between the 16S ribosomal RNA and the 23S ribosomal RNA genes; the melting curve analysis of the PCR product used a high-resolution melt fluorescent dye. The real-time PCR-HRM assay was able to distinguish M. arginini, M. bovigenitalium, M. bovis, M. bovirhinis, M. canadense, M. cynos, M. spumans, M. iowae, M. meleagridis, and M. agalactiae reference strains. The real-time PCR-HRM assay developed was evaluated by testing field isolates of M. bovis, M. arginini, M. bovirhinis, M. bovigenitalium, M. iowae, and M. spumans with results consistent with those of the fluorescent antibody test.