Development of real-time PCR test systems for detection of Mycoplasma bovis and M. bovigenitalium DNA

Currently, the causative agents of cattle mycoplasmosis caused by Mycoplasma bovis and M. bovigenitalium are prevalent in livestock farms all over the world, including the Russian Federation. Clinical signs observed in cattle mycoplasmosis are upper respiratory tract infection, serous catarrhal inflammation of the lungs, arthritis, rhinitis, pneumonia in young cattle, abortions in pregnant animals, vulvovaginitis, mastitis and birth of dead or non-viable litter. The economic damage includes mortality, involuntary slaughter, loss of live weight and offspring, treatment costs, prevention, etc. In this regard, the development of methods for detection of these pathogens is of high significance for the control of mycoplasma infection. This study is aimed at the development and optimization of methods for detection of M. bovis and M. bovigenitalium genomes using real-time polymerase chain reaction. For this purpose, we analyzed the genetic sequences of the GenBank database and selected the fusA locus for M. bovis and the 16S-23S rRNA locus for M. bovigenitalium . The optimum concentrations of the reaction components: magnesium chloride 5.5 mM for M. bovis and 6.0 mM for M. bovigenitalium , deoxynucleoside triphosphates 300 mM for M. bovis and 200 mM for M. bovigenitalium , primers 0.4 mM for M. bovis and 0.3 mM for M. bovigenitalium , fluorescent probes 0.05 mM for M. bovis and 0.15 mM for M. bovigenitalium ; the temperature and time conditions for M. bovis and M. bovigenitalium were as follows: 10 min at 95 °С (reaction mixture heating), then 40 PCR cycles consisting of DNA denaturation for 10 s at 95 °С, primer annealing and cDNA elongation for 60 s at 60 °С. A high analytical specificity, detection limit of mycoplasma DNA, which was 25-50 CFU/ml, and amplification efficiency of 90.94 and 91.85% for M. bovis and M. bovigenitalium , respectively, were established. Therefore, as a result of these studies, specific and reproducible test systems based on real-time polymerase chain reaction for detection of M. bovis and M. bovigenitalium DNA for routine diagnosis of bovine mycoplasmosis have been developed.