Mycoplasma detection by triplex real-time PCR in bronchoalveolar lavage fluid from bovine respiratory disease complex cases

Background In this study we evaluated the RespoCheck Mycoplasma triplex real-time PCR for the detection in bronchoalveolar lavage fluid (BALF) of Mycoplasma (M.) dispar , M. bovis and M. bovirhinis , all three associated with bovine respiratory disease (BRD). Primers and probes of the RespoCheck Mycoplasma triplex real-time PCR are based on the V3/V4 region of the 16S rRNA gene of the three Mycoplasma species. Results The analytical sensitivity of the RespoCheck triplex real-time PCR was, as determined by spiking experiments of the Mycoplasma strains in Phosphate Buffered Saline, 300 colony forming units (cfu)/mL for M. dispar , and 30 cfu/mL for M. bovis or M. bovirhinis . The analytical sensitivity of the RespoCheck Mycoplasma triplex real-time PCRwas, as determined on purified DNA, 10 fg DNA per assay for M. dispar and 100 fg fo r M. bovis and M. bovirhinis . The analytical specificity of the RespoCheck Mycoplasma triplex real-time PCR was , as determined by testing Mycoplasmas strains ( n = 17) and other bacterial strains ( n = 107), 100, 98.2 and 99.1% for M. bovis , M. dispar and M. bovirhinis respectively. The RespoCheck Mycoplasma triplex real-time PCR was compared with the PCR/DGGE analysis for M. bovis , M. dispar and M. bovirhinis respectively by testing 44 BALF samples from calves. Conclusion In conclusion, the RespoCheck PCR assay can be a valuable tool for timely and accurate detection of three Mycoplasma species associated with in bovine respiratory disease.