Abstract
BackgroundIn 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.ResultsThe 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 rM. 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.ConclusionIn 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.
Highlights
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)
Highly sensitive and species-specific PCRs are used in the diagnosis of Mycoplasma-associated diseases for M. dispar [14, 15], M. bovis [4, 16] and M. bovirhinis [17] in BALF or nasal swabs
4E−43 - 6E−45) to 31 complete genome or 16S ribosomal partial sequence, and for the amplicon of M. bovirhinis we found a 99–100% identity (E-values 5E−50 - 1E-51.) For the M. bovirhinis amplicon a 97% identity (3E-40) was found for 11 hits for Mycoplasma canis (Taxid:29,555)
Summary
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). In BRD, differential diagnosis of these pathogens with rapid turnaround time procedure is essential to implement appropriate treatment and intervention measures in a timely manner. Rapid detection of these pathogens at the early stage of outbreak can contribute substantially to minimize the spread of infection and increase treatment efficiency. Highly sensitive and species-specific PCRs are used in the diagnosis of Mycoplasma-associated diseases for M. dispar [14, 15], M. bovis [4, 16] and M. bovirhinis [17] in BALF or nasal swabs. A DNA microarray assay was developed for the parallel detection of 37 Mycoplasma species [19], in which species-specific probes derived from the 23S rRNA and tuf genes were used for species differentiation
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