Detection ofMycoplasma boviswith an improved pcr assay

Abstract

A Mycoplasma bovis species-specific PCR assay has been developed with improvement of a previously described method (Ghadersohi et al., 1997). This test and its semi-nested version (Hayman and Hirst, 2003) did not function at all in our hands. A new reverse primer (Mbr2) was designed using previously published sequence data. For testing specificity, DNA was extracted from the most frequently occurring mycoplasma species and bacteria of bovine origin. The new PCR detected only Mycoplasma bovis. Moreover, no cross-reaction was observed with the genetically closest relative species, M. agalactiae. The target organism could be detected in a dose as low as 150 CFU ml -1 in broth cultures using ethidium-bromide-stained agarose gels.

Agreement Among 4 Sampling Methods to Identify Respiratory Pathogens in Dairy Calves with Acute Bovine Respiratory Disease

“…Speciation of Mycoplasma isolates to identify Mycoplasma bovis was completed by PCR as previously described . Briefly, DNA was extracted from Mycoplasma isolates with a commercially available kit according to the manufacturer's directions.…”

“…Briefly, DNA was extracted from Mycoplasma isolates with a commercially available kit according to the manufacturer's directions. The PCRs were prepared in 25 μL volumes with M. bovis ‐specific primers described . The PCR assays were performed with an initial cycle at 94°C for 5 minute; 35 cycles of denaturation at 94°C for 20 second, annealing at 52°C for 1 minute, and extension at 72°C for 1 minute; and extension at 72°C for 5 minute.…”

Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex

“…Some of these samples were taken from cattle that showed symptoms of M. bovis infection, such as fever, arthritis, mastitis, conjunctivitis, and pneumonia. PCR amplification and commercial ELISA ( Mycoplasma bovis Antibody Test Kit, BioVet, Canada) were carried out separately on all samples [31], [32]. The serum samples were kept at –70°C for further study of screening immunogenic protein and establishment of a method of detection.…”

First report of Mycoplasma bovis infection in dairy cattle in Guangzhou, subtropical southern China

“…In recent years, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods have gradually replaced culture as the method of choice for detecting M. bovis. In particular, ELISA has been demonstrated as more reliable method for the herd diagnosis of M. bovis infection, as antibody levels detected by ELISA remain high for many months (Nicholas and Ayling, 2003;Tenk et al, 2004;Tenk et al, 2006;Ball and Nicholas, 2011;Maunsell et al, 2011).…”