A direct qPCR screening approach to improve the efficiency of Mycoplasma bovis isolation in the frame of a broad surveillance study

Prevalence and Risk Factors Associated With Antimicrobial Resistance in Bacteria Related to Bovine Respiratory Disease—A Broad Cross-Sectional Study of Beef Cattle at Entry Into Canadian Feedlots

“…Colonies were picked from TSA and stored at −80°C in BHI supplemented with 20% glycerol. For M. bovis , a high throughput PCR-based enrichment process was used for screening for the presence of M. bovis followed by bacterial isolation from positive suspensions ( 23 ).…”

“…For this, 3-5 colonies of pure cultures of P. multocida, M. haemolytica, or H. somni were suspended in 100 µL of TE buffer (10 mM Tris, 1 mM EDTA, pH = 8) and heat-lysed for 5 min at 95 • C. The suspension was centrifuged for 10 min, at 18,400 × g at 4 • C, and 2 µL of the supernatant was used as a DNA template. Presumptive M. bovis cultures were confirmed via directculture-PCR using 2 µL of liquid culture directly in the PCR master mix (23). The PCR amplicons were visualized either by agarose gel or capillary electrophoresis (QIAxcel, Qiagen).…”

Mycoplasma bovis Infections—Occurrence, Diagnosis and Control

“…In the real-time PCR assay [ 45 ], milk samples from dairies and lung tissue samples were culture-enriched in PPLO broth for 24 h before analysis. In another qPCR for M. bovis testing [ 46 ], the nasopharyngeal swabs were cultured for 3–5 days before the analysis. The molecular methods are optimized for the detection of M. bovis in nasopharyngeal swabs and milk samples, but they can be optimized to be used for the detection of M. bovis in different specimens [ 2 , 26 , 27 , 48 , 49 ].…”

“…It is also possible to detect M. bovis in processed semen [ 2 , 48 ]. The real-time PCR assays are characterised often by a low limit of detection (LOD) and specificity near to 100% [ 45 , 46 , 47 , 48 ]. Taking into consideration that the number of mycoplasmas that are shed during the infection is about >1 × 10 6 CFU/mL in milk [ 4 ] and the LOD for real-time PCR for M. bovis detection in milk is 1.3 × 10 2 CFU/mL [ 48 ], the probability of the detection of infected cow in a herd is high.…”

A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease

“…Briefly, DNPS were placed into 1 ml brain heart infusion broth containing 20% glycerol (Dalynn Biologicals, Calgary, AB) and vortexed for 1 min. Methods for TC-PCR detection of M. haemolytica, P. multocida, and H. somni were identical to those described by Stanford et al (15) with the following modifications: 100 µl of DNPS suspension was plated for M. haemolytica and P. multocida, 50 µl each of undiluted DNPS suspension and 10 −1 dilution were plated for H. somni and incubated for 48 h. Methods for TC-PCR detection of M. bovis were completed as described by Andrés-Lasheras et al (29). DNA was obtained from a 300 µl aliquot of DNPS suspension using the DNeasy Blood and Tissue kit (Qiagen, Toronto, ON, Canada).…”