Development of a Real-Time PCR for Detection of Mycoplasma Bovis in Bovine Milk and Lung Samples

Abstract

Abstract.A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6ЊC. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1ЊC. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 ϫ 10 4 and 7.7 ϫ 10 8 cfu/ml and the M. bovis culture-positive lungs between 1 ϫ 10 3 and 1 ϫ 10 9 cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

Isolation, identification and molecular characterization of Mycoplasma bovis in mastitic dairy cattle by PCR and culture methods

“…In this study culture results of milk samples showed 58 out of total 328 (17.69%) M. bovis bacteria with fried egg appearance under light microscopy. In a conducted study, comparing obtained isolates with existing isolates in Gene Bank based on the gene sequence 16SrRNA proved 100 percent similarity between isolates (Cai et al, 2005).…”

Mycoplasma Bovis Real-Time Polymerase Chain Reaction Assay Validation and Diagnostic Performance

“…4 Constitutive or ''housekeeping'' genes that encode for products that are necessary bacterial replication and survival are generally highly conserved and consequently are good candidates for genetic detection of the agent. 19 The deoxyribodipyrimidine photolyase uvrC gene, which is part of the excision DNA-repair system Uvr ABC, has been shown to be highly conserved and therefore may prove useful for M. bovis identification.…”

“…4 Briefly, 1-3 g of tissue was mixed with 5 ml of sterile saline; homogenized with a stomacher; and resultant fluid was collected. Two hundred microliters of the tissue or saline homogenate, fluid sample, broth culture, or milk sample was used for DNA extraction using a commercial kit j employing the kit's protocol according to the manufacturer's instructions.…”

Use of high-resolution melting curve analysis to identify Mycoplasma species commonly isolated from ruminant, avian, and canine samples

“…c DNA from lung and milk specimens was extracted as described previously. 1 Polymerase chain reaction was performed using a realtime PCR instrument d with a high resolution melting analysis kit.…”