Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

J Vet Diagn Invest. 2005 Nov;17(6):537-45.

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 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. 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 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 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.

Respected Sir,

It's possible.

Centrifuge 1.5-mL sample of the milk at 14 000 rpm for 15 min, then discard the supernatant and use the pellet for DNA extraction.

Primers  (5'CCCGCTGATGCAAGTGCC3') and L2 (5 'CCCGCACATCCCAACACC 3').

PCR reaction: 50 ,uL containing: 10 ,ul of DNA, Taq polymerase lx buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.3 mM MgCl2, and 0.001% of gelatine), 2.5 U Taq polymerase, 0.2 mM of each triphosphate nucleotide, and 20 pmol of each primer.

thermocycler  program  for 25 amplification cycles as follows: denaturation at 94°C for 1 min and annealing and extension at 70°C for 30 sec;

To what extent is it possible to detect M. bovis in milk samples by PCR? - ResearchGate. Available from: https://www.researchgate.net/post/To_what_extent_is_it_possible_to_detect_M_bovis_in_milk_samples_by_PCR [accessed Aug 20, 2016].

We are taking up a work on milk from peri-urban areas of Guwahati city. Although there are some literature on detection of M. bovisin milk samples, I am interested on application of molecular based techniques as well as other rapid methods. How far is it possible to detect M. bovis in milk by PCR? Other than PCR, is there any sensitive and specific rapid test for detection of M. bovisin milk samples?I shall be very happy to have the answers for food safety.