Development of a Nested PCR Method for Detection of Mycoplasma Bovis in Preserved Milk Samples

  • Cole Pinnow (Iowa State University)
  • Jenny Butler (Iowa State University)
  • Ricardo Rosenbusch (Iowa State University)
  • Leo L. Timms (Iowa State University)


A nested PCR method was developed and validated for detection of Mycoplasma bovis in milk samples preserved with bronopol. A previously described, randomly cloned DNA fragment was sequenced. This sequence information was used to develop a set of internal primers for a nested PCR assay. Cationic surfactant purification was used to eliminate milk protein substances that inhibit amplification. Serial diluted seeded milk samples were used to determine a sensitivity of 5.1 CFU equivalents/ml of milk. Specificity of the assay was confirmed testing against 7 other mycoplasma species and 11 other mastitis organisms. A comparison of culture, with blind passage, to the nested PCR reaction was performed on clinical field milk samples from M. bovis affected and M. bovis free herds. The nested PCR method was more sensitive than culture (~5 CFU/ml), specific for M. bovis, effective with preserved milk, and could be completed in less than one day. This may provide a practical, rapid, cost effective procedure to screen for clinical and subclinical M. bovis carriers using routinely obtained preservative-treated milk samples.

Keywords: ASL R1917

How to Cite:

Pinnow, C., Butler, J., Rosenbusch, R. & Timms, L. L., (2004) “Development of a Nested PCR Method for Detection of Mycoplasma Bovis in Preserved Milk Samples”, Iowa State University Animal Industry Report 1(1). doi: https://doi.org/10.31274/ans_air-180814-847

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Published on
01 Jan 2004
Peer Reviewed