COMPETITOR INTERNAL STANDARDS FOR QUANTITATIVE DETECTION OF MYCOPLASMA DNA

Homologous internal controls were used as competitor DNA in the polyme rase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucle otide sequences of the 16S rRNA gene of mycoplasma species. Amplificat ion of this gene was examined in five different mycoplasma species: My coplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pne umoniae. To evaluate the primers, a number of different cell lines wer e assayed for the detection of mycoplasma infections. All positive cel l lines showed a distinct product on agarose gels while uninfected cel ls showed no DNA amplification. Neither bacterial nor eukaryotic DNA p roduced any cross-reaction with the primers used, thus confirming thei r specificity. Internal control DNA to be used for quantitation was co nstructed by modifying the sizes of the wild-type amplified products a nd cloning them in plasmid vectors. These controls used the same prime r binding sites as the wild-type and the amplified products were diffe rentiated by a size difference. The detection limits for all the mycop lasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide -stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore acci dental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be usefu l in monitoring the progression and significance of mycoplasma in the disease process.