RESUMO
Recently, PCR is being used more frequently as a diagnostic method to detect M. pneumoniae. We used primer pairs reported by Van Kuppeveld, Leng, Lunerberg, and Bernet targeting 16s rRNA, P1 protein, tuf genes, and a short DNA sequence [MP5] to evaluate the sensitivity among different PCRs. Reoptimization experiments showed that tuf PCR had the highest sensitivity amongst these four PCRs, detecting 10 organisms. Detection limit for the rest of the PCRs was 100 copies of DNA. This study confirmed that 92°C would be the best dissociation temperature rather than higher temperatures that are still being used frequently in other studies. Besides, accurate optimizing of the annealing temperature and extension time had important roles on the sensitivity as well as using milli-Q distilled water rather than double distilled water. Experiments done on MP5 PCR proved that the non-specific products mentioned in previous studies were not eliminated by increasing the annealing temperature, although they disappeared on gel electrophoresis after careful optimization of extension time