Abstract:

Background and Aim: Mycoplasma is one of the smallest alive microorganisms, having the ability of transmitting from microbiological filters makes it to be a severe and important contaminant for cell culture, biological and biotechnological products. Using a rapid and sensitive method for diagnosing Mycoplasma’s infection is the first priority. This method should be able to discover typical Mycoplasma’s infection, but for meeting the standard we need to produce the internal control competitively in Mycoplasma spp PCR recognizing, which is the main aim of the project.

Materials and Methods: Using of special primers for IS6110 target, PCR test was optimized. Besides, the composite primers for IC- M. spp were designed then the PCR was optimized. The IC- M. spp which amplified in constringent condition, was ligated in pTZ57R plasmid, transformed  in E.coli JM107 and was cloned. Specification, sensitivity of test and the number  needed in each test. Also PCR with internal control were defermined. The number of IC, which is needed in each PCR reaction was scanned in the matter of dilution and PCR response with IC.

Results: PCR amplicon for Mycoplasma spp and IC- M. spp with special primers were 272bp and 660bp respectively, so there was a significant different between their size.

Conclusion: Despite the high speed and accuracy of PCR, false positive and negative results, which are caused because of PCR inhibitors, are the important problems of this technic that can reduce its efficiency. Using another DNA as an internal control can detect these inhibitors. Indeed, amplification of this DNA shows correct amplification and detection steps.