Detection of Mycoplasma orale contamination in cell culture by PCR method

Background and objectives: Mycoplasma contamination is one of the major concerns in cell culture technology. They can cause undesirable effects on cell cultures. Higher rate of cell culture contaminations by Mycoplasma orale is transmitted by laboratory personnel. Thus, the identification and rapid diagnosis, in controlling and prevention of this contamination are important. The aim of this study was to investigate the detection of Mycoplasma orale using a rapid, sensitive and specific polymerase chain reaction (PCR) method in cell cultures.Materials and Methods: A 16S rRNA-based Mycoplasma genus and specific primers PCR method for Mycoplasma orale were developed. The sensitivity and specificity of this method were determined.The PCR test was used after the cultured isolates were DNA extracted.Results: A total of 62 cell cultures sample were sent to the Mycoplasma Reference Laboratory at Razi Institute in Karaj, for detection of mycoplasma contamination. The specific PCR method reacted positively for the M. orale whereas no response was observed for, other species such as Mycoplasma S., mycoplasma H.,Mycoplasma arginine, mycoplasma L.,mycoplasma F., Mycoplasma pneumoniae, which showed the high specificity of this test. 42 out of 62 samples (67%) were scored positive by Mycoplasma genus. From these 42 samples, 8 (19%) samples were reacted positively in the M. orale specific PCR method. Conclusion: The obtained results are shown that PCR technique is a suitable and valuable tool for the detection of high percentage of mycoplasma contamination in cell cultures. So a large number of samples can be processed more rapidly during less than 24 hours . In conclusion, the PCR method is a rapid process with the high sensitivity, specificity and low cost. Therefore, it can be used routinely in cell culture laboratories.