Gopalkrishna, VVerma, HKumbhar, N STomar, R SPatil, P R2007-10-192009-05-282007-10-192009-05-282007-10-19Gopalkrishna V, Verma H, Kumbhar NS, Tomar RS, Patil PR. Detection of Mycoplasma species in cell culture by PCR and RFLP based method: effect of BM-cyclin to cure infections. Indian Journal of Medical Microbiology. 2007 Oct; 25(4): 364-8http://imsear.searo.who.int/handle/123456789/53585PURPOSE: A two-stage nested polymerase chain reaction (PCR) assay system was described that amplifies the 16S-23S rRNA spacer region sequences of Mycoplasma and Acholeplasma infections in cell cultures and virus stocks. METHODS: Established cell lines and virus stocks were screened for the presence of Mycoplasma by using nested PCR using two sets of outer and inner primers, amplifies 16S-23S rRNA. PCR and restriction fragment length polymorphism (RFLP) assay was used to detect and identify most of the species-specific Mycoplasmas involved in cell cultures and virus stock contaminants. Infected cultures detected by PCR-RFLP were further treated with BM-cyclin (5 microg/mL) and passaged for three times and tested for Mycoplasma infections by PCR-RFLP. RESULTS: Mycoplasma pirum and Mycoplasma orale infections were detected by nested PCR. Species specificity was identified by using RFLP of Vsp I, Cla I and Hin dIII restriction enzymes. Mycoplasma infections were cured by treatment with BM-cyclin. This was further confirmed by non-amplification of PCR amplimers in BM-cyclin treated vs. non-treated cultures. CONCLUSIONS: Regular monitoring of cell cultures for Mycoplasma infections and identification of species-specific Mollicutes will identify the source of contaminations. This approach can be used for quality control of the biological reagents used in cell culture and virology laboratories.engAnti-Bacterial Agents --pharmacologyCell Culture TechniquesDNA, Bacterial --geneticsDNA, Ribosomal Spacer --geneticsDiterpenes --pharmacologyMinocycline --pharmacologyMycoplasma --classificationPolymerase Chain Reaction --methodsPolymorphism, Restriction Fragment LengthQuality ControlVirology --methodsJournal Article