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 ORIGINAL ARTICLE
Year : 2007  |  Volume : 25  |  Issue : 4  |  Page : 364-368

Detection of Mycoplasma species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections


National Institute of Virology, P.O. Box 11, Pune - 411 001, India

Correspondence Address:
V Gopalkrishna
National Institute of Virology, P.O. Box 11, Pune - 411 001
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.37340

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Purpose: 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 μg/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.






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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04