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

Abstract

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 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.

• Authors: V Gopalkrishna, H Verma, N S Kumbhar, R S Tomar, P R Patil
• Journal: Indian journal of medical microbiology (Indian J Med Microbiol) Vol. 25 Issue 4 Pg. 364-8 (Oct 2007) ISSN: 0255-0857 [Print] United States
• PMID: 18087086 (Publication Type: Journal Article)

Chemical References

• Anti-Bacterial Agents
• DNA, Bacterial
• DNA, Ribosomal Spacer
• Diterpenes
• BM-cyclin
• Minocycline

Topics

• Anti-Bacterial Agents (pharmacology)
• Cell Culture Techniques
• DNA, Bacterial (genetics)
• DNA, Ribosomal Spacer (genetics)
• Diterpenes (pharmacology)
• Minocycline (pharmacology)
• Mycoplasma (classification, drug effects, genetics, isolation & purification)
• Polymerase Chain Reaction (methods)
• Polymorphism, Restriction Fragment Length
• Quality Control
• Virology (methods)