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.
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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)
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Chemical References |
- Anti-Bacterial Agents
- DNA, Bacterial
- DNA, Ribosomal Spacer
- Diterpenes
- BM-cyclin
- Minocycline
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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)
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