Detection of Mycoplasma species in cell culture by PCR and RFLP based method: effect of BM-cyclin to cure infections.
Indian J Med Microbiol
;
2007 Oct; 25(4): 364-8
Article
in English
| IMSEAR
| ID: sea-53585
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.
Full text:
Available
Index:
IMSEAR (South-East Asia)
Main subject:
Quality Control
/
Virology
/
Polymorphism, Restriction Fragment Length
/
DNA, Bacterial
/
Polymerase Chain Reaction
/
Cell Culture Techniques
/
DNA, Ribosomal Spacer
/
Diterpenes
/
Minocycline
/
Anti-Bacterial Agents
Type of study:
Diagnostic study
Language:
English
Journal:
Indian J Med Microbiol
Journal subject:
Microbiology
Year:
2007
Type:
Article
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