| Autor: |
Gopalkrishna, V, Verma, H, Kumbhar, NS, Tomar, RS, Patil, PR |
| Zdroj: |
Indian Journal of Medical Microbiology; October-December 2007, Vol. 25 Issue: 4 p364-368, 5p |
| Abstrakt: |
Purpose:A two-stage nested polymerase chain reaction (PCR) assay system was described that amplifies the 16S-23S rRNA spacer region sequences of Mycoplasmaand Acholeplasmainfections in cell cultures and virus stocks. Methods:Established cell lines and virus stocks were screened for the presence of Mycoplasmaby 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 Mycoplasmasinvolved 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 Mycoplasmainfections by PCR-RFLP. Results:Mycoplasma pirumand Mycoplasma oraleinfections were detected by nested PCR. Species specificity was identified by using RFLP of VspI, ClaI and HindIII restriction enzymes. Mycoplasmainfections 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 Mycoplasmainfections 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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