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.
