Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

<b>Purpose:</b> A two-stage nested polymerase chain reaction (PCR) assay system was described that amplifies the 16S-23S rRNA spacer region sequences of <i> Mycoplasma</i> and <i> Acholeplasma</i> infections in cell cultures and virus stocks. <b> Methods:</b> Established cell lines and virus stocks were screened for the presence of <i> Mycoplasma</i> 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 <i> Mycoplasmas</i> involved in cell cultures and virus stock contaminants. Infected cultures detected by PCR-RFLP were further treated with BM-cyclin (5 &#956;g/mL) and passaged for three times and tested for <i> Mycoplasma</i> infections by PCR-RFLP. <b> Results:</b> <i> Mycoplasma pirum</i> and <i> Mycoplasma orale</i> infections were detected by nested PCR. Species specificity was identified by using RFLP of <i> Vsp</i> I, <i> Cla</i> I and <i> Hin</i> dIII restriction enzymes. <i> Mycoplasma</i> 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. <b> Conclusions:</b> Regular monitoring of cell cultures for <i> Mycoplasma</i> 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