Detección de contaminación por Mollicutes en cultivos celulares mediante amplificación del gen 16S rARN

Abstract

Up to 80% of cell cultures have been reported to be contaminated with Mollicutes, causing unreliable experimental results and giving rise to unsafe biological products. The aims of this study were to estimate the frequency of mycoplasmas as contaminants in cell cultures, and to analyze the performance of a PCR assay targeting the 16S rRNA of Mollicutes. Thirty-nine cell cultures representing the most used lines of cell cultures and 17 primary cell cultures submitted for detection of mycoplasma contamination were studied between July and December 2005, Mycoplasmas were detected in 18/39 (46.2%,) cell line cultures, and in none of the primary cell cultures. Hpall analysis of the 16S-23S rRNA intergenic space of 6 positive cultures belonging to different laboratories gave two different restriction patterns. The sequentiation of each of the two patterns identified Mycoplasma hyorhinis and Mycoplasma salivarium as the contaminant mycoplasmas. The analytic sensitivity of the 16S rRNA PCR was 0.01 color-changing units per mL, as determined for dilutions of a Mycoplasma hominis culture, whereas the analytical specificity was 100%. In conclusion, these results reaffirm the importance of mycoplasmas as cell culture contaminants, and suggest that 16S rRNA PCR is a reliable method for detection of these organisms as cell culture contaminants.