Development of species-specific PCRs for the detection of mycoplasmas cell culture contaminants

Abstract

Mollicute contamination in cell cultures is common. Species such as Mycoplasma arginini, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii are identified as causing 95 % of the contaminations. The present work is aimed at developing species-specific PCRs for the identification of the most frequent contaminating mycoplasma species in cell cultures. The critical parameters of PCR were optimized and the analytical sensitivity and specificity were determined. The optimum temperature for hybridization was set at 55ºC; magnesium concentration at 1,5 mM. A range of 0,3-0,5 µM was selected for the primers. The analytical sensitivity of PCR was 1 pg/µL, while its analytical specificity was 100 %. Species-specific PCR was performed on 58 cell culture samples where the presence of Mollicutes was previously detected. As a result, Mycoplasma orale was detected as the most frequent species, followed by Mycoplasma fermentans, Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma hyorhinis, and Mycoplasma salivarium. The results of this study confirm that the species-specific PCR assays developed are feasible to use for the identification of mycoplasma species in cell cultures.