A polymerase chain reaction based method for detecting Mycoplama/Acholeplasma contaminants in cell culture

a detection system that utilizes a primer mixture in a nested polymerase ch ain reaction for detecting Mycoplasma contaminants in cell cultures is desc ribed. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sam ple. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, w hich include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhi nis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were co nsistently amplified. Mycoplasma contaminants generated a single DNA band o f 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic t wo-band pattern of 426 and 219 bp amplicons. Species identification could b e achieved by size determination and restriction enzyme digestion. Minor cr oss-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycop lasma contaminants in cell cultures has been developed based on this approa ch. (C) 2000 Published by Elsevier Science B.V. All rights reserved.