Species-specific PCR for identification of common contaminant mollicutes in cell culture

Mycoplasma arginini, M fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in c ontaminated cell cultures. Previous studies have shown that the published P CR primer pairs designed to detect mollicutes in cell cultures are not enti rely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region , and the 5 ' end of the 23S rRNA gene, as a whole, are promising targets f or design of mollicute species-specific primer pairs. We analyzed the 16S r RNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5 ' end of t he 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminan t mollicutes. Previously published, putative mollicute-specific primers amp lified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remainin g 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicu tes in specimens and for identification, if required.