PCR Mycoplasma Test Kit I/C utilizes the polymerase chain reaction (PCR), which was established as the method of choice for highest sensitivity in the detection of mycoplasma and acholeplasma contamination in cell cultures and other cell culture derived biologicals. Detection requires as little as 10-15 fg of mycoplasma DNA corresponding to 10-15 mycoplasma genomes per sample volume. The primer set is specific for the highly conserved rRNA operon, or more specifically, the 16S-rRNA coding region in the mycoplasma genome. This allows for detection of many Mollicutes strains such as M. orale, M. hyorhinis, M. arginini, M. fermentans, M. salivarium, and M. hominis, which are usually encountered as contaminants in cell cultures, but also Acholeplasma laidlawii and many other species (see complete list below). The kit does not detect the clinically relevant M. pneumoniae and Ureaplasma urealyticum which however are not known as cell culture contaminants. Eukaryotic and bacterial DNA is not amplified by using the kit. Only one protocol is needed for the detection of all mycoplasma species. The detection procedure can be performed within 3 hours. PCR Mycoplasma Test Kit I/C also provides internal control DNA (as negative control or for verifying a successful PCR run) as well as a positive control. When running the PCR with the internal control DNA, a successfully performed reaction is indicated by a 479 bp band on the agarose gel. The positive control yields a band at 270 bp.
All components required for the PCR (primer, nucleotides, control DNA, and Hot-Start Taq polymerase) come lyophilized in PCR tubes included in the kit (lyophilized Master Mix). For setting up the PCR, simply cut off the number of tubes required, rehydrate lyophilized Master Mix with provided Rehydration Buffer and add the sample.
Samples can be easily prepared by boiling and centrifuging the cell culture supernatant.