i would like to ask you all, for the method you kindly use for the detection of mycoplasma contamination in your cell cultures, or vaccine batches.
in our laboratory we are/were using a commercial kit : e-myco plus mycoplasma PCR detection kit - iNtRoN Biotechnology,
this kit is a ready to use one, conventional PCR method ( the run takes 1:45 mins ) and it has 3 controls :-
internal for the PCR inhibition, extraction control and a positive control one.
the kit covers a very wide range of mycoplasmas as the primers are designed for the highly conserved 16S rRNA with considering that some mycoplasma species have a difference .
knowing that the conventional PCR method, will still -comparing to nested and qPCR- have a limitation in the detection,
we moved to a nested PCR method according to the paper : Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions, Res. MicrobioL 1993, 144, 489-493.
applying the method used in this paper : ended in a positive result in everything we try to detect! except the no-template-control ...
the viral DNA/RNA extracted samples, previousely tested negative samples using qPCR method by another laboratory, etc etc ...
so it seems that the primers anneal to certain regions in templates non-specifically!
searching for a real-time PCR method, we only found a SYBR Green based method ... which we don't prefer at all !
we were desperate to find a probe-based assay ...
if any of you is applying any "good" assay, please kindly share it ...