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assay for the detection of mycoplasma contam in vaccines, cultures ... etc

PCR or qPCR

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#1 nightingale

nightingale

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Posted 25 June 2014 - 11:57 PM

Dear All, 

 

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 ... 

 

grateful

 


" The more you learn, the more you realize how little you know ... "




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