I am using PMA (Propidium monoazide) dye for the differentiation of live and dead bacteria.
Ideally, on treating dead bacteria with PMA, one should not get any amplification in the reaction tube as dye penetrates the membrane of dead cells and intercalates with the DNA of dead cell.
However, after treating the dead bacteria (heat killed) with PMA and isolating its DNA, I am not getting a significant Ct value difference between live cells and Dead cells by performing real time PCR. It's near about Ct value of live bacteria. I have tried my best to optimize conditions (dye incubation time, light exposure time) but still getting the same results.
What can be the possible reasons of getting amplification in PMA treated dead cells?
Dear Neetika Rani,
did you put negative control also? Are you using filtered tips and laminar hood for to perform the reaction? I suspect the reason is cross contamination.
Second possibility is that the PMA binds covalently only after sufficient expose to visible light. if the exposure is not proper then there would be amplification.
best of luck
Thanks for the reply.
Yes, m including negative control in my experiment and performing all experiments in sterile conditions.
Have repeated the experiments also,,, still the same.
In my case, exposure of treated samples to the visible light source (500W at a distance of 20 cm) is also proper.
So your negative controls or template controls not showing any band??
i am performing molecular beacon based qPCR to detect the target bacteria using PMA in order to discriminate between live and dead.
Not getting any amplification in NTC (No template contol) .
Ct difference between live cells and PMA treated dead cells is only about 2 or less.
performing experiments on biorad icycler.
Hi
I have experience in vPCR methods and their optimization, you can find a review in my profile if you're interested in to understand the key points for the optimization of a vPCR procedure. Moreover, If you can send me more information about Dye concentration, microorganims and amplicon size I'll he able to help you.
But currently I think that there are several critical points
1) dye concentrations / level of dead cells: 50-100 uM for 1E6 dead cells
2) amplicon size...vPCR needs large amplicon ( >300 bp)
3) in some cases buffer is critical, at least use PBS instead SS
4) dark incubation: high but non bactericidal temperature, 10 or more minutes, agitation...
best regards
Jung-Lim Lee and Robert Levin have a number of publications supporting use of EMA as opposed to PMA for this purpose. It might be worth your time to review their studies via Google Scholar.
Depending on the type of bacteria and the sufficient killing of the bacteria this won't materialize. I had the same problem but after some time I realised that the main point was the bacteria I was using and the time and type of light exposure which have made an impact on the qPCR results. Repeat the experiment and check different methods of killing of bacteria. E.g. In the case of MTB, if it has managed to go into the vesicles of amoeba, it can be resistance to some chemical killing for some extent.
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