Hi,
I have been trying to optimize a viability PCR using propidium monoazide (PMA) chemical. The idea is heat killing bacterial samples treating them with PMA and comparing the qRT-PCR results to non heat killed bacterial samples. PMA is supposed to bind to DNA and would inhibit the PCR of the bacteria. Also it is expected that PMA is non-membrane permeable, thus it will bind only to dead and/or membrane disturbed cells.
In reality one would expect less bacterial detection (if any) when the samples were killed compared to non-killed one. However I have been getting exactly the opposite results for almost a month now. I changed the PMA stock, tested different concentration, also tested on different life bacteria concentrations, time of PMA exposure, human error (made a more experienced colleague to perform the experiment) and I have been getting the same results.
Does anyone had problems in working with vPCR before? And if you have suggestions on why I am getting those results and what else I can do to find out where things are getting wrong, if will be very helpful.
Thank you.
Dear Hristina
I have some experience in this field, and I well known that sometimes different experimental artefacts can introduce huge bias during in vitro experiments.
In my recent works with Legionella and Salmonella we are introducing some changes during sample manipulation that increases a lot the vPCR performance. If you can share more detailed information about the whole experiment I'm sure that I can help you. Meanwhile I recommend you a quick read of my last works.
best regards
Hi,
In short all I am trying to do is to validate the assay for Chlamydia trachomatis. In order to do so, I have Infectious Chlamydia stock diluted in media or PBS and I split it in 4. 2 of the aliquots I heat kill at 95C for 15 mins and then treat one of them with PMA as per protocol. The other 2 I don’t heat them and again treat one of them with PMA. Further I just extract the DNA and run qRT-PCR.
The 2 non PMA treated samples have very similar Chlamydia load detect as expected. The problem I am having is that the heat killed, PMA treated sample has much more detected chlamydial DNA compare to the one which is not heat killed and PMA treated. This result doesn’t make sense what so ever. I have addressed every step in my protocol and things don’t change.
Any idea about why this might happen?
P.S. hope this explanation makes sense to you.
Hi
The facts are that
1) The PMA treatment don't work
2) The Killing procedure+ DNA purification protocol release more DNA for PCR than the control ( only DNA purification)
Since Article Viability-PCR shows That NAAT detects a high proportion of D...
demostrates that the vPCR works with this pathogen, several different approaches can be done now1) maybe you have some DNA adherence to tube wall and it's unavailable for PMA. Consider a tube change before photo-ctivation
2) review the cell lysis in your DNA purification protocol
best regards
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA