My lab is using PCR to detect bacteria in environment samples, however, PCR cannot distinguish between dead or live bacteria. I found that some papers reported that ethidium monoazide could help, but it seems no use in my experiment. Shall I use PMA instead of EMA for it?
My experiment setup is like this:
1) 1ml of bacterial culture was heat treated at 72c for 15mins (confirmed no growth in agar)
2) EMA (100mg/ml) added to culture
3) expose to 650W halogen light for 1 min
4) pellet the cell at 5000g x 5mins
5) extract DNA
6) PCR
However, still got a very bright band even I diluted culture to 0.1McF......
Hello Paul,
Do you know if the disease which causes the death of your animals is due to the bacteria itself, or its toxins? Some toxins might outlive the bacteria which produced them for a time, even after heat treatment (like cooking) for example.
If you get positive PCR, even after EMA treatment, but are not able to cultivate your bacterium, it might be that the bacteria present in your soil samples are viable (alive), but nonculturable.
@Stephen: Ethidium (EMA) or Propidium (PMA) MonoAzide are DNA binding dyes which selectively penetrate compromised membrane of dead cells but not intact membrane of viable cells and intercalate into DNA once inside the cell membrane. Upon exposure to intense visible light, the photoreactive azide group on the dye is converted to a highly reactive nitrene radical that cross-links with dead cell DNAs, making them unavailable for subsequent PCR amplification.
From my reading, it seems that PMA was demonstrated to be advantageous over EMA in terms of dead cells exclusivity. Moreover, EMA could be toxic to viable cells, whereas PMA did not show any lethal effect.
I found the paper by [Wang et al, 2009; Journal of Applied Microbiology 107:1719-1728] to be instructive and their protocol well planned and explained.
Hope this might be of some help.
I suggest you firstly try 2 killed cells conditions：95℃，5min or 70%Isopropanol ，10min；
If the follow-up experiment does not work well, I suggest you optimize the concentration of EMA。
Some data indicate that 25μg/mL or higher concentrations of EMA can inhibit DNA amplification of 2.0 × 107 CFU/mL Vibrio parahaemolyticus dead cell.
Personally, live or dead, there is going to be DNA there. A better approach would be to extract RNA (which should only come from live cells as it degrades quickly), made cDNA libraries, and then PCR.
We have the same problem in differentiating viable and dead fungal spores sampled from air. I've not used PMA or EMA but it might be simpler to switch to detecting RNA, which I understand is rapidly degraded and so is only detected in viable cells - but possibly this will be low in dormant cells that are nevertheless still viable. So I too willbe interested in other answers.
A colleague of mine did this exact experiment a few years ago, and so I will pull out the protocol for you when I get to work tomorrow.
I assume it is a metagenome analysis?ie, you want to detect all species that might be present?
In biochemistry (or chemistry) you rarely get a 0 or 1 response. Usually you are always somewhere in between (due to equlibrium constants). Degrading DNA form dead cells works well when you don't have to much. Some DNA will always be left behind, even at low concentrations, but in this case, it will probably be near or under limit of detection of your PCR. If you have high concentrations of extracellular DNA, raising EMA conc might improve your outcome, but I think some DNA will still be left. A similar problem is seen when trying to get rid of genomic DNA in RNA isolations. Usually a compromise must be accepted, e.g. if the amplification before and after EMA treatment differs by at least 6,6 or so cycles, than you can conclude that only 1% of amplification is due to dead cells. Of course, then you have the problem of variation of dead cells between samples and the efficieny of EMA treatment, but that's a whole new story...
some bacteria can heat (Gram-) but not effective much for Gram+. I recommend you heat at 95-100C for 5 if you want to heat and follow by many method for DNA ext. some time live cell cant show the sharp band because cell did not lyse by heating.
I think that this problem cab be solved by using ethidium monoazide and propidium monoazide and I suggest repeat your experiment by this method.
So the basic protocol would go something like....
- extract RNA directly from environmental sample
- reverse transcribe to make cDNA library
- amplify gene of interest using possibly limited cycle PCR (16S would be a good one as there is a lot of resources / databases out there that you can compare to)
the next bit might depend on technologies you have access to / money
option 1 (cheaper)
- clone PCR products into a sequencing vector, eg. pGemTEasy (promega)
- Sanger sequence as many clones as you can afford (>100)
- compare sequences to database (working out how many of the same sequene type will give a rough idea of abundance)
option 2 (more expensive)
- use next generation sequencing to sequence PCR amplicons (16S metagenomics using NGS is pretty big at the moment)
- compare to databases
Does that make sense?
Cheers,
Steve
Hi Steve, thanks for you suggestion, what I actually doing is, a gram negative bacterium that can found in soil and possible cause animal dead in my park. When I sample the soil around the park, I found many positive in PCR but negative in culture. So, I linked up this should be cause by dead or inactive target bacteria in soil, as even the bacterium dead, its DNA can still stable for period of time, and hence I got positive PCR result.
As culture method for this bacterium takes at least 12 days to complete, so I trying to find a possible PCR method to detect viable bacterium.
An alternative hypothesis could be is that you do not have the right culture conditions, and therefore cannot grow it in the lab? I would think it is more likely (obviously without knowing anything about your project), that this is the cause of your results, and that it is probably live (and dead) bacteria you are seeing in the soil, and no bacteria in the culture. That result is not surprising, given that the vast majority of bacteria cannot be cultured in the lab. It is also why culture independent screening methods are important. I am sure you can debunk my theory by telling us that this species can be cultured in the lab...?
What bacteria specie you would like to culture? Even it takes time to culture, the right condition can support bacterial growth. If numerous dead cell DNA contamination , mostly DNA amplification comes from dead cells. You may try to use special selective enrichment media to grow the target bacteria (to increase number) which the DNA of lived cell will enough to amplified and will overcome the dead cell DNA.
Reply Stephen and Narumon, i am doing Burkholderia pseudomallei, my lab is able to recover Burkholderia pseudomallei from soil sample, but it takes at least 10 days to complete. PCR is availableas well, but it cannot distinguish live/dead cell, as dead cell is no use in my situation, When I taking soil sample to perform culture and PCR, PCR is always positive while culture is negative, that mean no viable Burkholderia pseudomallei in soil, and the positive signal on PCR is from dead cell, am my hypothesis able to explain it?
I am not sure that it really makes sense to say that you can only PCR off dead cells...? Do you have a positive control to show that your cultured bacteria are really the species that you think they are? Another problem - if the bacteria in the soil are dead, how did you manage to culture them in the first place....? A quick Wiki search tells me they usually grow up within 24-48 hours... do you know why it takes 10 days? Also, what is the PCR target?
I am not going to PCR off dead cell....... I just want to have treatment before PCR to eliminate false positive from dead cell.
Yes, I have positive control from soil and water sample. Actually we can recover it from rain water in adverse weather.
'if the bacteria in the soil are dead, how did you manage to culture them in the first place' thats a good question, but how could you explain?
We are all research, theoretical is always different from practical, right?
I use type 3 secretion system gene, tat domain protein gene and 16sRNA
@Paul - If they are dead, they wont be able to be cultured. So it is much more likely that when you get a product from soil, then it is from live. However, a good way to solve this would be to follow the RNA route, as you will know it will come from live bacteria.
What is the basis for EMA or PMA in selectively inhibiting live v dead PCR products? All I can see is it is a fluorescent probe... is this correct?
@Jon - RNA would still be a good option for you I would think, but obviously not on the metagenome scale. Sounds interesting though - what are you detecting in your qPCR assay that forecasts plant disease? You mentioned fungal spores...?
Some authors showed that PMA can be applied to a wide range of species across the bacterial kingdom presenting a major advantage over ethidium monoazide (EMA).
Hello Paul,
Do you know if the disease which causes the death of your animals is due to the bacteria itself, or its toxins? Some toxins might outlive the bacteria which produced them for a time, even after heat treatment (like cooking) for example.
If you get positive PCR, even after EMA treatment, but are not able to cultivate your bacterium, it might be that the bacteria present in your soil samples are viable (alive), but nonculturable.
@Stephen: Ethidium (EMA) or Propidium (PMA) MonoAzide are DNA binding dyes which selectively penetrate compromised membrane of dead cells but not intact membrane of viable cells and intercalate into DNA once inside the cell membrane. Upon exposure to intense visible light, the photoreactive azide group on the dye is converted to a highly reactive nitrene radical that cross-links with dead cell DNAs, making them unavailable for subsequent PCR amplification.
From my reading, it seems that PMA was demonstrated to be advantageous over EMA in terms of dead cells exclusivity. Moreover, EMA could be toxic to viable cells, whereas PMA did not show any lethal effect.
I found the paper by [Wang et al, 2009; Journal of Applied Microbiology 107:1719-1728] to be instructive and their protocol well planned and explained.
Hope this might be of some help.
Hi Paul, I recomended you a washing step previous to PCR extraction. Both dyes can be used, there are some publications about the use of one or other depending on the bacterial genus used
Hi paul,
Have you found the exact solution to your problem (getting false positives in PMA based PCR from dead bacteria) as discussed.
I am also dealing with the same problem. Please suggest some possible solutions to overcome this.
This paper could be interesting for you.
http://www.ncbi.nlm.nih.gov/pubmed/22940102
best regards
Dear Paul,
Reading your experiment setup I did not find any incubation step, did you do it? You can extend the photoactivation time, but always controlling the sample temperature. Also you can check if your work temperature damages membrane cells, you can use live/dead kit for that.
Hope this might be of some help
HI Paul
What buffer are you using? pbs ? NaCl? Did you clean the sample with a previous centrifugation and re-suspension in PBS?
Some researchers opine that is very important agitation during dark incubation. And finally is a conventional bacteria or you are working with mycobacteria?
Hi Francesc, I am using 1x commercial BPS for washing and re-suspend cell pellet after centrifugation. Will try to use 0.85% NaCl later on.
Yes, I am using conventional bacteria - Burkholderia pseudomallei, and i do inverted the tube upside down several time during dark incubation.
Hi Paul Lee, I used PMA and it works fairly well. May I know what have you used as a 100% dead sample control for the EMA-PCR? Thanks
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