A longer step (10-20min.) at 95-100 degrees C might help, it will denature the proteases (in your lysate) which can degrade your Taq poly. These protease and other enzymes are removed in the DNA extraction protocols. Another problem you have is there will be a lot of ions in your lysate (also remove during DNA extraction) that can effect the PCR reaction. A dilution of 1/100 might help if 1/10 is not working, this will lower the conc. of ions coming from your lysate in the PCR rxt, but also lower the amount of viral DNA template. I am assuming your viral genome is DNA and not RNA. Depending how much material your have you may want to try a DNA extraction with a kit, just make sure the kit can isolate both genomic and viral DNA. What you are trying to do is not easy and may take a little bit of trial and error. Also try and call the manf. and see if they have any suggestion, sometimes the companies have really good tech.support. Hope this helps. Randy

If you're using DIFFERENT kits for "normal" and qPCR, you may need to optimize the reaction once more, for qPCR chemicals: Tm, MgCl2, and so on.

A longer step (10-20min.) at 95-100 degrees C might help, it will denature the proteases (in your lysate) which can degrade your Taq poly. These protease and other enzymes are removed in the DNA extraction protocols. Another problem you have is there will be a lot of ions in your lysate (also remove during DNA extraction) that can effect the PCR reaction. A dilution of 1/100 might help if 1/10 is not working, this will lower the conc. of ions coming from your lysate in the PCR rxt, but also lower the amount of viral DNA template. I am assuming your viral genome is DNA and not RNA. Depending how much material your have you may want to try a DNA extraction with a kit, just make sure the kit can isolate both genomic and viral DNA. What you are trying to do is not easy and may take a little bit of trial and error. Also try and call the manf. and see if they have any suggestion, sometimes the companies have really good tech.support. Hope this helps. Randy

Hi Jozef

I also agree with the suggestions above by Marcin and Randy.....since your using different types of templates (purified vs lysate) for analysis...you definitely need to optimize the conditions....meanwhile .apart from proteases...some other components can also inhibit the PCR per se or the DNAses may also degrade the primers...all types of possibilites are there.....in addition to the optimization of the reaction..

if some thing is interfering with your amplification when you use lysate...it can be validated by 'spiking' your purified template with the lysate (the amplification should be inhibited here too)...it will be evident...otherwise it may be 'optimisation' that will be required...

bye

Hi Jozef - For the most part your PCR conditions should be fine since the control works. The only thing you might change would be to increase the cycle number, since your product of interest might be at a low copy number relative to the positive control. To me the major issue sounds like the purity of your test samples. I would not expect a reaction to work the same on a control that is highly pure vs samples that are lysates. Proteins interfere with PCR. I would clean your test samples up with a phenol/chloroform treatment and precipitation (or use a nucleic acid purification kit). Good luck.

I agree with all of the above suggestions, regarding possible sources of PCR inhibition and/or non-amplification. I would add to this list the possibility that the DNA you are using for your standard, being derived from a different source, may actually contain one or more allelic variants in the primer binding domains. This could be occurring naturally within the virus you are assaying, or may be the product of Taq-polymerization error occuring in very early cycles of your test sample PCR amplification.

Also, a strong cautionary note...which Randy alludes to, as well. I think it's important to give very careful consideration to how you are really planning to interpret the data from your qPCR runs, if you are not producing your test samples and your standards using the same protocols/reagents. In our hands, we find that, especially if absolute quantification (target copy numbers per reaction) is your end objective, it's critical to isolate, purify and amplify both your standards and your test samples under identical (as much as acheivable) conditions.

Otherwise, even though you may be able to consistently reproduce statistically-similar results, comparing from dilution to dilution and run to run for a given DNA sample (or sample source), what you will end up with, without shadow of doubt, is consistently inaccurate copy number counts.

It looks like you are performing some kind of diagnostics.

Would you please elaborate on the term "lysate" i.e. what was the tissue source and how was the lysate prepared.

The best I could speculate here that you may try adding proteinase K to the Lysate and try the qPCR again.

Hope this helps?

Josef, failing all of the above, please could you confirm the primer concentrations you are using - they look extremely high. The final conc in the reaction should be 0.1-2uM. Also as SYBR mops up free MgCl2 (the quantitect kit does allow for this) but check, it should be in the range 2-5mM final (3mM usually works for most assays).

Failing that, try dropping your annealing temp (try 58C, then 55C - don't worry about specificity at this point) could be a mismatch in the viral genome compared with your PCR standard (ie incomplete primer binding) or the lysate is throwing the salts conc changing the apparent Tm of your primers.

You could rule out lysate inhibition by doing a dilution series on it (assuming your viral copy number is high enough) - a 1:100 dilution normally rules out sample inhibition.

Good luck,

Mark

Yes the the final concentration of primer pair should be 0.1 to 0.3 uM in reaction you can prepare this by taking 5ul + 45ul NF water of 100uM F and 5ul + 45ul NF water of 100uM R -> 10uM F and 10 uM R

10ul + 90 ul NFW of 10uM F and 10ul + 90 ul NFW of 10uM R -> 1 um F and 1um R

1 ul of 1uM F and 1 ul of 1uM R in a 10ul final reaction ie. (5ul sybr + 2ul primer + 3ul of template ) would give you more chances of getting a good reproducible reaction.

P.S you can scale up the reaction as per you needs.

Jozef - a couple of things I would like to point out before making my suggestions. The first being that I think you meant 50nM - 100nM primer concentration as 50uM - 100uM seems excessively high. You indicate you are using the Qiagen Quantifast qPCR kit. This kit is most effective when used in tandem with the Qiagen FastLane Cell cDNA Kit. The FastLane Cell cDNA Kit extracts gDNA from lysed cells, so the starting material for the Quantifast qPCR kit is actually not crude cell lysate but a more refined gDNA (http://www.qiagen.com/literature/qiagennews/weeklyarticle/08_09/e24/default.aspx).

I agree with my other colleagues above that qPCR inhibitory agents inherent within your unknown samples are most likely the cause of your reaction failures. A quick way to confirm this would be to spike your unknown templates with your standard and see if you are able to detect the standard (I am assuming this is not a multiplex reaction) in the presence of the inhibitory agents of your unknown sample. If this proves to be the case, I would implement one of the cheaper purification options mentioned above. I agree 100% with Randy and Larry in that you may need to setup your standards in a way that mimics the experimental conditions of your unknown sample. I hope this helps. Happy hunting!

Josef, do you sure about presence of the virus in your sample?may be the copy number of the virus is too low (below LOD of the assay).

How much of the 50uM primers stock you added in the reactions?what was the volume of your reactions?

As a standard amount, the final concentration should be between 50 to 200 nM.

for example in the following protocol the final concentration of the primers is 200nM.

Component Volume Per Reaction (µl)

Supermix (2 x) 10.000

10 µM Forward Primer (SYBR_target_1) 0.400

10 µM Reverse Primer (SYBR_target_1) 0.400

Template (1.00)

Water 8.200

Total Volume (Excluding Template) 19.000

If you can do, perform a routine DNA purification on the lysate that may contain PCR inhibitors.

Milad's question brings me to think about something else...if you've not done so, I would suggest re-quantifying both your standard DNA and your sample DNA specimens, using a method specific for dsDNA, such as PicoGreen or Hoescht Dye 33258. A260/A280/A320 (such as with NanoDrop) determination of [DNA] is greivously unreliable, particularly when you have high sample contaminations...your actual virus [DNA] could be much, much lower than you've measured, if using spectrophotometry alone. We recommend using dsDNA-specific concentration measurements for all qPCR work...

Sorry, i am not majoyed in biology, but in organic chemistry.

my recommend using dsDNA-specific concentration measurements for all qPCR work, and also try with normal mix for qPCR?

Jozef.. when we try with lysate for PCR amplification on qPCR there are chances that SYBR green dye is reacting with some other compound in ur lysate and not binding with ur DNA prep. I suggest you increase EDTA concentration to at least 50mM for preparing your lysate to stop any inhibitors present. Also you make sure to stop enzymatic reactions( especially lysosomal) which are active after lysis. Crude lysate will give good amplification in normal PCR but may not amplify in qPCR...As mentioned earlier it may not be problem with PCR optimization.. If you can, try purifying your lysate to remove as many contaminants as possible...

Good Luck..!

Completely agree with those who suggest to purify your unknown. If I were you I would first take the purification suggestion and then if it still does not work look into template conc., annealing temp etc...Or if you have enough sample you could try multiple things in parallel.

Thank you all so very much, you have all given here useful information, and I think that as most of you have pointed out, I need to perform a clean up of my unknowns. The problem is that I have around 600 samples that I need to do this with. I guess there is no other way around this, and I just have to take the chance that I will loose some virus DNA in the process. The experiment I did is virus competition over infection of a marine algal species, wehre I had cultures infected by virus A then cultures infected by virus B and finally cultures infected with both. Each virus has a gene I targeted that is not present in the other, so to answer the question, yes I am interested not in wxpression that much but in copy numbers as an indicator of competition between the two for infection. I combine this with flow cytometry data but there is no way to quantify the two viruses and distinguish them by FC so this is why I am doing the qPCR. Is there a crude fast easy phenol protocol that you can suggest? I have several protocols that give good results that I have used before to isolate the DNA for full genome sequencing but they all take a lot of time and I have only 4 weeks left before I start writing up my thesis. This is the last experiment. Any 96 well plate protocols?

A cheap, quick and easy way to get somewhat cleaner DNA is to use Chelex 100. Google to find a protocol. Test it and adapt it to plate format. I have done it on easier types of sample material (genomic DNA from blood) and it works nice. Not as clean as Boom Silica, but a lot cheaper. Good luck!

Hello Jozef,

I think that lysate contains PCR inhibitors and you have to chooce a different method of DNA isolation. You did not mention what type of material you use for isolation. Sometimes certain sample (tissue) contains unidentified inhibitors for PCR. Maybe you should dilute your DNA before amplification,

Good luck,

Jozef you can try a modified CTAB method, I have used this to screen virus presence in Banana samples. I have not proceeded with qPCR but i get good amount and high purity DNA. I have used it on more than 100 samples with good consistency. Its is based on fungi but i have used it well on plant, animal and bacterial tissues with best yield in bacteria.

"Modified Simple Protocol for Efficient Fungal DNA Extraction Highly

Suitable for PCR Based Molecular Methods"

Link- idosi.org/gjms/gjms5(1)/6.pdf

I hope it does the work you.

Good luck

Thanks a lot to everyone, I did some DNA extractions by using a phenol/chloroform method as most of you suggested. The good news is that I finaly got amplification. The bad news is that I must have lotst a lot of DNA during the extraction method, as the DNA started amplifying in much later cycles than my standards did and is now bellow the levels of accurate detection. Although I now have nice curves I dont know how relaible they are becusae for some reason my smaller dilutions of the standards are all over the place and do not seem to be very accurate (even after several repetitions). I also get amplification of the NTC towards the end of the cycles.

Try changing annealing temperature. Also you are using crude extract and that might be interfering with the primers and your target. Why not to try to purify the DNA and give a try to amplify your target region.

All the best.

A quick suggestion for you DNA isolation. We use the Epicentre Masterpure kit (you can also look up the references and do a home-made version). We get good, consistent yields of both viral and cellular DNA with a fairly quick method- good for large numbers of samples. In addition, the large cytomegaloviral DNA is intact enough to transfect into cells and get virus out- not true for many of the other DNA isolation methods/ kits that are out there. We routinely use this method to extract DNA for qPCR, and get good results. If you do this with transfected cells you will also get plasmid DNA- so if you are working with a small DNA virus it should also work.

Yeah you can use any good kit for the extraction purposes. I use Quiagen ;)

Likely, DNA isolation protocols used for detecting DNA in stools works well for your samples.There are several protocols most of them are very easy to perform.

See the folwing reference,

Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalis in stool specimens.

Nantavisai K, Mungthin M, Tan-ariya P, Rangsin R, Naaglor T, Leelayoova S.

J Clin Microbiol. 2007 Feb;45(2):581-3. Epub 2006 Nov 22.

Alternatively you can use the Quantum Prep® Gel Slice from BIORAD, simply mixing the algae extract with the 10ul of DNA binding beads, whasing and then eluting the DNA, using the aforementioned kit.

I also have the same opinion with the suggestions above, since your using different types of templates for analysis. you definitely want to optimize the conditions, mean while apart from proteases...some other mechanism can also inhibit the PCR per se or the DNAs may also degrade the primers...all types of potential are there.....in addition to the optimization of the reaction

Dear all, thank you very much for your comments, its great to have such a place on the Internet where one can get advice from the rest of the scientific community, and not only advice, but a good one that can be applied. I can now say that after extracting my DNA from the samples and purifying it I managed to amplify it on the qPCR and it was coparable to the standard. So it appears that your suggestions were of great help. I might even aknowledge you all in a paper if I get that far. Cheers. J.

Try FTA cards. Good for chicken and swine clinical samples. ILTV (dsDNA) CAV(ss-DNA) PCV2 (ssDNA)