Facing problem in PCR amplification of begomovirus from cotton (Gossypium hirsutum). We sampled typical symptomatic plants and extracted total genomic DNA. Genomic DNA quality and quantity is good. We tried different concentrations of template DNA and range of annealing temperature in PCR profile. But could not get amplification except in positive sample in which a clone plasmid was used as template.
HI, then explain to all of us what exactly your problem is? Cheers, Nadine
Okay, now you modified your question. I guess your plants have no begomovirus you are searching on. The symptoms of your plants could be the result of another virus or some bacteria or a lack of nutrients or... Can you analyse your samples via electron microscope to be sure that it is your special begomovirus?
Cheers, Nadine
I agree with Dr. Gund. It appears your G. hirsutum are free of begomovirus.
The begomovirus is not necessarily absent if it does not amplify in PCR, maybe it is present but just not abundant enough to become visible after PCR, or there could also be some PCR inhibitors present?
I disagree with Maryline. qPCR is not necessarily the solution. It costs way more and other troubleshooting steps can be done before that. Just a small questions, how much more concentrated is your positive control plasmid (in DNA) compared to your samples?
To edit my above answer a little bit:
Maybe the DNA extraction method is not optimal? For my current project I tested seven different DNA extraction protocols, and for each protocol I tried out different "modifications". This resulted in a different yield, but most important, an immense difference in abundance and diversity of specific bacterial groups.
So maybe the extraction method you are using now does not extract the DNA of the begomovirus, or it does extract it, but in very low amount, which makes it invisible after PCR. If you have time and money, other extraction methods are worth a try.
Something I also did several times, is a nested PCR (using the same primers). After a first round of PCR, no bands appeared. However, when doing a new PCR, starting from the previous (cleaned) PCR product, I got a nice band on agarosegel.
As for the range of annealingtemperatures and the PCR device, I noticed several times that there can be a big difference between PCR devices that heat/cool slow (9600 method) and the ones that heat/cool fast (9700 method).
In fact, for all genes I am investigating, I tried out a big range of annealingtemperatures, primer concentrations, DNA concentrations, inhibitor blockers, ... and noticed I could never really trust the previous literature. Sometimes I had to develop a complete new PCR program because the programs used in literature simply did not work. Small modifications can influence your result a lot! Just to give an example: a normal PCR did not work, but when adding a 3x cycle with longer durations of each step at the start of the PCR, my results went from negative to positive.
Dear khadim,
i have faced same problem with CLCuV in cotton, i have extracted DNA by modified CTAB method (Doyle & Doyle 1990) and amplified it but you first check the concentration of DNA template used if its near to positive control then problem with primer or PCR profile, do your work step by step to solve the problem, stay blessed
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