Weird duplex PCR results - (Feb/11/2006 )
I'm new but you all seem to know your stuff, so I'm hoping you can help. I am performing PCR diagnostics to determine if any viruses are present in shrimp.
I'm trying out a new primer for Reverse Transcriptase-PCR that is specific for a certain virus. It works very well. When I run it against a known negative, it gives negative results, and shows positive with known positives.
Here's the weird part: When I duplex the primer with an internal control (primers specific to a shrimp actin gene to show DNA is present) BOTH appear positive, with very distinct bands in their expected fragment size. Since the sample is a known negative for the virus, it should only show a reaction with the internal control primers.
I verified this by running the virus primers alone against the same samples and got the expected negative results. This doesn't look like a contamination issue. Any suggestions?
What sized bands are you looking for?
Have you run a primer diagnostic program comparing primers against each other (ie: what happens with forward virus vs forward actin or reverse actin etc..)
Is the shrimp genome known? have you tried blasting each primer to the known mRNAs and/or genome?
When you say the actin primers confirm there is DNA, you mean cDNA right? Do the actin primers span an intron etc.?
i assume the sample you are using contains the shrimp DNA only (which you wanted to check with the actin primers)....it looks to me that your shrimp actin primers amplify a second product in the sample of the same size of the expected viral amplicon but it is probably something else....although your actin shrimp primers are supposed to be very good and specific only for actin. run the sample just with the actin primers. on the opposite, your new primers (supposed to be specific for the virus) are amplifying some sequence in the shrimp and giving you a product of approximately same size. blast the new primers against the shrimp genome.
Wow, fast replies!
Pardon the mistake... I did mean cDNA. One gives a 441bp and the other gives a 211bp amplicon. I actually use this internal control set for both DNA and RNA samples, and haven't had a problem yet, even with multiplexing.
I have compared the primers against each other and the viral sequence. Everything good there. I suppose it is possible that the virus primers are reacting with some part of the shrimp genome to give the same size as the virus amplicon, but I haven't run it against the entire genome. I figured that if the virus primers were to react with any part of the shrimp genome then that would appear when I run that set alone, without the internal control (which nothing shows up when it's alone except for the positive control--as expected).
If it were anything I think it would be the internal control reacting with something in the virus genome, but a problem wasn't detected in the program. This sample hasn't tested positive for any other virus.
Now that I'm thinking about it a bit more, I bet if I just sequence these weird bands then I should have a better clue.
Thanks for your suggestions!
I forgot to say that I did enter the new primers in blast and did not have a match with the shrimp genome.
I have also run these samples separate from each other. This is a sample that is virus free. When I run them separately, I get the expected results: negative for the virus and positive for the Shrimp RNA. But when I run them together, positive for both--it's reproducible!
Anyway, like I said, the sequence should give me a better clue to this mystery...
I still welcome comments, if you think you have the answer or any other suggestions for me to try!
actin primers+shrimp DNA=positive ok
new primers+shrimp DNA=negative ok (blast was also negative)
actin primers+new primers+shrimp DNA=2 bands weird
when you duplex do you change any condition (annel. temp; number of cycles; MgCl2 conc; etc) as compared to single run?
the best,as you said, is to sequence the product or adjust the conditions of the run or make sure this sample is shrimp only or redesign the new primers! good luck