Hello,

Try to put some DNA of your control on some samples and you do the PCR. If you have amplifications you can say that in your samples there is no inhibitor of the reaction and for that's I suggest you to review your primers maybe they will not allow you to amplify your strain as you are convinced that the virus is present.

best

Hello.

Use positive and negative amplification controls, use nano drop to control initial DNA quantity, large amount of initial DNA in sample also can inhibit the amplification. If positive control is working that means no problems with primers.

For checking the inhibition you can add positive control to one of your sample and see the results if you see amplification after that it means problems with DNA in your sample no inhibition. If you do not see any amplification you should perform clean up of your sample again to receive more pure DNA.

You can try doing some dilutions of your original samples. If inhibitors are presents their will be diluted and the PCR works.

In addition to previous answers, if you still don’t see results from controls and any amplifications from your samples. You should check amplification temperature and other things relevant to PCR cycle. If control samples work, so your strain does not amplify within the bounds of possibility.

Maybe your primers are degraded, you could try to use new primers.

From your description, it seems your primers are not degraded since positive controls are showing DNA amplification. Try to dilute your sample by 1:10 or 1:50, this usually reduces the amount of inhibtors in your sample and your sample. If this does not work, try to use kits especially those used by forensic scientists eg PowerClean® DNA Clean-Up kit, DNA IQ™ System, Chelex®-100 methods. Of these three I highly recommend PowerClean because it removes quite a number of inhibtors

I think others have mentioned need to dilute your DNA. This is important but also are the primers the right ones? Are they specific or general primers?

Review the primers

If all of the suggestions above fail, I will question the specificity of the primers. There might be a different strain of the virus in the samples.

Sometimes, it may be some changes in ur samples (sequence) which forward or reveverse primer is not binding. So, try new primer set with different sequence of that region.

When i was trying to amplify beta satellite of begomovirus the same was happening to me, positive control was giving amplification but my sample wasn't so I tried gradient PCR and set different annealing temperature and this worked for me.