nested PCR for low viral load- HBV patient sample - (Jul/02/2014 )
I have some problems with nested PCR for low viral load- HBV patient sample.
Kit is Expand High Fidelity PCR kit from Roche.
Samples is total NA from serum of HBV patient sample, extracted by MagNA Pure 96 system. Viral load is from 105 -109
I got not bad results in high Viral load108 -109 However, the lowers are not detected (Picture 1).
In addition, I got dark smear.
The annealing temp. is 67-68 oC
Please take a look at electrophoresis photos and give me some advice for improve the result.
I also try with gradial annealing temp. from 55-68 oC for medium viral load sample, but it did not improve.
2% DMSO add to reaction mix also did not help (Picture 2)
Thank you so much for your advices.
what´s the expected amplicon size for round 1 and 2, what are the cycling conditions, how much dan are you adding to 1st round?, How much of template you are using in the second round?
Based on your result, I have to say it's really bad.
For my case, Even I don't use nested PCR, the amount of HBV DNA I can detect is as low as 10^4 copies/ml serum.
Why you have to use nested PCR? To increase the sensitivity, I think you should try traditional PCR. It worked to me.
and why the PCR product size is so high? I guess you might to detect mutation later, right? ^-^
My suggestion is:
you should try traditional PCR with optimal PCR condition like high Taq or Mg++ concentratrion, or use more DNA template in the reaction. I used 10ul DNA template for V25 o4 V50. It works all the time.
Redesign primer. I saw your PCR1 has two bands, it is not good.