Failure SYBRGREEN PCR - (Aug/29/2013 )
Hello! I want to know if someone can help me with this.
We have a sample that we ran in a qPCR using Sybrgreen PCR technology. The result was negative. We have positive and negative controls and they worked fine, both in the amplification curve and in the melting curve.
However, based on the story of the patient, we conducted a nested conventional PCR. In this case the result was positive, which corresponds with the clinical evolution of the patient.
We have repetead 3 times the qPCR and always is negative.
I only know that this patient was receiving antimicrobials, so I wanted to know if a possilibity can be that the antimicrobials are inhibiting the real time PCR but not the nested PCR? Can that be possible??
I know that another option is that the primers are failing to detect the DNA fragment (primers sequences are different from the real time PCR and the nested PCR). However these primers are actually for a conserved region, and have been tested in other parts of the world, so I am actually not sure what can be happening.
Any input or idea will be greatly appreciated.
Did you test the qPCR primers yourself (at least gradient PCR) or did you just rely on the fact that they have already been used/published? Because I've already had primers that were published and didn't work for me anyway...
Thanks Tabaluga for your input. Actually I have been using these primers for a while, and this is the first time this has happened. But I guess this sample in particular might have a polymorphism. I have contacted the company to check if there is a way that an inhibition must have happened. Will let you know, thanks!
You can test inhibion of specific sample by running other qPCR assay on it or by adding a validated positive template to the reaction. Other way is to do a serial dilution of a sample, but it can be expected that if in only comes positive in nested PCR the template concentration is possibli quite low.
However inhibition happens to a polymerase enzyme, which is basically same for classic and qPCR. SYBR green chemistry itself may contribute to the inhibition of qPCR sample, but not that much.
Single nucleotide polymorphisms may reduce binding of primers, but usually not that much it would run negative (but I have experience it shifted Ct values over ten cycles, so if the expected Ct is already high, it may seem negative then).
Actually if it's a matter of inhibition it's quite possible you got results from nested PCR not because of increased sensitivity of the nested system, but because you diluted the original template. You can dilute it and see if you get a product in the first round.
So probably the first question is, what are you detecting, gDNA, pathogen, mRNA or what?
And the second would be, if you run this assay before, what would be the expected Ct value for this sample?
Do you use the same mastermix for the nested PCR and qPCR?
Is the nested PCR targeting a different gene or are the primer sets (qPCR and nested) related?
Is the end goal to quantify the amount of organism in your sample or are you just concerned that your qPCR assay may not be as comprehensive as you previously thought?