Real time PCR results-interpretation - (Nov/06/2009 )
Hello
Have a question regarding real time PCR in virus detection.
I´ve recently developed a real time PCR system for detection of this particular virus. It works well in cell supernatants and it is specific and sensitive.
The aim was to develop this method to be used in clinical samples as well. I´ve ran serum samples (which were antibody negative at the time) now and only a few of them are positive. And the ones that are have Ct-nr over 36 and only one of the duplicates is positive. It is known that viremia is quite short in this virus infection and the amount of virus in serum may be quite little. Virus isolation has never been succesful in from serum samples but from whole blood it works well.
So, if only one of the duplicates is positive with high Ct-nr, is it correct to conclude that the amount of virus in serum samples is very little? What are the usual reasons for having only one of the duplicates/triplicates etc positive?
Thanks!
Usually when only one of the replicates calls positive and with a high Ct, it means either contamination by other positive samples or that there is really only a trace amount of template, that the distribution in the replicates becomes uneven. I'd would say there is really very little of the virus in the sample.
But when trying to develop a system for virus detection, you should first determine clinical relevance of your findings. That means those that were detected, did have any clinical signs of infection or not? Modern PCR methods can be more sensitive that is really relevant, so you need to set a treshold to say, "Ct this low means a danger to the patient, higher is safe".
Jka83 on Nov 6 2009, 02:26 PM said:
Have a question regarding real time PCR in virus detection.
I´ve recently developed a real time PCR system for detection of this particular virus. It works well in cell supernatants and it is specific and sensitive.
The aim was to develop this method to be used in clinical samples as well. I´ve ran serum samples (which were antibody negative at the time) now and only a few of them are positive. And the ones that are have Ct-nr over 36 and only one of the duplicates is positive. It is known that viremia is quite short in this virus infection and the amount of virus in serum may be quite little. Virus isolation has never been succesful in from serum samples but from whole blood it works well.
So, if only one of the duplicates is positive with high Ct-nr, is it correct to conclude that the amount of virus in serum samples is very little? What are the usual reasons for having only one of the duplicates/triplicates etc positive?
Thanks!
From my experience. i would suggest you to add more template(RNA or DNA) in to your reaction PCR.
But first i would like to know that
1. Does it RNA or DNA virus?
2. How do you extraction?
3. Which type of realtime that you used....sybergreen or probe detection??
If RNA virus and extraction by trizol or kit and use probe detection, you can add more template (about 5ul in total 20 ul)
If RNA virus and extraction by trizol or kit use sybergreen detection,
that mean you have to convert RNA to cDNA.
the problem may be because the mistake with your RT-PCR. (dilute of cDNA about 1:2 is better than undilute)
If DNA virus, I believe you can put more DNA template. And careful with your realtime reaction. what is your realtime reaction????