Real time PCR results-interpretation - (Nov/06/2009 )
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?
The serum preparation may well damage any DNA floating around in solution.
Your results indicate that there is pretty much no virus in the serum. However, it may be incorrect to conclude that there is no/little virus in the serum, have you looked for viral proteins as well? How is the serum prepared? stored? doe it have PCR inhibitors? Did you try to extract DNA or just use serum? could these things affect the results?
Having one replicate positive with a Ct of greater than 36 indicates that there is probably a contamination of that replicate.