Genotype ratio with quantitative PCR - (May/11/2005 )
We would like to measure the ratio of two strains of a virus present in our samples using quantitative PCR.
Has anyone had experience with setting up a qPCR with one primer pair and two different probes (a FAM probe and a VIC probe) in the same reaction mixture, and then taking the Ct value of each probe in the sample to form a ratio of the concentration of one virus to the other? Our probes have a 3 basepair difference.
We were planning on running a curve of mixtures of the 2 virus strains' RNA at various proportions, such as 100:0, 90:10, 75:25, 50:50, 25:75, 10:90, 0:100.
Does this sound feasible to get accrurate ratios? Or would it be better to run separate reactions with each probe?
-IHNV-
QUOTE (IHNV @ May 11 2005, 05:49 PM)
We would like to measure the ratio of two strains of a virus present in our samples using quantitative PCR.
Has anyone had experience with setting up a qPCR with one primer pair and two different probes (a FAM probe and a VIC probe) in the same reaction mixture, and then taking the Ct value of each probe in the sample to form a ratio of the concentration of one virus to the other? Our probes have a 3 basepair difference.
We were planning on running a curve of mixtures of the 2 virus strains' RNA at various proportions, such as 100:0, 90:10, 75:25, 50:50, 25:75, 10:90, 0:100.
Does this sound feasible to get accrurate ratios? Or would it be better to run separate reactions with each probe?
Has anyone had experience with setting up a qPCR with one primer pair and two different probes (a FAM probe and a VIC probe) in the same reaction mixture, and then taking the Ct value of each probe in the sample to form a ratio of the concentration of one virus to the other? Our probes have a 3 basepair difference.
We were planning on running a curve of mixtures of the 2 virus strains' RNA at various proportions, such as 100:0, 90:10, 75:25, 50:50, 25:75, 10:90, 0:100.
Does this sound feasible to get accrurate ratios? Or would it be better to run separate reactions with each probe?
This is feasible. Here is a paper using 2 TaqMan Probes with a one base pair difference to detect Factor V Leiden.
http://www.clinchem.org/cgi/content/full/50/4/787
-dobbiewalton-