I am testing HBV DNA (unknown copy number) along with HBV quantitative controls 1, 2, 3 and 4, with values 2e7, 2e6, 2e5 and 2e4 IU/ml respectively, using 3 different primer-probe concentrations and got different Ct values. When I used the values obtained for quantitative standards with each of the primer-probe concentrations and extrapolated them to the unknown HBV DNA, the copy number values obtained for unknown HBV DNA were different for each of different primer-probe concentrations. How would I confirm if which copy number is correct for the unknown DNA?
1st primer-probe gave a slope of -3.177, efficiency : 106% and Re2 is 0.99
2nd primer-probe gave a slope of -4.131, efficiency : 74% and Re2 is 0.997
3rd primer-probe gave a slope of -4.239, efficiency : 72% and Re2 is 0.999.
If the 1st results are good then is it the correct primer-probe concentration that I have to proceed further with? Is this the way to optimize the assay?
The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 103 to 109 HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision.
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