Quantitative polymerase chain reaction using capillary electrophoresis with laser-induced fluorescence detection: Analysis of duck hepatitis B.
We report an accurate and reproducible DNA quantitation method using the polymerase chain reaction (PCR). The amount of PCR product is monitored after each PCR cycle by capillary electrophoresis. To ensure accurate quantitation, a non-amplified internal standard is added to each PCR-amplified electrophoresis sample to correct for variations in injection volume. Quantitation of the sample is based on the number of cycles necessary to generate a predetermined amount of PCR product. Duck hepatitis B virus genome was used as a model in this study. The genome was quantified with a linear relationship between cycle number and logarithm of sample DNA for amounts of sample DNA between 30 and 3.1 x 10(8) copies ( r(2)>0.999). The relative standard deviation for the corrected capillary electrophoresis signal was 2.7%, while the relative standard deviation for the overall assay was 3.0%. Results from a single-blind study generated a relative error of 1.3%.
D000818:Animals; D004279:DNA, Viral / Q000032:analysis*; D004372:Ducks; D019075:Electrophoresis, Capillary / Q000379:methods*; D015611:Hepatitis B Virus, Duck / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral