Quantitative analysis of hepatitis C virus RNA by competitive nested polymerase chain reaction

Abstract

Abstract A method that allows the quantitation of hepatitis C virus (HCV) RNA is described. The RNA was extracted from serum samples and reverse transcribed. Target cDNA was then co‐amplified by nested polymerase chain reaction with a known amount of competitive template at various concentrations. Since this internal control DNA uses the same primers as those of the target and is distinguishable from the target cDNA after amplification by size, the initial concentration of the target could be estimated by comparing the intensity of the two bands of amplified DNA fragments. That is, if the starting amount of the cDNA and the competitive template are equal, the intensity of the two bands should also be equal. Using this method the amount of HCV RNA in serum samples obtained from 85 patients with chronic hepatitis type C was determined. There was as much as a 100 000‐fold difference in the levels of HCV RNA from patient to patient. A rapid decrease of HCV RNA in a patient treated with interferon is also described.