Use of a simple polymerase chain reaction combined with a heteroduplex mobility assay to characterize the non coding 5 ' region of hepatitis C virus

Background: One of the most used methods for the characterization of hepati tis C virus strains is the use of a nested polymerase chain reaction (PCR) with a restriction fragment length polymorphism (RFLP) assay. Sometimes, RF LP results do not differentiate new strains. There are other more complex m ethods and only the sequencing of the PCR fragment allows a correct charact erization of the strain. Aim: To report the detection of hepatitis C virus using a single PCR assay of the 5' non codifying region. Material and metho ds: Thirty five serum samples coming from patients with chronic hepatitis o r blood donors were assayed for hepatitis C virus. Results: The reported me thod increases the PCR sensitivity through the combination of polyacrylamid e gel electrophoresis and silver staining of amplified products. This allow ed the semi quantitative estimation of viral load and the characterization of amplified products through their electrophoretic motility. These PCR pro ducts were used in a heteroduplex motility assay, allowing the discriminati on between sequences of different genotypes. Conclusions: Heteroduplex assa ys can be used to characterize the 5' non codifying region of the hepatitis C virus for routine laboratory purposes.