Amplification of NS3 and NS5A in hepatitis C virus by the multiplex nested polymerase chain reaction

Resistance‑associated substitutions in patients with hepatitis C virus infection have been shown to decrease the efficacy of direct acting antivirals and lead to treatment failure. Amino acid mutations in the NS3 and NS5A regions have been found to be associated with the treatment failure of NS3 and NS5A inhibitors. A method enabling the detection of these mutations would be useful for identifying patients who are at risk of treatment failure. The standard method used to detect mutations is based on nested polymerase chain reaction and nucleotide and amino acid sequencing. The aim of the present study was to simultaneously amplify NS3 and NS5A of HCV by using the multiplex nested PCR method. A set of primers was designed and optimized specifically for the NS3 and NS5A regions, and the multiplex nested PCR conditions were also optimized. A total of 20 serum samples were processed to evaluate the validity of this method. Both the amplified NS3 and NS5A fragments were detected within 14 of the samples (70%) using optimized multiplex nested PCR. Both specific amplification fragments could be detected in a cDNA dilution as low as 1:8. On the whole, the present study demonstrates that optimized multiplex nested PCR is a simple, rapid and effective method that accomplishes the simultaneous amplification of NS3 and NS5A in HCV.