Optimisation and sensitivity of single-stranded conformation polymorphism for the detection of hepatitis C virus quasi-species

Single-stranded conformation polymorphism (SSCP) is a technique used widely for the detection of differences in DNA sequence based on PCR technology. Developed by geneticists for the detection of mutations causing disease, it has been adopted more recently for the analysis of the quasi-species of vi ral genomes, such as hepatitis C virus (HCV). The rigorous standardisation and determination of the limit of detection of the technique has rarely bee n shown. Variants within the quasi-species of the hypervariable region of H CV were cloned into pUC119 and the resulting plasmids quantitated and used as templates to optimist SSCP. Variables studied included the number of var iants detected, the sensitivity of detection of variants in the minority, t he electrophoresis temperature, methods of generation of single-stranded DN A, effect of numbers of cycles of PCR and use of DNA polymerase with proof- reading ability. It was demonstrated that the optimised method could detect at least five variants within a quasi-species and that variants could be d etected down to a level of 2% of the quasi-species. Electrophoresis at room temperature for 18 h was highly reproducible. Generation of single-strande d DNA using a single primer with Taq polymerase for 20 cycles gave an accur ate reflection of the quasi-species make-up and use of Pfu polymerase reduc ed sensitivity of detection of minor bands. This SSCP method provides an ac curate tool to evaluate HCV quasi-species. (C) 2001 Elsevier Science B.V. A ll rights reserved.