METHOD FOR DETECTING AND/OR QUANTIFYING MINORITY VARIANTS WITHIN A HETEROGENEOUS VIRAL POPULATION IN A BIOLOGICAL SAMPLE

摘要

The invention concerns a method for detecting and/or quantifying minority variants bearing at least a mutation in a heterogeneous virus population contained in a biological sample. The invention is characterised in that it comprises the following steps: (a) extracting nucleic acids contained in the biological sample; (b) PCR amplification of the nucleic acids from step (a) with a pair of primers bordering a viral nucleic acid sequence possibly bearing the mutation; (c) preparing a standard by PCR amplification with the same pair of primers as at step (b) of a nucleic acid sequence bearing the mutation; (d) real time PCR amplification of the amplification products of step (b) and of the standard of step (c) with a pair of highly preserved primers not discriminating the sequences bearing or not bearing the mutation, and quantifying the number of sequences of viral nucleic acids bearing or not bearing the mutation present in the sample; (e) real time PCR amplification of the amplification products of step (b) and the standard of step (c) with a pair of primers specific of a sequence bearing the mutation, and quantifying the number of viral nucleic acid sequences bearing the mutation present in the sample; (f) comparing the quantifications of steps (d) and (e) to determine the proportion of virus bearing the mutation in the sample.