ANALYSIS OF THE GENOMA ALMOST COMPLETE OF THE HCV DRUG RESISTANCE.

摘要

An analysis to identify a mutation in the genome of an HCV present in a sample, the analysis comprising the successive stages of: a) extraction of viral RNA from the sample containing the HCV; b) determination of the genotype and subtype of HCV; c) synthesis of the partial cDNAs of the HCV genome in three separate reverse transcription reactions, the first reverse transcription reaction is initiated from a first external antisense primer selected to specifically hybridize with a 3 'UTR sequence of a genome HCV prototype of the same genotype and subtype, the second reverse transcription reaction is initiated from a second external antisense primer selected to specifically hybridize with a sequence of the NS4B-NS5A region of the prototype HCV genome and the third transcription reaction Reverse is initiated from a third external antisense primer selected to specifically hybridize with a sequence of the NS2 region of the prototype HCV genome; d) synthesis of the second strand and amplification of the partial cDNAs of step c) in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction, the first external antisense primer and a first primer external effector selected to specifically hybridize with a complementary sequence of the NS4B-NS5A region of the prototype HCV genome, the second PCR reaction comprising an aliquot of the second reverse transcription reaction, the second external antisense primer and a second external effector primer selected to specifically hybridize with a complementary sequence of the NS2 region of the prototype HCV genome, and the third PCR reaction comprises an aliquot of the third reverse transcription reaction, the third external antisense primer and a third external effector primer selected to specifically hybridize with a sequence co Complementary to the 5 'UTR region of the prototype HCV genome, where the second external antisense primer hybridizes with a sequence of the NS4B-NS5A region of the prototype HCV genome that is located 3' from the region that is complementary to the first primer external effector and where the third external antisense primer hybridizes with a sequence of the NS2 region of the prototype HCV genome that is located 3 'with respect to the region that is complementary to the second external effector primer; e) further amplification of the partial cDNAs of step d) in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and the first internal antisense and effector primers, the second reaction comprising PCR nested an aliquot of the second PCR reaction and the second internal antisense and effector primers, and the third PCR reaction nested an aliquot of the third PCR reaction and the third internal antisense and effector primers, where the internal primers are not overlap with the external primers, the second internal antisense primer hybridizes with a sequence of the NS4B-NS5A region of the prototype HCV genome that is located 3 'from the region that is complementary to the first internal effector primer and the third internal antisense primer hybridizes with a sequence of the NS2 region of the prototype HCV genome that is located 3 ' with respect to the region that is complementary to the second internal effector primer; f) sequence analysis of the additionally amplified cDNAs of step e); and g) comparison of the sequences obtained from step f) with that of the prototype HCV.