Determination of HCV Genotypes by RFLP

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Abstract

Several different methods have been developed for the typing of HCV variants: direct sequence analysis, slot-blot hybridization analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products using cDNA probes specific to each HCV genotype and PCR amplification using type-specific primers that are designed to match only virus sequences of a defined HCV type and will fail to amplify sequences of other types. An alternative method is the nonselective amplification of virus cDNA using conserved primers followed by restriction fragment length polymorphisms (RFLP) The coding regions of the HCV genome are highly variable, so reliable amplification of different HCV genotypes with the same set of primers is difficult. For this reason, typing methods based on analysis of amplified DNA are more reliable if carried out in the highly conserved 5′ noncoding region (5′ NCR). Different typing assays are discussed in Chapters 14, 16, and 17. The RFLP assay method described in the following section has been developed to enable the detection HCV genotypes 1–6 (1).The advantages that this particular method offers are that it is relatively easy to carry out, the apparatus required is generally available in most laboratories or is inexpensive to buy and a large number of samples can be genotyped at the same time as those being tested by PCR in the laboratory. The major problem associated with the technique is the possible misidentification of novel subtypes of HCV type 6 as type 1 variants (2).