Direct blotting electrophoresis for sequencing and genotyping hepatitis C virus.

ABSTRACT

Many methods have been used to differentiate the hepatitis C virus (HCV) genotypes based on, for example, type specific primers, probes and restriction fragment length polymorphism. However, determination of the nucleotide sequence remains the reference. Therefore, a simple non-radioactive cycle sequencing technique was developed for clinical tests. PCR-amplified products of the 5' non-coding region (from position -274 to -31) were sequenced using a 5' digoxygenin-labeled primer. After denaturation, the samples were loaded on a direct blotting electrophoresis system (GATC 1500). Sequencing products were blotted onto a nylon membrane during the electrophoresis. The DNA fragments were then UV-cross-linked, incubated with phosphatase-labeled anti-digoxygenin antibody and stained with a precipitating substrate. Reading the sequence of six samples were possible within 2 days. In 41 different samples, five different genotypes were found by sequence analysis from position -245 to -69, of which 17 were type 1a, 7 type 1b, 5 type 2a, 8 type 3a, 3 type 4 and 1 type 5. These results agreed with those obtained by reverse hybridization assay. Direct blotting electrophoresis offered a good non-radioactive method of performing clinical sequencing on a medium scale, with a minimum of investment.