Hepatitis C virus heteroduplex tracking assay : application to genotype determination, quasispecies analysis, and molecular evolution studies

The heteroduplex tracking assay (HTA) is a tool that can be used for determining genotype, quasispecies analysis, molecular evolution, and epidemiological studies (1, 2, 3, 4, 5, 6, 7). By hybridizing a labeled, single-stranded DNA probe to colinear, reverse transcriptase (RT) PCR products from a sample of interest, the probe will either form a homoduplex with identical molecules or a heteroduplex with nonidentical sequences. The hybridization products are separated on MDE or polyacrylamide gels and visualized. Delwart et al., the developers of the HTA technique (1,2,7), have previously shown that the migration of heteroduplexes relative to the homoduplex on gels are approximately proportional to the percent nucleotide divergence between two species, and therefore, the genetic distance between two species can be determined. Genetic rearrangements, deletions, and/or insertions can alter the migration of heteroduplexes in a manner that disturbs the direct relationship between relative migration and genetic distance. Typically, heteroduplexes of 0.176–1.8 kb containing >1.4–3% to ~30% nucleotide substitutions, which lack genetic alterations, can be identified as unique species on MDE gels (1,4,6). The number and distribution of unique bands indicates the genetic complexity of viral species in each sample

Hepatitis C Virus Heteroduplex Tracking Assay for Genotype Determination Reveals Diverging Genotype 2 Isolates in Italian Hemodialysis Patients

A heteroduplex tracking assay (HTA) was developed for genetic analyses of the hepatitis C virus (HCV) using single-stranded probes from the core (C)/E1 region. Nucleotide sequencing of reverse transcriptase (RT)-PCR products from 15 Italian dialysis patients confirmed the specificity and accuracy of the HTA genotyping method, which identified 5 of 15 (33.3%) 1b, 7 of 15 (46.7%) 3a, and 3 of 15 (20%) type 2 infections. The genotypes of an additional 12 HCV antibody-positive blood donors from different geographical locations were also in agreement with the genotypes determined by the Inno-LiPA HCV II kit (Innogenetics) and/or restriction fragment length polymorphism (RFLP). Isolates which had between 35 to 40% nucleotide divergence from control subtype 1a, 1b, 2a, 2b, or 3a standards could be typed. Surprisingly, HTA detected one 1b-2 coinfection which was missed by DNA sequencing. Three samples that were designated non-2a or 2b type 2 by HTA were found to be type 2a by both RFLP and direct nucleotide sequencing of the 5′ untranslated region. The genetic distance between patient type 2 and control 2a, 2b, and 2c isolates indicated that a new subtype was present in the population being studied. Serotyping (RIBA serotyping strip immunoblot assay kit) of 23 dialysis patients showed that the genotype could be determined in 6 of 8 (75%) C/E1 RT-PCR-negative and 15 of 23 (65.2%) RT-PCR-positive samples, indicating that the two tests complement each other