Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis

Abstract

We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.

Use of Base Excision Sequence Scanning for Detection of Genetic Variations in St. Louis Encephalitis Virus Isolates

“…CFLP has recently been used for hepatitis C virus genotype determination (14), and seems to be a promising technique, but as for the SSCP and HMA techniques, precise localization of the mutations is not possible. Moreover, the pattern provided presents many bands, and computerized analysis may be required when numerous isolates are investigated.…”

Genetic Methods for Assessing Antimicrobial Resistance

“…One must be cautious, however, in attributing all mutations in these genes to antiretroviral resistance; further clinical studies are required to validate these associations. Although a direct mechanistic link between nucleotide sequences in hepatitis C virus (HCV) genomes and the effect of immunotherapy (interferon) has not been proven, such an association appears to predict response to this therapy (9,10,33,62,83) and so this combination is included in the Table 1. In the subsequent discussion, examples of genetic testing methods which have been developed for these purposes are described.…”

“…(iii) PCR-CFLP analysis. A genetically engineered structure-specific endonuclease, Cleavase I, has been used to detect mutations in the katG (5) and rpoB (20) genes of M. tuberculosis and to determine HCV genotypes (33). The Cleavase I enzyme recognizes the folded structures which DNA strands form after denaturation and cleaves at the junction of the single-stranded and duplexed areas (Fig.…”

Mutational analysis of human p53 gene in human gliomas by CleavaseR fragment length polymorphism

“…CFLP also has more advantages than SSCP; for example, the pattern of objective electrophoresis roughly defines a mutative position of objective DNA. [14][15][16][17][18]…”

“…It also has more advantages such as the fact that the pattern of objective electrophoresis roughly defines a mutative position of a subjective DNA. [14][15][16][17][18] Therefore, in an attempt to analyze genetic mutations present in human glioma cells, we have employed the CFLP method. The principal aim of this study was to evaluate the efficiency and accuracy of CFLP for mutation detection of human glioma tissues.…”