Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis.
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.
Marshall
D J
DJ
Third Wave Technologies, Inc., Madison, Wisconsin 53711, USA.
Heisler
L M
LM
Lyamichev
V
V
Murvine
C
C
Olive
D M
DM
Ehrlich
G D
GD
Neri
B P
BP
de Arruda
M
M
D001483:Base Sequence; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005821:Genetic Techniques; D014644:Genetic Variation; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000453:epidemiology / Q000821:virology; D006801:Humans; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D014481:United States / Q000453:epidemiology
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral