Colorimetric microwell plate reverse-hybridization assay for detection and genotyping of hepatitis C virus.
This study describes a colorimetric method for detecting and genotyping hepatitis C virus (HCV) in which four different oligonucleotide probes are fixed onto microwell plates and hybridized separately with biotinylated PCR amplification products derived from clinical samples. The first probe capable of hybridizing with all seven known HCV genotypes was used for overall detection, and the remaining probes were used to recognize specifically genotypes 1-3. When combined with an improved silica-based RNA extraction method, the sensitivity of the test was 50 IU/mL. Eighty-five of the 86 samples analyzed (98.8%) yielded results in agreement with reference detection methods. The remaining sample was HCV-RNA positive in the COBAS Amplicor qualitative assay, but was negative using the reverse-hybridization method. The usefulness of the new genotyping test was confirmed by comparison with direct sequencing of PCR products: 98% of samples tested (54/55) were in agreement using the two methods (21, 7 and 27 from genotypes 1-3, respectively). The single discrepancy might have been due to a mixed HCV infection. The new method is an alternative to the use of commercially available genotyping kits and should be particularly convenient in developing countries where genotypes 1-3 represent a high proportion of HCV isolates.
Costi
Cintia
C
Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde (CDCT/FEPPS), Porto Alegre, Brazil.
da Silva
Cláudia Maria Dornelles
CM
Da Fré
Nicole Nascimento
NN
Grandi
Tarciana
T
Hamester
Fernanda Irma
FI
Zaha
Arnaldo
A
Niel
Christian
C
Rossetti
Maria Lucia Rosa
ML
D019921:Biotinylation; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic