Colorimetric microwell plate reverse-hybridization assay for detection and genotyping of hepatitis C virus

Abstract

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.

A phase I study to evaluate safety, pharmacokinetics, and pharmacodynamics of respiratory syncytial virus neutralizing monoclonal antibody MK‐1654 in healthy Japanese adults

“…This finding suggests a lack of PK differences based on race and indicates that subsequent infant studies do not require a dose adjustment between Japanese and non-Japanese infants because there are no major differences in the distribution of body weight by age in children born in Japan 24 compared to those born in the United States. 25 MK-1654 demonstrated linear PK and geometric mean half-life values ranging from 76 to 91 days in healthy Japanese adult men, which is a similar finding to that reported in the non-Japanese cohort (73-88 days). 19 This half-life is approximately four-fold longer as compared to palivizumab in adults.…”

“…After adjusting for the lower body weight observed in Japanese compared to non‐Japanese participants, GMRs suggest that exposures were largely comparable between the two cohorts. This finding suggests a lack of PK differences based on race and indicates that subsequent infant studies do not require a dose adjustment between Japanese and non‐Japanese infants because there are no major differences in the distribution of body weight by age in children born in Japan 24 compared to those born in the United States 25 …”

Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

“…All the experimental microarray-based methods for HCV genotyping use immobilized oligonucleotides from the 5Ј untranslated region of the HCV genome. They can therefore identify only a small number of subtypes (1a, 1b, 2a/2b/2c, 3a, 3b, and 6a), although their determination of genotypes is reported to be almost 100% (6,22,34). In this work, use of probes complementary to subtype-specific sequences of the NS5B region enabled us to identify more than 20 HCV subtypes in the specimens tested.…”