Development of new IL28B genotyping method using Invader Plus assay.

IL28B polymorphism is associated with the response to pegylated interferon-α with ribavirin (PEG-IFN-α/RBV) treatment in chronic hepatitis C patients. As a genotyping assay for IL28B single nucleotide polymorphisms (SNPs) in clinical practice, the Invader Plus assay was developed. The accuracy, intra-assay, inter-assay precision, and the limit of detection of the Invader Plus assay were evaluated. Two SNPs (rs8099917 and rs12979860) associated with IL28B were genotyped by the Invader Plus and TaqMan assay in 512 Japanese patients. In comparison with direct sequencing, the Invader Plus assay showed 99% accuracy in rs8099917 and 100% accuracy in rs12979860. Intra-assay and inter-assay precision were sufficient to use in clinical practice and the detection limit was 1ngDNA/assay. Genotyping by rs8099917 showed that 361 (71%), 144 (28%) and seven (1%) of the patients were major homozygous, heterozygous and minor homozygous types, respectively. Five of the 512 cases (1%) had haplotype differences, but none showed differences between the two genotyping methods. For patients with HCV genotype 1, the prevalence of responders in the major homozygous type was 83.3%, and that of non-responders in the minor heterozygous/homozygous type was 72.5%. A convenient IL28B genotyping method using the Invader Plus assay could be useful to predict the treatment outcome in clinical practice.

Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea.