When two or more elements within the same group had signals differing from one another by less than 1.5-fold, then all such signals Sixj were selected as positive. As a result, a set of elements from the various groups ix1, iy1, ix2, iz2, ix3, etc., were detected whose signals were at least 1.5 times the rest of the signals in their groups. If the number of elements homologous http://www.selleckchem.com/products/kpt-330.html to one subtype in such a set, for example, ix1 and ix3, or ix1 and ixy2, exceeded the number of elements corresponding to other subtypes by at least 1, the conclusion was that the analyzed specimen belonged to subtype x of genotype i. When the elements of different groups in the set so obtained corresponded to a different subtype, for example, ix1, iy2, and iz3, or ix1 and iyz3, the sets of signals corresponding to individual subtypes were compared to each other.

If the signal of an element corresponding to subtype x in one group was 3 or more times stronger than the strongest signals from the groups corresponding to other subtypes, the conclusion was that the analyzed specimen belonged to subtype x. If the ratio of signals Six1/Siy2 was 3 or less, we concluded that the subtype could not be determined. Similarly, if the probe specific for two subtypes was the strongest signal in the group, for example, ixy1, and there were no valid signals in other groups of the elements, the conclusion was that the subtype could not be determined. Finally, if the signals of subtype-specific groups of elements did not pass the primary signal filtration relative to Iref, the conclusion was that the subtype of the analyzed specimen could not be determined.

Statistical analysis. The kappa coefficient was measured using Stata SE 9.2 (StataCorp LP, College Station, TX) to evaluate the concordance between the HCV subtypes determined by NS5B sequencing and the NS5B biochip assays. The overall proportions of HCV subtypes determined by NS5B sequencing and the NS5B biochip assays were checked using the chi-squared test. P values of <0.05 were considered significant. RESULTS Determining the genotype/subtype by biochip Entinostat analysis of the NS5B region. HCV genotyping based on analysis of the NS5B region was performed by hybridization on the biochip. The procedure consisted of three steps: (i) RT-PCR to amplify the NS5B region fragment, (ii) asymmetric PCR to obtain fluorescently labeled predominantly single-stranded DNA fragments, and (iii) hybridization of the labeled product on the biochip with gel elements carrying immobilized oligonucleotides. Figure Figure2B2B shows an example of hybridization pattern and distribution of normalized signals of biochip elements resulting from analysis of a subtype 1b sample.