68). The signals in other groups containing genotype-specific probes were close to background (the deviation from Iref did not exceed 0.12). The conclusion was that this HCV sample belonged to genotype 1. Further processing of the groups of selleck elements containing specific probes for genotype 1 subtypes produced the following results. In group 1, the strongest signal was obtained from element 1bd1 (4.98). In group 2 it was 1b2 (3.72), and in group 4 it was 1b4 (2.46). Group 3 contained no elements with positive signals (see corresponding histogram in Fig. Fig.2B).2B). Consequently, this specimen was identified as subtype 1b. Additional examples of fluorescence patterns by hybridization with different HCV samples are shown in Fig. 3A to I.

All the genotype-specific probes hybridized with the corresponding target genotypes without cross-reacting with the other genotypes. Analysis of some samples, for instance, 4d (Fig. (Fig.3G),3G), resulted in cross-hybridization with oligonucleotides specific for subtypes of genotype 2. However, the data processing algorithm uses only the elements with subtype-specific probes of the genotype that was determined in the previous step regardless of the signals in other biochip elements. As a result, the subtypes for most samples were identified unambiguously. FIG. 3. Hybridization patterns obtained using HCV samples belonging to subtype 1a (A), 1b (B), 2a (C), 2i (D), 3a (E), 4a (F), 4d (G), 5a (H), and 6x (I). The groups of elements containing genotype- and subtype-specific oligonucleotides corresponding to the analyzed …

The ability of biochips to identify mixed HCV infections was examined. Specimens having 1a, 1b, 3a, and 4a subtypes were each adjusted to an equal HCV RNA concentration and subsequently mixed at different proportions as follows: 1a + 4a, 1b + 3a. The mixed infections were identified successfully as long as the amount of the minor species was no smaller than 20% (data not shown). The lower concentration of the minor subtype in a mixed sample led to decrease of signals in the corresponding groups of elements, and such a sample was identified as one that contained the dominant subtype only. Analytical sensitivity and specificity. The analytical sensitivity of this method was estimated by assaying 10-fold serial dilutions (with seronegative plasma) of a plasma standard containing 5.2 �� 106 IU/ml of HCV subtype 1b.

Four replicates were used for each dilution. The hybridization results obtained with the 2.0 �� 102-IU/ml concentration of viral RNA were unambiguous. The specificity of the procedure was tested using 24 seronegative plasma samples. All were GSK-3 identified as negative samples. With at least three replicates of each sample analyzed, the deviations of signals of genotype- and subtype-specific elements for identical samples remained within 20% of the average background signal (Iref).