The degenerated LNA-containing fw-primer and the long degenerated dInosine-containing biotinylated rev-primer generated a single band of the correct size, 502 nt, when analyzed by electrophoresis using EtBr-stained agarose gel in all five clinical samples (data not shown). All four microsphere-bound probes detected the consensus synthetic rotavirus target as well as the amplified rotavirus nucleic acid from all five clinical samples. Interestingly, the four probes hybridized almost equally well within each sample. It was found that an asymmetric PCR of the clinical samples was necessary in order to obtain an MFI of reasonable strength from the probes (data not shown). This was probably because the complementary strand of the PCR product outcompeted the probe due to an affinity between the two strands that was higher than that between a dInosine-containing degenerated probe and the target strand. The data in Figure 5B are from one of the experiments using samples run in duplicate. The PCR products were sequenced (Supplementary Table S4 ), revealing that the Ino14_w4 and Ino11_w7 probes, which belonged to the same haplotype, covered the variations in all positions in all samples. However, the other pair of probes (Ino8_w4 and Ino5_w7) had one mismatch against clinical samples 1 and 2 and two mismatches against clinical samples 3, 4 and 5, as well as the consensus synthetic rotavirus target (magenta colored boxes in targets in Figure 5C , Supplementary Table S4 ). In conclusion, the long dInosine-containing degenerated probes worked well as variation-tolerant probes, covering variations, accepting a few mismatches, and still remaining specific (neither of the Rotavirus probes hybridized with the InflA target).