Detection and Identification of Common Food-Borne Viruses with a Tiling Microarray

Chen, Haifeng; Mammel, Mark; Kulka, Mike; Patel, Isha; Jackson, Scott; Goswami, Biswendu B.

Hybridization of an oligonucleotide to target sequence is disrupted by a mismatch; decrease in hybridization in a Figs. (2, 3) , hybridization signal intensities of tiling probes matching the sequence of reference strain HM175 18f were compared. The ratios of hybridizations of the reference strain HM175 18f (perfect matches) to two test strains (HM175 wt and HAS15) were plotted. Peaks indicate decreased hybridization of the test strains due to nucleotide changes. The corresponding sequence alignment shown (Figs. 2B, 3B) validates the peaks of maximum destabilization by identifying the nucleotide changes in HAS 15. In contrast, comparison of HM175 and 18f, which differ by only 28 nucleotides over their entire 7.4 kb genome, show only three areas of nucleotide changes (red line) within the VP1/2A junction. In Fig. (4A, B) , we show that the analysis can be expanded over thousands of nucleotides of multiple viral genomes, where the first 3700 nucleotides of each virus strain were interrogated. Unlike in Figs. (2, 3) , where the ratio of hybridization of each 25mer oligonucleotide probe of a reference strain to a test strain was plotted to detect the location of a mutation, in Fig. (4A, B) , the average signal value of all oligonucleotide probes from a virus strain was plotted against the position of that probe in the array, creating a unique profile for that virus (HAS 15 or PA21) to all other HAV strains we tested (data not shown). The results clearly show that signal strength was highest with multiple peaks in the region where HAV probes are positioned. There was no other area over the entire surface of the array where the target from any HAV strain produced a meaningful signal. This analysis was carried out with all HAV and CXKV strains listed in Table 1 (Supplement) and some norovirus (Fig.1D, E) .