A Microarray Based Approach for the Identification of Common Foodborne Viruses

Ayodeji, Mobolanle; Kulka, Michael; Jackson, Scott A; Patel, Isha; Mammel, Mark; Cebula, Thomas A; Goswami, Biswendu B

In order to complete this analysis, the two different HM175 strains designated clone 1 and the cytopathic 18f strain were again subjected to hybridization and the total normalized intensities of all probes belonging to the different HAV probe groups were plotted as in Fig. (3) . Again, we found no overall differences in the hybridization profile but rather found peaks of hybridization intensities with the strongest hybridization intensities for the group 1 (HAV1Cb) consensus sequence following calculation of average probe intensity (data not shown). The fold-change in intensity between clone 1 and 18f targets was calculated for each probe in the probe set HAV1Cb. As shown in Fig. (6) , ten well defined peaks were observed over the range of the HAV1Cb probe set and the probe number that corresponds to each peak was identified. It is important to note that due to the initial size of the graphical analysis output, it was necessary to compress the scale of the x-axis (HAV1Cb probe number) in order to fit all data points within a smaller graph. As a result, analysis of the hybridization (signal) values revealed two features not readily discernable on the graph; i) a probable single peak at probe 109 rather than what appears as two adjacent (overlapping) peaks, and ii) a possible second overlapping peak adjacent to probe 441. Since the HAV1Cb probe set (group 1) is a consensus sequence developed from the alignment of seven strains assigned to this group (Fig. 1) , there are nucleotide differences between each group member and the consensus sequence. Plotting the fold-change in intensity between clone 1 and 18f would potentially identify nucleotide sequences in a probe that are identical to clone 1 but not identical to 18f. Indeed, upon comparative analysis of clone1 and 18f amplified target sequences with HAV1Cb probes set sequence synonymous with the target sequences, one would predict a total of 11 peaks to occur by this method of analysis. We then sought to determine whether the "peak" probes contained nucleotide differences that could be mapped to nucleotide differences [e.g. single-nucleotide po- Fig. (5) . Comparison of hybridization profiles for three HAV genotype Ib strain targets. Average signal probe intensities were calculated and plotted following hybridization of targets generated as PCR products from reverse transcription of RNA derived from either in vitro transcribed pHAV/7 (black bar), HAV HM175 clone 1 infected cells (white bar) or clarified supernatant from HAV 18f infected cells (grey bar). lymorphisms (SNPs), deletions, or insertions] that exist between clone 1 and 18f (and the probe set). As shown in Table 5, we were able to conservatively detect 10 out of 11 predicted nucleotide changes in the 18f genome identifiable by this method of analysis. It is important to note that these nucleotide changes represent mutations arising in the 18f virus during its emergence as a cytopathic strain from the HM175 noncytopathic strain which were identified by direct sequencing [21] . These results demonstrate a strong correlation between results obtained by direct sequencing and array hybridization and strongly suggest that tiling arrays can be used to detect nucleotide changes instead of sequencing amplified PCR products over a much longer span of the genome in a single experiment.