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.

All microarrays used in this study were customer ordered to be fabricated by Affymetrix Inc. (Santa Clara, CA) using a photolithographic array synthesis technology [19] . The tiling microarray was designed to detect common food-borne and fecally transmitted viruses including hepatitis A virus, norovirus, sapovirus, coxsackievirus, astrovirus, rotavirus, as well as hepatitis E virus. All viral genetic sequence data were obtained from the GenBank database of viral genomes. The tiling microarray chips were composed of overlapping 25mer oligonucleotides with a 2-nucleotide spacing whose sequences were derived from the 5' end viral genomes (3,700 nucleotides) of each virus except rotavirus. Sequence representation for this virus was derived from individual genome segments and ranged from approximately 2,300 to 3,300 nucleotides. Multiple sequences of partial genomes were also used as in the case, for example, of the HAV VP1-P2A junction. The genogroup (or genotype, serotype, group or partial genome) identification and the number of oligomer probes representing that particular group of genomes on the array is given in Table 1 . Microarray Hybridization, Scanning, and Data Analysis. Microarray hybridization was performed using the Affymetrix protocol. Briefly, biotinylated cDNA in the presence of Affymetrix hybridization buffer was hybridized to the microarray chip in a total volume of 120 l. Before application to the array, the samples were heated to 98 °C for 1 min, cooled at 45 °C for another 5 min, and centrifuged at 12,000 x g for 5 min. The microarray chip was then incubated at 45 °C for 16 hrs in a hybridization oven. Following hybridization, the wash and stain procedures were carried out by the Fluidics station (Affymetrix, CA). All arrays were imaged by using Affymetrix microarray scanner at a resolution of 10 m per pixel. Signal intensity of the hybridization was extracted by using Affymetrix power tools, and the subsequent data analysis was performed using MS Excel. For each viral genome represented on the array, the average signal intensity for all the probes within that genome was determined. The average intensity is first determined as described by Jackson et al. [20] , and Ayodeji et al. [15] . Each average genome intensity was then normalized by the average intensity of all the probes represented on the array. To minimize effects of nonspecific hybridization, an empirical cutoff value of 3.0 was considered as a threshold value for a positive signal. Microarray hybridization data were then converted to color visualization schemes in which hybridization signal intensity is reflected by the color scale of vertical strips.