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

lymerase (Exo -). Labeled products were purified by spin column chromatography, and concentrated by centrifugation through Microcon ® (Millipore, Billerica, MA) filters. Biotinlabeled DNA was denatured in a total volume of 20 l of hybridization solution containing 5XSSC, 0.1%SDS, 5 g poly A, and 5 g human Cot-1 DNA and 6 l used per hybridization reaction per well of a 12 well sample pod (Nim-bleGen Systems, Inc.). The microarray slide (NimbleGen) was laid on top (oligonucleotide side down) of the sample pod and held in place in a metal cassette provided by the manufacturer. Hybridization was carried out for 12h at 42 °C. The slides were washed sequentially with 2XSSC/0.1%SDS, and 0.1XSSC/0.1%SDS at 42 o C then distilled-deionized water at room temperature. The slides were then stained with a Cy3-streptavidin conjugate (Amersham Biosciences, Piscataway, NJ) as described in Jackson et al. [15] . Data Extraction and Analysis. Hybridized, Cy3-stained microarrays were scanned using an Axon GenePix ® 4200A scanner at 5 m resolution using a 532 nm laser. Fluorescence intensities of each feature (oligonucleotide probe) were extracted utilizing NimbleScan TM software (NimbleGen Systems Inc), and all subsequent data analyses were performed using MS Excel. Data were analyzed independent of comparison to a reference strain assuming that each virus strain is unique. Following normalization for background fluorescence, the fluorescent intensity of each probe (normalized probe intensity) was plotted against the genome position of each probe to generate a hybridization profile for each viral strain [15, 17] . To generate the average probe intensity for each probe set per hybridized virus strain, the sum of all normalized probe intensities for individual probes within a probe set (i.e. set of probes derived from an individual strain or group sequence) was divided by the number of probes within that set [15] .