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

Different strains of HAV and many enteric viruses show variable sequence diversity [23, 24, 26] . This allows easy identification of a virus at the genotype level by sequencing discrete segments of the viral genome amplified by RT-PCR. Ideally, sequencing should be done on amplicons that are known to have multiple nucleotide differences between strains. However, designing PCR primers that will capture a significant number of members of that group requires significant sequence homology, and therefore, a relatively variable region flanked by conserved regions is needed for sequence based identification. While for some virus groups such as HAV it is relatively easy to find PCR primers that can capture many members, it is much more difficult with CV genomes due to extreme sequence diversity. The length of the amplified region is another constraint for sequence based identification. Sequencing an amplicon larger that 500 bp generally will require designing multiple primers for sequence walking. Although automated sequencing techniques currently available can be used for rapid sequencing of a moderate sized amplicon, the process is still too time consuming to be used on a routine basis where a quick identification is needed. a Detection of (putative) nucleotide differences by array hybridization between clone 1 and 18f was initially based on the hybridization profile in Fig. (6) . b Probe numbers are from Fig. (6) and represent the oligonucleotide probes whose sequence contains nucleotide change(s) between clone 1 and 18f when the clone 1 target sequence is identical to the probe sequence. Nucleotide changes were indentified (grey boxed) based on comparison of the clone 1 and 18f GenBank sequences (accession numbers in Fig. (1) ) used to develop the HAV1Cb group 1 consensus probe set (Fig. 1) . The probe nucleotide range numbering is defined by the 29-mer probe and corrected to 18f nucleotide numbering from Lemon et al. [21] . c The nucleotide change and position between clone 1 and 18f as reported by Lemon et al. [21] . d Probe 120 defines a 26 nucleotide base region of 18f due to the three-base GAT deletion. e This probe identified as a potential peak overlapping with the peak at probe 441. Fig. (7) . Comparison of hybridization profiles of three CV strain targets: average probe intensity. Viral genomic RNAs isolated directly from clarified tissue culture supernatants of infected cells were used for RT followed by PCR amplification and labeling prior to array hybridization as described in Materials and Methods. The hybridization data (normalized probe intensities) were converted to average probe intensities [15] and plotted vs each individual probe set following hybridization with either CVB1 (panel A), CVA3 (panel B) or CVA5 (panel C). The underlined identifies the human enterovirus species (HEA, HEB and HEC) represented by a CV probe set.