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.

Microarray Design and Fabrication. Throughout this manuscript, "probes" are used to mean oligonucleotides immobilized on the solid support, and "targets" are labeled cDNA sequences of the sample virus. Table 1 lists the selected enteric viruses and the number of probes for each strain of virus that were synthesized directly on the FDA_EVIR microarray by photolithography, whereby the total number of probes on the array is 91542. Selected regions of several enteric viral genomes were tiled at two nucleotide spacing, which means each succeeding oligo probe starts from the third nucleotide of the preceding oligo probe. Thus the approximately 91,000 probes can interrogate a total of more than 180,000 nucleotides. Since most enteric viruses have rather small genomes (approx. 7400 nucleotides), we were able to scan the genomes of a number of viruses. We also laid on the array a number of partial sequences in the database, particularly for norovirus. Each probe is 25 nucleotides long and there is a 23 base-pair overlap between consecutive probes for the same virus genotype; therefore, the complete array covers 183,084 nucleotides of viral genomic sequence. The number of probes for each group of virus range from 1113 for rotavirus (RV) group C segment IV to 18736 for norovirus (NV) genogroup II. The large range is due to the number and extent of independent genome sequences available for NV from GenBank. Norovirus genogroup II is represented by the largest number of probes due to a preponderance of partial sequences for these strains in GenBank. Generally, the short size of oligonucleotide probes on the array ensures that their binding to target sequences are sensitively affected by single base mismatches within a test genome that could be detected by a decrease in hybridization signal. Therefore, the tiling array design would allow detection of nucleotide changes by interrogating multiple neighboring probes.