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

Molecular methods based on viral RNA amplification by RT-PCR have evolved as rapid alternatives to cell culture for the detection and identification of viral strains [3] . For *Address correspondence to this author at the Division of Molecular Biology, Office of Applied Research and Safety Assessment (OARSA), Food and Drug Administration, 8301 Muirkirk Road, HFS-025, Laurel, MD, 20708, USA; Tel: [phone]; E-mail: [e-mail] example, the differential identification of strains within a species is possible based on the difference in the size of the amplified PCR product (amplicon) detectable by gel electrophoresis ( [4] or single-strand conformational polymorphism (SSCP) [5] . Indeed, we have utilized agarose gel electrophoresis following RT-PCR using primer pairs straddling a 14 base insertion at the non-coding region of some HAV genomes to identify specific cytopathic strains from noncytopathic strains of HAV [4, 6] . We also reported the use of SSCP analysis following Alu 1 or Hinf 1 digestion of amplicons generated from the 3' end of the viral genome to provide differential identification of multiple HAV strains [5] . However, SSCP is a multi-step procedure involving radiolabeling of restriction fragments prior to electrophoretic separation of individual DNA strands. Consequently, this procedure works best when the restriction fragments are small enough to provide sufficient single-stranded DNA separation for effective strain identification. For genetically wellconserved viruses such as HAV, the region to be amplified for SSCP analysis has to be carefully chosen in order to represent areas of reasonable diversity [7, 8] . Due to these considerations, it has been preferable to sequence the PCR amplified DNA fragment in order to specifically identify the genotypes or strains of the viruses. While sequencing amplified PCR products is considered a precise technique for identification, PCR amplification of a mixed population of target sequences may be biased in favor of a dominant (by copy number) target such that subsequent sequence analysis may not reveal the presence of other closely related target sequences in starting populations. Putative mixed virus populations (e.g. of the same or different species) can exist in isolates obtained from environmental and infected-host samples particularly those resulting from RNA virus replication that is known to generate a sub-population of "quasi-species" [9] . Therefore, a threshold number of RNA molecules must have the same specific mutation in order to be unambiguously detectable by RT-PCR and sequencing, due to possible inhibition of amplification of a less abundant template by template competition [10] . Conversely, the dominant mutation present in a population may be preferentially amplified, and therefore, sequence analysis would represent the dominant mutant [11] . Therefore, while sequencing remains a "goldstandard" for target sequence identification, the identification of multiple viral species or tracking species mutations necessitated the development and application of a broader approach to identification prior to undertaking sequence analysis.