The detection and identification of microbial contaminants in food are essential for prevention and investigation of foodborne illnesses. There is increasing demand to develop methods for the rapid and reliable detection of foodborne pathogens. Currently, nucleic acid-based detection remains the method of choice. Microarray analysis, one such nucleic acid-based detection, has been applied to simultaneously detect and genotype multiple foodborne pathogens.
Purpose:
The purpose of this study was to develop a new generation custom DNA microarray for detecting and identifying foodborne pathogens from multiple sources of samples. The new array is aimed to achieve better sensitivity and broader pathogen coverage than the previous tiling array with Affymetrix GeneChip design.
Methods:
The new, high-density peg format design is based on individual gene sets of selected virus strain containing a central nucleotide mismatch for paired-complimentary probe set. In addition to common foodborne viruses, the design of the new array specifically increased the sequence density for human norovirus and selected surrogates detection, as well as internal (RNA) controls for food processing.
Hepatitis A virus (HAV) strain HM175/18f and norovirus (NoV) strain MD145 are used as viral targets for protocol optimization and performance evaluation. Viral RNA is either extracted from culture supernatant or synthesized by in vitro transcription. Microarray analysis is performed following the modified Affymetrix GeneAtlas protocol.
Results:
The new microarray can detect and identify HAV HM175/18f as a subtype IB strain and NoV MD145 as GII.4 genogroup. Multiple approaches to data analysis are being assessed. Assay specificity was confirmed by the complete absence of cross reactivity observed between and/or among unrelated viral species.
Significance:
We demonstrate the development and application of a new custom DNA microarray for detection of foodborne pathogens. This method is being developed in order to address multi-virus detection and identification in FDA surveillance and outbreak investigations.