Towards standardization of microarray-based genotyping of Salmonella

Genotyping is becoming an increasingly important tool to improve risk assessments of Salmonella. DNA microarray technology is a promising diagnostic tool that can provide high resolution genomic profile of many genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a tool in source attribution models for comparable characterization of isolates across laboratories and countries. The reproducibility of data was evaluated for a simple and single-dye DNA microarray (Huehn et al., Appl Environ Microbiol, 2009, 75:1011-1020) for genotyping of Salmonella at two different laboratories. The low-density array contained 281 of 57-60-mer oligonucleotide probes for detecting a wide range of specific genomic markers associated with antibiotic resistance, cell envelope structures, mobile genetic elements and pathogenicity. Several test parameters that differed between the two labs were identified: printing facilities and equipment, choice of hybridization buffer, wash buffers used following hybridization and the choice of procedure for purifying genomic DNA. These critical parameters were randomized in a four-factorial experiment and statistical measures of inter-lab consistency and agreement were performed based on the kappa coefficient. A high level of agreement (kappa = 0.7-1.0) was obtained even when using different printing and hybridization facilities, different procedures for purifying genomic DNA and different wash buffers. However, less agreement (Kappa = 0.2-0.6) between microarray results were observed when using different hybridization buffers, indicating this as the most critical factor for standardization between laboratories. In conclusion, this study indicates that it is possible to set up an international standard for a decentralized and simple-to-implement DNA microarray as part of a pan-European source-attribution model for risk assessment of Salmonella.