Microarray-based genotyping of Salmonella: inter-laboratory evaluation of reproducibility and standardization potential.
Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variation among different isolates in order to control pathogen-induced infections. Microarray technology is a promising diagnostic tool that provides genomic information on many genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a routine method for characterizing Salmonella isolates across borders and laboratories. A comparative study was designed in which the agreement of data from a DNA microarray assay used for typing Salmonella spp. between two different labs was assessed. The study was expected to reveal the possibility of obtaining the same results in different labs using different equipment in order to evaluate the reproducibility of the microarray technique as a first step towards standardization. The low-density array contains 281 57-60-mer oligonucleotide probes for detecting a wide range of specific genomic marker genes associated with antibiotic resistance, cell envelope structures, mobile genetic elements and pathogenicity. Several critical methodology parameters that differed between the two labs were identified. These related to printing facilities, choice of hybridization buffer, wash buffers used following the hybridization and choice of procedure for purifying genomic DNA. 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) in microarray results was obtained even when employing 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 parameter as being highly critical when transferring a standard microarray assay between laboratories. In conclusion, this study indicates that DNA microarray assays can be reproduced in at least two different facilities, which is a pre-requisite for the development of standard guidelines.
Grønlund
Hugo
H
National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
Riber
Leise
L
Vigre
Håkan
H
Löfström
Charlotta
C
Folling
Liselotte
L
Huehn
Stephan
S
Malorny
Burkhard
B
Rådström
Peter
P
Rudi
Knut
K
Hoorfar
Jeffrey
J
D019411:Clinical Laboratory Techniques; D005516:Food Microbiology; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D012475:Salmonella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification