National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
Int J Food Microbiol. 2011 Mar 1;145 Suppl 1:S79-85. doi: 10.1016/j.ijfoodmicro.2010.08.007. Epub 2010 Aug 14.
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.
人类由沙门氏菌引起的细菌性食源性感染被认为是一个至关重要的食品安全问题。因此，在评估沙门氏菌的风险时，考虑不同分离株的基因组变异对于控制病原体引起的感染非常重要。微阵列技术是一种很有前途的诊断工具，可以同时提供许多基因的基因组信息。然而，在将 DNA 微阵列分析用作跨国界和实验室之间鉴定沙门氏菌分离株的常规方法之前，需要对其进行标准化。本研究设计了一项比较研究，评估了用于鉴定沙门氏菌的 DNA 微阵列检测在两个不同实验室之间的数据一致性。该研究旨在揭示在不同实验室使用不同设备获得相同结果的可能性，以便评估微阵列技术的可重复性，作为标准化的第一步。该低密度阵列包含 281 个 57-60 -mer 寡核苷酸探针，用于检测与抗生素抗性、细胞包膜结构、移动遗传元件和致病性相关的广泛特定基因组标记基因。确定了两个实验室之间存在几个差异较大的关键方法学参数。这些参数与打印设施、杂交缓冲液的选择、杂交后使用的洗涤缓冲液以及基因组 DNA 纯化程序的选择有关。在一个四因子实验中对关键参数进行了随机化，并基于 Kappa 系数对实验室间一致性和一致性的统计度量进行了分析。即使使用不同的打印和杂交设施、不同的基因组 DNA 纯化程序和不同的洗涤缓冲液，微阵列结果也具有很高的一致性（Kappa=0.7-1.0）。然而，当使用不同的杂交缓冲液时，微阵列结果的一致性较低（Kappa=0.2-0.6），这表明该参数在将标准微阵列检测从一个实验室转移到另一个实验室时非常关键。总之，这项研究表明，DNA 微阵列检测可以在至少两个不同的设施中重现，这是制定标准指南的前提条件。
