Maynard Christine, Berthiaume Frédéric, Lemarchand Karine, Harel Josée, Payment Pierre, Bayardelle Paul, Masson Luke, Brousseau Roland
National Research Council of Canada, Biotechnology Research Institute, 6100 Ave. Royalmount, Montreal, Quebec, Canada H4P 2R2.
Appl Environ Microbiol. 2005 Dec;71(12):8548-57. doi: 10.1128/AEM.71.12.8548-8557.2005.
A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.
设计了一种小型寡核苷酸微阵列原型，其探针针对废水中常见的几种病原体的通用16S rRNA和cpn60基因。除了这两个靶标外，微阵列中还包含wecE特异性寡核苷酸探针，以增强其在肠杆菌科内的鉴别能力。使用通用PCR引物直接扩增大肠杆菌和鼠伤寒沙门氏菌基因组DNA混合物（二元）、大肠杆菌、鼠伤寒沙门氏菌和小肠结肠炎耶尔森氏菌基因组DNA混合物（三元）或废水总DNA中的16S rRNA、cpn60和wecE基因的可变区。扩增产物用荧光标记并在原型芯片上杂交。对于鼠伤寒沙门氏菌，通过PCR结合微阵列杂交，检测灵敏度估计为总DNA的0.1%（10⁴个鼠伤寒沙门氏菌基因组）左右。通过杂交由针对细菌亚群特异性基因（如wecE基因）的PCR产生的扩增子，而不是通用分类扩增子，可以提高原型的灵敏度。然而，有证据表明，当向测试样品中加入越来越多的不同病原体时，PCR偏差会影响给定病原体的检测限。这些结果证明了使用DNA微阵列检测混合群体中水源性病原体的可行性，但也提出了此类实验中PCR偏差的问题。
