Myers K M, Gaba J, Al-Khaldi S F
Division of Microbiological Studies, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD 20740-3855, USA.
Mol Cell Probes. 2006 Apr;20(2):71-80. doi: 10.1016/j.mcp.2005.09.006. Epub 2005 Dec 5.
A DNA microarray chip of four virulence genes and 16S ribosomal DNA gene conserved region among all Gram negative species, including Yersinia, as a positive control was developed and evaluated using 22 Yersinia enterocolitica isolates. Eight different oligonucleotide probes (oligoprobes) with an average size of 22 bp, complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all five genes, and single stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer of all genes. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed specificity of genotyping Y. enterocolitica using multiple microarray-based assays. Final validation of the chip's ability to identify Y. enterocolitica genes from adulterated pasteurized whole milk was confirmed and successful. The limit of chip detection of virulence genes in pasteurized whole milk was found to be 1000 CFU per hybridization.
开发了一种包含四个毒力基因和所有革兰氏阴性菌（包括耶尔森氏菌）中16S核糖体DNA基因保守区域的DNA微阵列芯片作为阳性对照，并使用22株小肠结肠炎耶尔森氏菌分离株进行评估。设计了八个平均长度为22 bp、与每个基因独特序列互补的不同寡核苷酸探针（寡探针），并将其固定在化学修饰载玻片的表面。采用多重PCR同时扩增所有五个基因的DNA靶区域，并在所有基因的一个引物存在下，通过扩增子的引物延伸制备用于微阵列分析的单链DNA（ssDNA）样本。通过将代表微阵列基因特异性寡探针不同样本的荧光标记ssDNA进行杂交来确定小肠结肠炎耶尔森氏菌中基因的存在，并通过PCR进行确认。研究结果表明，使用基于微阵列的多种检测方法对小肠结肠炎耶尔森氏菌进行基因分型具有特异性。芯片从掺假巴氏杀菌全脂牛奶中鉴定小肠结肠炎耶尔森氏菌基因的能力得到最终验证且获得成功。发现芯片对巴氏杀菌全脂牛奶中毒力基因的检测限为每次杂交1000 CFU。
