Quiñones Beatriz, Parker Craig T, Janda John M, Miller William G, Mandrell Robert E
U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Produce Safety and Microbiology Research Unit, Albany, CA 94710, USA.
Appl Environ Microbiol. 2007 Jun;73(11):3645-55. doi: 10.1128/AEM.02984-06. Epub 2007 Apr 6.
To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; the signal intensities detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold higher than the background levels. Specific identification of A. butzleri, C. coli, and C. jejuni was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the C. jejuni isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the use of C. jejuni sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of C. jejuni in whole chicken carcass samples. C. jejuni was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.
为了探索利用DNA微阵列检测食品中的病原体，我们制作了DNA寡核苷酸阵列，以同时测定零售鸡肉样本中嗜水气单胞菌和弯曲杆菌的存在情况。选择了针对布氏嗜水气单胞菌以及耐热空肠弯曲菌和结肠弯曲菌中管家基因和毒力相关基因的探针。这些微阵列显示出高度的探针特异性；布氏嗜水气单胞菌、结肠弯曲菌或空肠弯曲菌探针检测到的信号强度比背景水平至少高10倍。无需PCR扩增步骤即可实现对布氏嗜水气单胞菌、结肠弯曲菌和空肠弯曲菌的特异性鉴定。通过采用一种采用膜过滤和选择性培养基的分离方法，在富集之前从全鸡胴体的包装液中回收了空肠弯曲菌分离株。延长富集时间导致了布氏嗜水气单胞菌的分离并增加了空肠弯曲菌的回收率。通过使用针对脂寡糖（LOS）生物合成位点的另一组探针进一步对空肠弯曲菌分离株进行分类。我们的结果表明，大多数空肠弯曲菌分离株可能具有B类、C类或H类LOS。验证实验表明，该DNA微阵列的检测灵敏度阈值约为10,000个空肠弯曲菌细胞。有趣的是，使用空肠弯曲菌序列特异性引物标记基因组DNA提高了该DNA微阵列检测全鸡胴体样本中空肠弯曲菌的灵敏度。在全鸡胴体的包装液和富集肉汤中都能直接高效地检测到空肠弯曲菌。
