Li Yong, Mustapha Azlin
Department of Food Science, 256 WCS Wing, Eckles Hall, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
J Food Prot. 2004 Jan;67(1):27-33. doi: 10.4315/0362-028x-67.1.27.
With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.
利用三对引物，建立了一种多重PCR检测方法，用于同时检测大肠杆菌O157:H7、沙门氏菌和志贺氏菌。在优化条件下，该检测方法分别从大肠杆菌O157:H7扩增出252 bp的产物，从鼠伤寒沙门氏菌扩增出429 bp的产物，从福氏志贺氏菌扩增出620 bp的产物。当将多种目标微生物的DNA提取包含在同一反应中时，可观察到两个或三个不同大小的相应扩增子。在特异性试验中，10株大肠杆菌O157:H7菌株和1株大肠杆菌O157:NM菌株显示出预期的252 bp扩增子。其他七株大肠杆菌菌株未产生信号。此外，从覆盖16个血清型的20株沙门氏菌中产生了429 bp的扩增子，而从覆盖4个种的11株志贺氏菌中产生了620 bp的扩增子。用其他48株细菌的DNA未观察到非特异性扩增。经过24小时的增菌后，所建立的检测方法能够同时检测苹果酒、哈密瓜、生菜、番茄和西瓜中初始接种水平约为8×10⁻¹CFU/g（或CFU/ml）以及苜蓿芽中8×10¹CFU/g的这三种病原体。整个过程可在30小时内轻松完成。该多重PCR检测方法有可能成为一种简单、快速且高效的工具，用于对苹果酒和农产品进行推测性同时筛查，以检测是否受到大肠杆菌O157:H7、沙门氏菌和/或志贺氏菌的污染。
