Johnston Lynette M, Elhanafi Driss, Drake Maryanne, Jaykus Lee-Ann
Department of Food Science, College of Life Science and Agriculture, North Carolina State University, Raleigh, North Carolina 27695-7624, USA.
J Food Prot. 2005 Nov;68(11):2256-63. doi: 10.4315/0362-028x-68.11.2256.
The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.
美国食品药品监督管理局认识到生豆芽是食源性疾病的一个重要病因，目前建议对用过的灌溉水或最终产品进行沙门氏菌和大肠杆菌O157:H7检测，以此确保供食用产品的安全性。为了简化此类检测工作，研究了一种从豆芽和用过的灌溉水中预富集病原体的简单方法，以便利用聚合酶链反应（PCR）技术直接（无需事先进行培养富集）检测病原体。分别以10⁻¹至10⁶CFU/g或CFU/ml的浓度将肠炎沙门氏菌鼠伤寒血清型和大肠杆菌O157:H7接种到苜蓿芽和用过的灌溉水中。将样品匀浆（仅针对豆芽），然后高速离心以使总细菌群体沉淀。对沉淀物进行处理以分离DNA、进行PCR扩增，并通过Southern杂交确认扩增子。两种基质中两种病原体离心后的平均病原体回收率在96%至99%之间。使用针对鼠伤寒沙门氏菌invA基因和大肠杆菌O157:H7 stx基因的引物，能够在接种浓度低至10 CFU/g的苜蓿芽中检测到这两种病原体。用过的灌溉水中两种病原体的PCR检测限均为10⁻¹CFU/ml，相当于每升水100 CFU。由于用过的灌溉水成分简单，特别适合通过简单的离心步骤进行细菌浓缩。在本研究中，朝着开发一种快速、廉价且灵敏的检测与豆芽相关病原体的方法取得了进展，该方法与当前的行业实践和需求相关。
