Kim Tae-Hoon, Hwang Hyun Jin, Kim Jeong Hee
1 Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University , Seoul, Korea.
2 R&D Center , Ahram Biosystems, Inc., Seoul, Korea.
Foodborne Pathog Dis. 2017 Oct;14(10):580-586. doi: 10.1089/fpd.2017.2290. Epub 2017 Jul 11.
Salmonella enterica serovars Enteritidis and Typhimurium are the most common causative agents of human nontyphoidal salmonellosis. The rapid detection and timely treatment of salmonellosis are important to increase the curative ratio and prevent spreading of the disease. In this study, we developed a rapid multiplex convection polymerase chain reaction (PCR) method to detect Salmonella spp. and differentiate Salmonella Enteritidis and Salmonella Typhimurium. We used the invA gene for Salmonella spp. detection. Salmonella Enteritidis-specific primers and Salmonella Typhimurium-specific primers were designed using the insertion element (IE) and spy genes, respectively. The primer set for Salmonella spp. detection clearly detected both Salmonella Enteritidis and Salmonella Typhimurium after a 21-min amplification reaction. Serovar-specific primer sets for Salmonella Enteritidis and Salmonella Typhimurium specifically detected each target species in a 21-min amplification reaction. We were able to detect Salmonella spp. at a single copy level in the singleplex mode. The limits of detection for Salmonella Enteritidis and Salmonella Typhimurium were 30 copies in both the singleplex and multiplex modes. The PCR run time could be reduced to 10.5 min/15 cycles. The multiplex convection PCR method developed in this study could detect the Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium in artificially contaminated milk with as few as 10 colony-forming unit/mL after 4-h enrichment. The PCR assay developed in this study provides a rapid, specific, and sensitive method for the detection of Salmonella spp. and the differentiation of Salmonella Enteritidis and Salmonella Typhimurium.
肠炎沙门氏菌血清型肠炎亚种和鼠伤寒亚种是人类非伤寒型沙门氏菌病最常见的病原体。沙门氏菌病的快速检测和及时治疗对于提高治愈率和预防疾病传播至关重要。在本研究中，我们开发了一种快速多重对流聚合酶链反应（PCR）方法来检测沙门氏菌属，并区分肠炎沙门氏菌和鼠伤寒沙门氏菌。我们使用invA基因检测沙门氏菌属。分别使用插入元件（IE）和spy基因设计了肠炎沙门氏菌特异性引物和鼠伤寒沙门氏菌特异性引物。用于检测沙门氏菌属的引物组在21分钟的扩增反应后能清晰地检测到肠炎沙门氏菌和鼠伤寒沙门氏菌。肠炎沙门氏菌和鼠伤寒沙门氏菌的血清型特异性引物组在21分钟的扩增反应中能特异性地检测到各自的目标菌种。我们能够在单重模式下以单拷贝水平检测沙门氏菌属。肠炎沙门氏菌和鼠伤寒沙门氏菌在单重和多重模式下的检测限均为30拷贝。PCR运行时间可缩短至10.5分钟/15个循环。本研究开发的多重对流PCR方法能够在4小时富集后，检测人工污染牛奶中低至10个菌落形成单位/毫升的沙门氏菌属、肠炎沙门氏菌和鼠伤寒沙门氏菌。本研究开发的PCR检测方法为沙门氏菌属的检测以及肠炎沙门氏菌和鼠伤寒沙门氏菌的区分提供了一种快速、特异且灵敏的方法。
