School of Agriculture, Food Science, and Veterinary Medicine, UCD Veterinary Sciences Centre, University College Dublin, Dublin, Ireland.
Foodborne Pathog Dis. 2011 Jul;8(7):769-80. doi: 10.1089/fpd.2010.0768. Epub 2011 Mar 7.
A two-step real-time SYBR Green I multiplex polymerase chain reaction (PCR) assay with melting curve analysis was developed for rapid detection of 19 Salmonella serotypes frequently encountered in humans, animals, and animal-associated meat products within the European Union. The first-step single-tube reaction (Multiplex PCR I), consisting of five primer pairs, classified an initial test panel of eight Salmonella serotypes into five groups on the basis of characteristic amplicon melting temperatures produced by each strain. Following designation into groups, two subsequent triplex reactions (Multiplex PCR II-G1 and II-G3) allowed for further identification of five Salmonella serotypes by their melting peak temperatures. Primers for serotype differentiation were designed to target the genes encoding either phase 1 and 2 flagellar antigens fliC and fljB or unique serotype-specific loci. In addition, the assay simultaneously screened for the presence of the ampicilin-amoxicillin, chloramphenicol-florfenicol, streptomycin-spectinomycin, sulfanomides, and tetracycline (ACSSuT)-type multidrug resistance pattern, indicated by the floR gene, and for the Salmonella virulence plasmid encoded by the svp operon in Salmonella serotype Typhimurium. The established multiplex assays were successfully tested on 97 isolates, comprising 37 distinct Salmonella serotypes and 12 non-Salmonella strains. The two-step assay correctly detected 19 of 37 Salmonella serotypes and all non-Salmonella strains produced negative results. Of the 19 serotypes detected in the assays, 7 serotypes, including Salmonella serotypes Ohio, Goldcoast, Livingstone, Kedougou, Enteritidis, Kentucky, ACSSuT-type Salmonella serotype Typhimurium DT104 and DT104b, as well as non-ACSSuT-type Salmonella serotype Typhimurium strains, were definitively identified. The developed multiplex real-time SYBR Green I PCR assay represents a more rapid and reliable method for identification of large numbers of Salmonella serotypes prevalent throughout the European Union than assays using phenotypic serotyping methods.
建立了一种两步实时 SYBR Green I 多重聚合酶链反应（PCR）检测方法，并结合熔解曲线分析，用于快速检测欧盟范围内常见的 19 种人类、动物和动物源性肉产品相关的沙门氏菌血清型。第一步单管反应（Multiplex PCR I）包含 5 对引物，根据每个菌株产生的特征扩增子熔解温度，将初始测试面板中的 8 种沙门氏菌血清型分为 5 组。分组后，进行两个后续的三重反应（Multiplex PCR II-G1 和 II-G3），根据熔解峰温度进一步鉴定 5 种沙门氏菌血清型。血清型分化的引物设计靶向编码 1 相和 2 相鞭毛抗原 fliC 和 fljB 的基因，或独特的血清型特异性基因座。此外，该检测方法还同时筛查了氨苄西林-阿莫西林、氯霉素-氟苯尼考、链霉素-壮观霉素、磺胺类药物和四环素（ACSSuT）型多药耐药模式，该模式由 floR 基因指示，以及沙门氏菌 Typhimurium 中由 svp 操纵子编码的沙门氏菌毒力质粒。建立的多重检测方法成功地检测了 97 株分离株，包括 37 种不同的沙门氏菌血清型和 12 种非沙门氏菌菌株。两步检测法正确检测到 37 种沙门氏菌血清型中的 19 种，所有非沙门氏菌菌株均产生阴性结果。在检测到的 19 种血清型中，包括沙门氏菌血清型 Ohio、Goldcoast、Livingstone、Kedougou、Enteritidis、Kentucky、ACSSuT 型沙门氏菌 Typhimurium DT104 和 DT104b 以及非 ACSSuT 型沙门氏菌 Typhimurium 菌株在内的 7 种血清型被明确鉴定。开发的多重实时 SYBR Green I PCR 检测方法代表了一种比基于表型血清分型方法更快速、可靠的方法，可用于鉴定欧盟范围内流行的大量沙门氏菌血清型。
