Journal of Chromatography B ( IF 2.8 ) Pub Date : 2018-06-30 , DOI: 10.1016/j.jchromb.2018.06.057 Luqi Zhu , Yating Zhang , Pingang He , Yan Zhang , Qingjiang Wang
Foodborne bacteria are some of the most important human pathogens and can cause many diseases. In this study, multiplex PCR amplification combined with microchip electrophoresis (MCE) was studied to simultaneously and sensitively detect Staphylococcus aureus, Proteus mirabilis, and Enterobacter sakazakii. In order to simultaneously and accurately detect the aim bacteria, three pairs of primers were specially designed for the multiplex PCR amplification of the target genes of three bacteria, which were the specific genes corresponding to these bacteria respectively. After the DNA fragments of three bacteria were simultaneously extracted, the multiplex PCR amplification was performed by adding the three pairs of specific primers in the mixed DNA fragments solution. The multiplex PCR products of the three food-borne pathogens were analyzed by MCE and the limits of detection of target DNA fragments were 1.2–2.2 ng μL−1, (S/N = 3). The limits of detection of the aim bacteria were calculated as 53 CFU mL−1 for Enterobacter sakazakii, 32 CFU mL−1 for Proteus mirabilis, 28 CFU mL−1 for Staphylococcus aureus, respectively. Satisfactory results were obtained when this method was applied to detect the three foodborne bacteria in milk samples. The experimental results show that this method has the advantages of quickness, less sample consumption, high selectivity and high sensitivity.
中文翻译:
多重PCR扩增策略与微芯片电泳相结合,可同时灵敏地检测三种食源性细菌
食源性细菌是人类最重要的病原体之一,可引起多种疾病。在这项研究中,多重PCR扩增结合微芯片电泳(MCE)进行了研究,以同时,灵敏地检测金黄色葡萄球菌,变形杆菌和阪崎肠杆菌。。为了同时准确地检测出目标细菌,特意设计了三对引物,用于对三种细菌的靶基因分别进行多重PCR扩增,这三种基因分别对应于这些细菌的特异基因。同时提取三种细菌的DNA片段后,通过在混合的DNA片段溶液中添加三对特异性引物进行多重PCR扩增。通过MCE分析了三种食源性病原体的多重PCR产物,目标DNA片段的检出限为1.2–2.2 ngμL -1,(S / N = 3)。检测目标细菌的极限计算为53 CFU毫升-1为阪崎肠杆菌,32 CFU毫升-1对于奇异变形杆菌,金黄色葡萄球菌分别为28 CFU mL -1。当该方法用于检测牛奶样品中的三种食源性细菌时,可获得令人满意的结果。实验结果表明,该方法具有快速,样品消耗少,选择性高,灵敏度高的优点。