Abstract

Rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. However, traditional detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 4–7 days to complete. In this study, we described a high-flux polymerase chain reaction (PCR) method for simultaneous detection of nine targeted genes (rfbE, stx1, stx2, invA, oprI, tlh, trh, tdh, and hlyA) with multiplex strains. The designed primers were highly specific for their respective target gene fragments. As the selected primers follow the principles of similar melting and annealing temperature, all the targeted genes could be detected for one strain with the same PCR program. Combining with 96-well PCR plate, by adding a single different gene to each well in each row, both the ATCC strains (E. coli, Salmonella spp., V. parahaemolyticus, L. monocytogenes, P. aeruginosa, S. aureus) and the clinical strains (E. coli, P. aeruginosa, S. aureus) were simultaneously detected to carry their specific and virulence genes. Therefore, using 96-well PCR plate for PCR amplification might be applied to high-flux sequencing of specific and virulence genes.

中文翻译：

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高通量同时筛选常见食源性病原体及其致病因子

摘要

食源性病原体的快速灵敏检测技术对食品工业至关重要。但是，传统的检测方法依赖于细菌培养和生化测试，该过程通常需要4-7天才能完成。在这项研究中，我们描述了一种高通量聚合酶链反应（PCR）方法，用于同时检测9个靶基因（rfbE，stx1，stx2，invA，oprI，tlh，trh，tdh和hlyA）的多重菌株。设计的引物对其各自的靶基因片段具有高度特异性。由于选择的引物遵循相似的解链和退火温度原理，因此可以使用相同的PCR程序检测一种菌株的所有靶基因。结合96孔PCR板，通过向每行的每个孔中添加单个不同的基因，将两种ATCC菌株（大肠杆菌，沙门氏菌，副溶血性弧菌，单核细胞增生李斯特菌，铜绿假单胞菌，金黄色葡萄球菌）和临床菌株（大肠杆菌，铜绿假单胞菌，金黄色葡萄球菌。同时检测到）携带其特异性和毒力基因。因此，使用96孔PCR板进行PCR扩增可用于高通量测序特异性和高毒力基因。