College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, Shaanxi, China; College of Food Science and Pharmacy, Xinjiang Agricultural University, No. 311, Nongda East Road, Shayibake District, Urumqi, 830052, China.
College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Anal Biochem. 2022 Oct 1;654:114797. doi: 10.1016/j.ab.2022.114797. Epub 2022 Jul 4.
Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template. Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 (E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 μg/μL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 10 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 10 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 10 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.
聚合酶链反应（PCR）是快速监测食源性病原体最常用的方法之一；然而，它需要纯化的核酸作为模板。传统的核酸纯化是一个耗时且费力的过程。为了克服这个问题，我们开发了聚多巴胺纳米球（PDA NPs）辅助的直接 PCR 来检测大肠杆菌 O157:H7（E. coli O157:H7）。PDA NPs 与 PCR 试剂（包括聚合酶和引物）具有强烈的相互作用，极大地提高了 PCR 效率，从而能够调节 PCR 性能。PDA NPs 的最佳浓度和直径分别为 0.10μg/μL 和 504nm。PDA NPs 辅助的直接 PCR 在检测大肠杆菌 O157:H7 方面具有很高的灵敏度。PDA NPs 辅助的直接 PCR 的检测限为 6.7×10 CFU/mL，比直接 PCR（6.7×10 CFU/mL）低 10 倍。此外，该传感器对大肠杆菌 O157:H7 具有出色的选择性，对其他八种常见病原体的反应为阴性。最重要的是，PDA NPs 辅助的直接 PCR 可以检测到牛奶、牛肉和西瓜样品中 10 CFU/mL 数量级的大肠杆菌 O157:H7。整个过程无需进行文化富集，耗时不到 3 小时。因此，PDA NPs 辅助的直接 PCR 在病原体的快速灵敏检测方面具有巨大的潜力。
