关键词: 食源性致病菌；大肠杆菌O157；水凝胶；数字化聚合酶链式反应；荧光检测

Abstract: In this study, we developed a direct hydrogel digital polymerase chain reaction (Direct-hdPCR) technique for the detection of Escherichia coli O157 in grape juice. In this method, samples were directly mixed with a hydrogel PCR system, followed by release of bacterial DNA through thermal lysis, and in situ digital PCR amplification of target DNA was achieved by taking advantage of the three-dimensional network structure of the hydrogel material. The amplified products were visualized as fluorescent dots, whose number of dots corresponded to the number of DNA molecules, enabling precise quantitative analysis. PCR primers specific to E. coli O157 were designed, and the developed method was validated for feasibility, sensitivity and specificity and was applied to real samples. The results demonstrated that this method exhibited high sensitivity, with a detection limit as low as the single bacterium level, without any need for DNA extraction or purification. It also showed strong specificity to the target bacterium and excellent anti-inhibition effects, without any need for sample pretreatment. The total detection time, from sample preparation to result reporting, was reduced to less than 40 minutes. Using the Direct-hdPCR method, all contaminated grape juice samples were directly detected with a positive detection rate of 100%, which is consistent with the results obtained by the dilution plating method. The average recoveries ranged from 96.9% to 112.0%, with a relative standard deviation (RSD) of 3.6% to 12.4%. This method offers advantages such as rapidity, high sensitivity, no need for sample pretreatment, and simple operation, making it suitable for on-site testing.

Key words: foodborne pathogens; Escherichia coli O157; hydrogel; digital polymerase chain reaction; fluorescence detection