Establishment of a Droplet Digital PCR System for the Detection of Diarrheagenic Escherichia coli in Ready-to-Eat Fruit and Vegetable Products

The traditional test for diarrheagenic Escherichia coli is time-consuming. The droplet digital PCR system established by constructing plasmid DNA standard candidates containing the characteristic genes of diarrheagenic Escherichia coli bfpB, ipaH, and stp achieved rapid detection. After the optimisation of primer probe concentration and annealing temperature by droplet digital PCR, the linear correlation and precision of the method were assessed using gradient-diluted plasmid DNA as template, and the specificity of the method was verified by adding non-target genes invE and sth. The ddPCR method was used for the actual detection of ready-to-eat fruit and vegetable products. The results showed that the optimal primer final concentration of bfpB, ipaH, and stp plasmid DNA was 400 nmol/L, and the optimal probe final concentrations were 200, 100, and 200 nmol/L, respectively; the optimal annealing temperatures of bfpB, ipaH, and stp plasmid DNA were 60, 58, and 58 °C, respectively. Moreover, the characteristic genes bfpB, ipaH and stp were linearly correlated in the concentration ranges of 8.87~38700.00, 3.33~36533.33, and 6.57~61833.33 copies/μL, respectively, with the linear correlation coefficients of the concentration gradients all greater than 0.99. The limits of quantification (LOQ) for bfpB, ipaH and stp genes were 15.33, 7.97 and 15.00 copies/μL, respectively, and the limits of detection (LOD) for bfpB, ipaH and stp genes were 8.87, 3.33 and 6.57 copies/μL, respectively. Additionally, the precision was less than 5% and the specificity was good. The results of the actual sample tests showed that the non-spiked samples were not detected, and the spiked samples and positive controls could be detected, and the results of the ddPCR method were consistent with the international standard method. The established method can greatly reduce the detection time of diarrheagenic Escherichia coli and provide a technical reference for the rapid detection of diarrheagenic Escherichia coli in food.