A universal dual-mode hydrogel array based on phage-DNA probe for simultaneous rapid screening and precisely quantitative detection of Escherichia coli O157:H7 in foods by the fluorescent/microfluidic chip electrophoresis methods

Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL-1 by the FL signal and 6 CFU mL-1 by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.