A sensitive visual DNAzyme-based strategy for Cronobacter sakazakii in PIF by aRPA

Abstract

Cronobacter sakazakii, as a most important foodborne pathogen in powdered infant formula (PIF), can cause diseases with high mortality in infants and young children, and has become a key monitoring target in dairy industry. In this study, a visual detection strategy based on DNAzyme and asymmetry recombinase polymerase amplification (aRPA) was developed for monitoring C. sakazakii in PIF. A large amount of single-stranded DNA (ssDNA) rich of guanine (G) was produced in this process and ssDNA could bend and fold into intramolecular parallel G-quadruplex in the presence of potassium chloride. The complex of G-quadruplex and hemin, described as DNAzyme, has strong peroxidase-like activity and catalyze the reaction of H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB), making the color of solution blue. To our knowledge, this is the first attempt in the detection of C. sakazakii based on aRPA and DNAzyme. The limits of detection (LOD) of this method for C. sakazakii were as low as 2.2 CFU mL−1 in pure culture and 5.4 × 101 CFU g−1 in artificially contaminated PIF, respectively. Besides, good stability of the method was shown in the on-site simulation evaluation, which was realized by only warm water, indicating that such strategy may have bright application prospects for field test without heating equipment.