Naked-Eye Detection of Food-Borne Pathogens Using Multiplex Hyperbranched Rolling Circle Amplification and Magnetic Particles

Abstract

Food safety is a significant public health issue in both developed and developing countries. Previous detection methods struggle to meet the current demands. We have proposed a new way to detect pathogens, allowing detection to be visualized by the naked eye. Using our newly developed assay, when target genes are present in the reaction, corresponding padlock probes form closed-loop molecules. Each reaction tube contains a pair of universal primers for identifying target genes. The ring padlock probes and corresponding universal primers start hyperbranched rolling circle amplification (HRCA) under the action of the polymerase, so as to gain branched chain amplification products, which are irreversibly entangled with magnetic particles to form aggregated magnetic particle clusters, and the detection results are visible to naked eyes. On the contrary, by using linear probes, the clustering of magnetic particles will not be produced. This method was applied to the detection of five food-borne pathogens enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC) and Escherichia coli (E. coli), with detection limits of 1 × 103, 1 × 104, 1 × 103, 1 × 104 and 1 × 102 CFU/mL, respectively. This method can realize multiplex automatic detection of nucleic acid and shows great development potential in the field of molecular diagnosis.