Development of a one-step sample to answer multiplex Hyper-branched Rolling Cycle Amplification (pR@FRET- MB@mHSRCA) with tunable hybrid signaling (pR@FRET-MB) for simultaneous pathogen detection in food

Abstract

Abstract Salmonella and Staphylococcus foodborne diseases are the majors causes of human diseases and food losses impacting negatively society's well-being and economy. It is primordial to establish a simple and efficient method for specific detection and identification of bacteria. Hence the necessity of the isothermal amplification method. We designed a triple-probe multiplex rolling circle amplification procedure for the simultaneous detection of Salmonella and Staphylococcus. The long padlock probes were designed to target InvA and GlnA of respectively salmonella and staphylococcus and tagged with fluorophores. A pH-based detection method was then established for the visual detection of the targeted pathogens while the Fluorescence signal was used to efficiently identify the pathogen. After optimization of the detection procedure conditions, the one-step mHSRCA could be conducted at room temperature (30°C) for 3 hours. It shows specificity for both staphylococcus and salmonella with a detection limit of 0.039 µM/µl when the fluorescence signal is concern and 0.078µM/µl for the colorimetric signal when the synthetic bacteria gene target is used after 30minutes of test. The simulative test of the present method for effective detection of both bacteria spiked in milk show after 3 hours of amplification a detection limit of 10 CFU/ml and 5CFU/ml for respectively Staphylococcus and Salmonella when the fluorescent signal is concerned. The colorimetric signal the detection limit was 10x101 CFU/ml and 5x101 CFU/ml. In summary, the triple-probe-multiplex rolling circle amplification method could be effectively used for screening food against foodborne pathogens within hours with good specificity, high sensitivity, and easy result reading.