Modification-Free Oligonucleotide-Utilized Lateral Flow Assay System for On-Site Detection of Foodborne Pathogens

Abstract

Foodborne infectious diseases are a major public health and economic issue worldwide, warranting an effective food safety management system that can rapidly detect foodborne pathogens. We developed a modification-free lateral flow assay for the specific hybridization (M-FLASH) system using modification-free oligonucleotides that specifically recognize the single-stranded loop region of loop-mediated isothermal amplification (LAMP) products for sensitive and on-site detection of foodborne pathogens. To increase its suitability for field diagnosis, four unique strategies were introduced: (i) thermal lysis for DNA extraction, (ii) direct LAMP without any additional DNA purification steps for DNA amplification, (iii) LFA system where the capture probe is immobilized by our salt-mediated immobilization of nucleic acids (SAIoNs) method, and (iv) reporter probe-gold nanoparticle (AuNP) conjugates prepared by the microwave-assisted heating-dry method. The proposed system relies on sequence-specific hybridization of LAMP products with both the CP in the LFA strip and the reporter probe in AuNPs. It exhibited high specificity without any interference from nontarget pathogens; furthermore, modifications to DNA oligonucleotides were unwarranted, significantly reducing production and assay costs. The system successfully detected Escherichia coli O157:H7 down to 10¹ CFU/mL with an assay time of <2 h and was effective in analyzing target pathogens in artificially contaminated food samples. It also demonstrated universal applicability for detecting Listeria monocytogenes and multiplex detection of E. coli O157:H7 and L. monocytogenes. This system has high potential for use in resource-limited settings and can be used as the core platform for the detection of various pathogenic bacteria or viruses.