Selective visual detection of multiplex PCR amplicon using magnetic microbeads

Abstract

Nucleic acid amplification tests (NATs), such as genetic tests using polymerase chain reaction (PCR), are sensitive methods for detecting pathogens and food contamination. The rapid, easy, and inexpensive detection of amplicons, DNA, or RNA is key to realizing on-site NATs. We have previously developed a novel amplicon detection method using magnetic microbeads based on the hydrophobicity of DNA in deionized water. In this study, we aimed to expand the method for the detection of multiplex DNA amplicons. Tagged primers and probes for selective attachment were used to detect amplicons from two strawberry pathogens. The amplicon-labeled magnetic microbeads were placed in the round-bottom well of a hydrophilic glass substrate. The attachment of amplicons to the magnetic microbeads changed their surface from hydrophilic to hydrophobic. The magnetized microbeads concentrated at the bottom when the substrate was placed on a permanent magnet, and the concentrated microbeads were easily recognizable by the naked eye. Microbeads without amplicons were adsorbed over a broad area of the bottom of the glass well owing to their hydrophilicity. The appropriate tag probe was attached to specific amplicons for detection, and each amplicon from multiplex PCR was selectively detected within approximately 15 min. Notably, this method requires no electric power and contributes to the realization of on-site NAT detection. This study presents a simple and rapid method for the selective detection of multiplex PCR amplicons using DNA–DNA hybridization.