Solid-phase PCR based on thermostable, encoded magnetic microspheres for simple, highly sensitive and multiplexed nucleic acid detection

Abstract

Multiplexed detection of nucleic acids is important for pathogen identification and disease diagnosis. Though real-time quantitative PCR allows for simultaneous amplification of different genes in a single test, the interferences between various primers inevitably cause difficulty in assay design, and pose limitation on multiplexing degree. Conventional solid-phase PCR (SP-PCR) on plenary microarrays can assess more targets than real-time quantitative PCR. However, they are suffering from low reaction efficiency, high background noise, and requiring special equipment and expertise for reliable analysis. Here, we addressed the challenges by performing multiplex PCR on thermostable, tailor-made host-guest encoded magnetic microspheres (Beads based PCR, BB-PCR), which were constructed by coupling magnetic host microspheres with fluorescent guest nanoparticles. As proof of concept, a 3-plex barcoded microbeads with high thermostability and low non-specific fluorescence absorption were selected as model BB-PCR carriers to amplify and detect three gene targets in Salmonella Enteritidis. After BB-PCR reaction, the barcoding and reporting signals were easily identified through flow cytometry. Attributed to the effective interactions between reagents and microspheres during PCR reaction, this method showed high sensitivity of 10 copies/reaction, which was comparable to that of liquid PCR. Thus, BB-PCR developed here allowed for simple, rapid, highly sensitive and high throughput detection of nucleic acid targets, which paves the way for routine multiplexed molecular diagnostics in clinical laboratories.