Multiplexed Detection of Pathogens Using Solid-Phase Loop-Mediated Isothermal Amplification on a Supercritical Angle...

Area of Science:

• Molecular biology
• Biotechnology
• Pathogen detection

Background:

• Point-of-care (POC) and onsite testing demand advanced molecular techniques for multiplexed pathogen detection.
• Loop-mediated isothermal amplification (LAMP) is promising for POC due to its features, but multiplexing is challenging due to non-defined amplicon sizes.

Purpose of the Study:

• To develop a solid-phase loop-mediated isothermal amplification (SP-LAMP) technique to enable multiplexed pathogen detection.
• To address the challenge of identifying specific LAMP amplicons in multiplexed assays.

Main Methods:

• Developed SP-LAMP by integrating LAMP with supercritical angle fluorescence (SAF) micro-optic structures as solid supports in an array format.
• Optimized parameters including SS primer length, primer-binding region length, surface density, and primer cross-reactivity.
• Validated detection of multiple pathogens including *Salmonella* spp., *Campylobacter* spp., avian influenza virus (AIV), and internal control (IC).

Main Results:

• SP-LAMP with SAF enabled spatial separation of LAMP amplicons for multiplexed detection.
• Singleplex detection of *Salmonella* spp., *Campylobacter* spp., AIV, and IC was achieved.
• Demonstrated multiplexing capacity of SP-LAMP using AIV and IC with promising results.

Conclusions:

• SP-LAMP successfully addresses the challenge of multiplexed pathogen detection.
• The developed technique shows promise for creating multiplex POC systems for rapid, simultaneous pathogen identification.
• This advancement opens new avenues for developing advanced diagnostic tools for infectious diseases.