Probe-Based Multiplex LAMP Enables High-Precision Point-Of-Care Diagnostics in PANPOC system

Standard LAMP reactions are widely used in point-of-care (POC) diagnostics due to their simplicity and efficiency. Conventional LAMP assays use intercalating dyes where the fluorescence results from the presence of any double-stranded DNA, making it impossible to distinguish between multiple targets in a single reaction. This becomes a major obstacle in multiplexed diagnostics requiring the detection of multiple targets in a single test, which results in with faster, more efficient and more informative diagnostics. Target specificity is essential for multiplex detection.

In the PAIR project, to address this challenge within our PANPOC system, we implemented a probe-based detection strategy that enables target-specific signal discrimination, even in multiplex settings. We selected molecular beacons for their ability to deliver both high sensitivity and sequence-specific fluorescence.

Molecular beacons are specially designed, hairpin-shaped oligonucleotides labelled with a fluorescent dye at the 5’ end and a quencher at the 3’ end. In the absence of the target, the hairpin remains intact and the signal remains quenched. Upon hybridisation with the specific target sequence, the beacon opens, separating the fluorophore and quencher, thereby emitting a fluorescent signal. This allows for real-time, target-specific detection within a multiplex LAMP reaction.

To further enhance binding affinity and specificity, we incorporated locked nucleic acids (LNAs) into the molecular beacons. The LNA-modified beacons targeting influenza A and the external control EGFP (enhanced green fluorescent protein) demonstrated strong binding and robust signal output, with amplification consistently detected within 20 minutes.

Following successful validation of each assay in singleplex, we advanced to a duplex format, combining influenza A and EGFP into a single-tube reaction with their respective primers and molecular beacons. While multiplexing is a key advantage for POC applications, it introduces complexities such as cross-reactivity, signal interference, and reagent interactions. Despite these challenges, preliminary results from the duplex system are promising, showing clear and target-specific amplification signals.

“At present, we are focused on duplex reactions for each of the three PAIR targets – Influenza A, B and beta-CoV – alongside an external control” states Maria Dimaki from the Technical University of Denmark (DTU). “However, our overarching goal is to fully multiplex all four targets within a single well, significantly enhancing the efficiency and capability of POC diagnostics”.

Further optimization is currently underway to fine-tune reaction conditions and reagent concentrations, ensuring reliable performance and signal accuracy regardless of assay variability.

This probe-based approach represents a key step toward more reliable, efficient, and scalable point-of-care diagnostics, paving the way for fully integrated multiplex testing in the PANPOC system.

With the contribution of Maria Dimaki and Ankita Mishra (DTU)

Photo by Fusion Medical Animation on Unsplash