Multiplexing with LAMP: Lessons for all researchers from Optimizing Multi-Target Assays

Over the past 24 months, DTU has been working on optimizing a multiplex LAMP assay, that detects four different targets simultaneously in a single reaction tube. The chosen targets are Influenza A, Influenza B, SARS-CoV-2, and an internal control (eGFP). The aim has been to develop a fast, robust assay suitable for point-of-care diagnostics, while exploring the practical limits of multiplexing using LAMP technology.

Lesson n°1: the design of the two inner primers is critical to the success

Multiplexing is inherently challenging, even for PCR-based assays. With LAMP, the complexity increases substantially, as each target requires six primers that must work together to initiate and sustain amplification. The two inner primers are particularly critical, as they drive formation of the characteristic stem–loop structures that enable self-priming. Mutations in primer binding regions—especially in the inner primers—can significantly reduce amplification efficiency.

Lesson n°2: Test the right concentration of primers to avoid unequal amplification

This issue is especially pronounced for Influenza A and SARS-CoV-2, both of which exhibit high sequence variability. In contrast, Influenza B and the eGFP control are more genetically stable. In a four-target multiplex reaction, these differences translate into unequal amplification dynamics, with mutated viral targets amplifying more slowly and competing poorly for shared reaction components. As a result, primer design and fine-tuning of the relative concentrations of all 24 primers become critical.

Lesson n°3: Use assimilating probes for a simpler and cost-effective amplification approach

Our initial strategy employed molecular beacons for target-specific detection. While this approach performed well in singleplex and duplex reactions, it consistently failed in higher-order multiplex assays. Influenza A proved particularly difficult to accommodate, likely due to the combined effects of primer mismatches and late amplification onset, which negatively impacted assay sensitivity.

To overcome these limitations, we transitioned to assimilating probes as an alternative fluorescence-based detection strategy. Assimilating probes consist of a fluorophore-labelled strand paired with a quencher strand. Upon target recognition, the probe opens and integrates into the LAMP stem–loop structure, generating a fluorescent signal. This approach proved both simpler and more cost-effective than molecular beacons, while also delivering faster signal generation.

Using assimilating probes, single-target reactions performed comparably to assays based on intercalating dyes. However, even with this improved chemistry, fourplex reactions including Influenza A remained unreliable. Based on these results, we restructured the assay design into two complementary formats: a duplex assay targeting Influenza A and eGFP, and a triplex assay targeting Influenza B, SARS-CoV-2, and eGFP.

Results from the lessons learned

This configuration yielded robust and reproducible results, with amplification times of approximately 20 minutes for all targets. Although the limits of detection remain somewhat lower than those achievable with RT-PCR, they are well within the range required for point-of-care applications.

We are now moving forward with validation of the current duplex and triplex setup. In its present form, the system enables testing of six samples per cartridge for all three viral targets. For veterinary applications, where Influenza A detection is the primary focus, up to 14 reactions per cartridge can be performed.

With the collaboration of Danmarks Tekniske Universitet