Boosting Sensitivity and Accuracy of the Lamp Covid Test

The covid test, now widely available for detecting coronavirus from saliva, can be used in the same way that a tetanus shot is — to ward off a potential infection. However, the rapid expansion of the test isn’t without its challenges. While many tests have been rushed to market for the pandemic, some haven’t passed muster in terms of sensitivity, and others have been found to be inaccurate or unreliable.

Some point-of-care tests, like the ones developed by HiberGene Diagnostics and a team in Racine, Wisconsin, are using an approach known as LAMP, or loop-mediated isothermal amplification. LAMP works by enlisting DNA polymerase and a set of four to six primers that recognize specific sequences on the target DNA or RNA. Those primers then displace the original DNA strands, creating new DNA copies and forming loops that are amplified because of complementary sequences on the outermost loops. That amplification and the resulting colorimetric signal allow for detection, all without requiring highly specialized or costly equipment.

Unlike the polymerase chain reaction-based methods that are currently the standard of care, which require repeated heating and cooling of the sample at precise intervals in an expensive machine called a thermocycler, LAMP is isothermal and operates at a constant temperature. It’s a more labor-intensive process, but it can be performed by an ordinary hot plate instead of more specialized – and costlier – thermal cycling equipment.

Ameliorating the test’s sensitivity has been an ongoing project. One key step was ensuring that the assay wasn’t overpowered by any non-specific background DNA or RNA. Another was identifying and optimizing a pair of primers that can detect the virus with the highest sensitivity, and yet still yield a good result when multiple different primer pairs are tested.

Testing the triplexed assay with both synthetic RNA in water and spiked saliva directly in the sample also boosted sensitivity (Supplementary Fig. 3a, b). Further optimization by increasing the number of primer sets and improving their mismatch tolerance and allelic discrimination further increased sensitivity.

The resulting assay, now known as Vivid COVID-19 LAMP, has demonstrated excellent performance in comparison to RT-qPCR on a panel of variant-specific synthetic controls and low viral load patient samples with sequencing-confirmed lineage status (Fig. 8b, c). It also performs well when compared with other point-of-care LAMP kits that use a similar detection system, namely the Harmony COVID-19 LAMP kit from Life Technologies.

Using the same reagents and a portable analyzer, HiberGene’s system is designed to allow rapid screening of direct saliva samples from patients with suspected VC-19 infection. The test is also designed to be easy to scale up to larger volumes, allowing screening groups to operate out of any room that can accommodate a 96-well plate. A smartphone app can be used to objectively read and report the colorimetric results, making it suitable for widespread use by individuals without a laboratory setting. For that reason, it’s already being implemented in a range of settings around the world: from an unused corner of city hall to a classroom lab.