(348c) Portable and Label-Free Quantitative Loop-Mediated Isothermal Amplification (LF-qLamp) for Reliable COVID-19 Diagnostics in Three Minutes of Reaction Time

Loop-mediated isothermal amplification (LAMP), and particularly colorimetric LAMP, has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based colorimetric diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based and label-free quantitative LAMP-based amplification system (qLAMP) assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction.

The discrimination between negative samples and positive samples with medium to high viral loads (i.e., at least 100 copies) was feasible by direct comparison of the pH (i.e., the electric potential trajectories (ΔP)). However, samples containing a low copy number (i.e., less than 10 gene copies) cannot be distinguished from negative samples by only comparing trajectories (Figure 1). The behavior of the trajectory of the amplification reaction in positive and negative samples can be better discriminated during the first stage of the amplification (i.e., the first three minutes of the amplification). Indeed, the value of the area under the curve or of ΔP versus t is distinguishably higher in positive samples than in negative samples during the first stage of the amplification.

We show that this simple system and analysis strategy enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well.

In addition, we analyzed a limited set of saliva samples (n=15) from volunteers displaying COVID-19 symptoms. We complemented this experimental set with saliva samples from 30 healthy volunteers. From these experiments with actual saliva samples, we determined a sensitivity >95% and a selectivity >92% for qLAMP.