Handheld Point-of-Care System for Rapid Detection of SARS-CoV-2 Extracted RNA in under 20 min

Abstract

The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (<20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against extracted RNA from 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 91% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 ± 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n = 52), showing average detection times of 12.68 ± 2.56 min for positive samples (n = 34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geolocalization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.

Figure 1

SARS-CoV-2 diagnostic workflow. (A) Sample collection and preparation illustrating nasopharyngeal swab and RNA extraction. (B) Nucleic acid amplification methods for SARS-CoV-2 RNA detection used in this study (RT-qPCR, RT-qLAMP, and RT-eLAMP). Thermal profiles are illustrated for comparison of the assays. (C) Point-of-care diagnostics by RT-eLAMP showing the proposed handheld LoC platform including the microfluidic cartridge with control and sample inlets, and the smartphone-enabled application for geolocalization and real-time visualization of results.

Figure 2

Phylogenetic analysis and LAMPcov assay design. (A) Reference sequence NC_45512 SARS-CoV-2 showing priming regions. (B) Phylogenetic tree showing the specificity of the amplicon for SARS-CoV-2 detection. Clades shadowed in blue include the reference sequence NC_45512. Clades highlighted in light red include HKU1, SARS, and MERS, all distant from the inclusivity clade. (C) Sequences of primers of the LAMPcov assay. One mismatch was introduced in F2 to avoid hairpin formation of the primer (in red). (D) Standard curve with RT-qLAMP using a control RNA at concentrations ranging between 10¹ and 10⁹ copies per reaction. (E) Comparison between our assay (blue bars) and the published assay by Zhang et al. (stripped bars). Concentrations (dilution factor) of a clinical sample are plotted against TTP (min).

Figure 3

Clinical validation using RT-qPCR, RT-qLAMP, and RT-eLAMP. (A) Correlation between RT-qPCR and RT-qLAMP based on the estimated sample concentration (copies/μL of eluted sample volume), n = 115. (B) Correlation between RNase P and viral RNA concentration of the positive clinical samples (copies/μL of eluted sample volume) to indicate quality of extraction across all of the cohort, n = 127. (C) Boxplot distribution of RNase P concentration (copies/μL of eluted sample volume) across negative (n = 56) and positive (n = 127) clinical samples by RT-qPCR. The calculated p-value between both groups was below 0.05 (p-value = 8.09 × 10^–6). (D) Boxplot distribution of TTP for the RT-qLAMP and RT-eLAMP demonstrating the performance of the LoC platform (n = 34 positive samples). The calculated p-value between both groups is higher than 0.05 (p-value = 0.32). It is important to note that 18 negative samples were confirmed by RT-qLAMP and RT-eLAMP. (E) Example overview of data processing steps for sample 179 to extract amplification curves from sample and control wells on the LoC platform during RT-eLAMP. The spatial image illustrates the microchip ISFET sensing array output (4368 sensors, 2 × 4 mm) on the single-use cartridge where the averaged sensor signals in each well demonstrate, respectively, amplification with a TTP of 10.63 min (sample) and no amplification (control).