Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

Keywords: RT-LAMP; SARS-CoV-2; biodetection; point-of-care.

Figure 1

Sample concentration with magnetic wand and paramagnetic wand. The paramagnetic beads bind to viral RNA in the lysed sample. The magnetic wand is used (1) to collect these RNA-bound beads away from the rest of the sample volume and (2) to transfer them to the loop-mediated isothermal amplification (LAMP) reaction cartridge. To release the beads from the magnetic wand, the magnet is slid from the 3D-printed sheath. In the absence of a magnetic field, the paramagnetic beads are no longer magnetized and are able go into solution in water at the top of the LAMP reaction cartridge.

Figure 2

Easy to use Accessible LAMP-Enabled Rapid Test (ALERT) cartridges. One-step reaction-ready cartridge layering: cartridge layers at (a) room temperature and (b) at 64 °C. Demonstration of magnetic wand; (c,d) wand accumulates beads and (e) is placed into the reaction cartridge where (f) the magnet is removed to release beads, reagents have dyes added for demonstration purposes: primer mix is orange and the enzyme droplet is black.

Figure 3

Two-step reaction-ready cartridge layering. Cartridge layers at (a) room temperature, (b) 55 °C and (c) at 63 °C.

Figure 4

(a) Proteinase K lysis efficacy on nasal mid-turbinate (NMT) swabs spiked with 0, 1000, 500 and 250 pfus of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 100 copies of synthetic SARS-CoV-2 RNA, 50 copies of synthetic SARS-CoV-2 RNA and non-template water controls.; (b) TCEP/EDTA lysis efficacy on NMT swabs spiked with inactivated SARS-CoV-2. Bottom strip shows Twist Biosciences SARS-CoV-2 RNA and negative water controls.

Figure 5

(a) Detection of SARS-CoV-2 using proteinase K, magnetic wand extraction and a one-step cartridge, NMT swabs spiked with inactivated SARS-CoV-2; (b) same tubes visualized with the GMO Detective.

Figure 6

LoD improvement with a 2-step system (a) 1-Step RT-LAMP limit of detection (LoD) confirmed to be at ~500 copies of Twist Biosciences SARS-CoV-2 control as reported by Zhang et al. 2020; (b) 2-step RT/LAMP cartridge with increased sensitivity with LoD down to 25 copies of Twist Biosciences SARS-CoV-2 control.

Figure 7

(a) BPIFA1 Primers were designed to traverse exon-exon junctions to be mRNA specific; (b) BPIFA1 RT-LAMP primer set specificity using New England Biolabs Colorimetric RT-LAMP kit: 1. Nasopharyngeal swab, TRIzol/chloroform extraction 2. Nasopharyngeal swab, Qiagen Viral RNA Mini kit extraction 3. Saliva RNA 4. Blood RNA 5. Nasopharynx flush-through (NFT), Qiagen Viral RNA Mini Kit extraction 6. Human (male) gDNA 7,8. Non-template water control, (c)—BPIFA1 detection across 11 individuals spiked and non-spiked with SARS-Cov-2, 500 PFU of SARS-CoV-2 and non-template water controls.

Figure 8

Multiplexed reactions allow for the detection of different viral targets or internal controls: (a) green fluorescence indicates BPIFA1 amplification, red fluorescence indicates Twist Bioscience SARS-CoV-2 control amplification, and orange fluorescence indicates both BPIFA1 and Twist Bioscience SARS-CoV-2 control amplification, (NP—nasopharynx); (b) multiplexed reactions for BPIFA1 and SARS-CoV-2, 11 Nasal pharynx flush thru (NFT) samples and also spiked with 500 PFU of SARS-CoV-2 and extracted with TRIzol chloroform method, 50 cp Twist synthetic controls, 25 cp Twist synthetic controls and non-template water controls; (c) green fluorescence indicates influenza B (IB) RNA amplification, red fluorescence indicates SARS-CoV-2 amplification, and orange fluorescence indicates both influenza B and SARS-CoV-2 amplification.

Figure 9

Assay sensitivity on blinded XPRIZE samples: The set of blinded XPRIZE samples comprised 123 samples with an unknown concentration SARS-CoV-2 Twist synthetic RNAs in water or SARS-CoV-2 ZeptoMetrix particles in phosphate-buffered saline (PBS), nasal sample, or saliva sample. True positive rate (y-axis) is shown for different concentrations (x-axis) across the different matrices. The number of samples tested at each concentration for a sample type is represented by the values above the x-axes.