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.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

“…In the current OmniLAMP R assay, we included human b-actin RNA (rACTB) as internal control. Other constitutive targets for SARS-CoV-2 RT-LAMP include BPIFA1 (Bektaş et al, 2021) Similar to its high sensitivity, obtained in this work and by other studies, the SARS-CoV-2 RT-LAMP specificity is undoubtedly high and is frequently reported as 100% without any cross-reactivity with other respiratory or SARS-CoV-unrelated viruses (Chow et al, 2020;Mohon et al, 2020;Park et al, 2020;Aoki et al, 2021;Nawattanapaiboon et al, 2021). We also confirm that the SARS-CoV-2 RT-LAMP solution presented here is highly specific and does not cross-react with Brazilian occurring seasonal influenza A and B, hRSV, or arboviruses.…”

“…In the current OmniLAMP ® assay, we included human b-actin RNA (rACTB) as internal control. Other constitutive targets for SARS-CoV-2 RT-LAMP include BPIFA1 ( Bektaş et al, 2021 ), human 18S RNA ( de Oliveira Coelho et al, 2021 ), and Statherin RNA ( Sherrill-Mix et al, 2021a ; Table 2 ).…”

“…The possibility of accessing results by the naked eye made RT-LAMP an exciting alternative that facilitates the use of COVID-19 molecular testing. Simple, scalable, cost-effective RT-LAMP–based alternatives for SARS-CoV-2 detection have emerged during pandemics including protocols for viral inactivation, quick run, RNA extraction–free and LAMP-associated CRISPR/Cas strategies ( Baek et al, 2020 ; Broughton et al, 2020 ; Chow et al, 2020 ; Dudley et al, 2020 ; Song et al, 2021 ; Godfrey et al, 2020 ; Joung et al, 2020 ; L’Helgouach et al, 2020 ; Park et al, 2020 ; Rabe and Cepko, 2020 ; Bektaş et al, 2021 ; Bokelmann et al, 2021 ). On April 14, 2020, the RT-LAMP received the emergency use authorization from the United States Food and Drug Administration (FDA) for SARS-CoV-2 detection in COVID-19 diagnostics.…”

A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test

“…For application in a dedicated diagnostic laboratory environment there are different protocols available using fluorescence detection, or absorbance readings as quantitative measurement 27 – 33 . These advanced detection methods are also possible in POCT diagnostics and several protocols are available 34 – 36 .…”

A Comparative Evaluation of Indirect Sequence-Specific Magnetoextraction-aided Fluorescence and Electrochemical LAMP with SARS-CoV-2 Nucleic Acid as the Analyte

“…The rest of the methods predominantly are either whole nucleic acid extraction or immunomagnetic capture followed by lysis. The former also included all magneto-extraction associated SARS-CoV-2 detection techniques using LAMP (entries 7, 10, 14, 24, 35, 48, Table S1 25,[33][34][35][36][37] ). Surprisingly, none of these studies listed in Table S1 utilized electrochemical readout despite its promising potential in limited resource detection.…”