Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2

Abstract

Over the course of the COVID-19 pandemic, several SARS-CoV-2 genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants is important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive qPCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons paired with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants which contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific, affordable and allows multiplexing with other SARS-CoV-2 LAMP primer sets.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons

“…The LAMP-BEAC provides an effective complement to SalivaDirect since it does not rely on commercial enzyme mixes, potentially providing more resilience to possible supply chain disruptions, and can be carried out as an end-point assay using a heat block and fluorescent viewer, thus bypassing the need for quantitative real-time PCR machines. Additionally, LAMP-BEAC allows identification of variants of concern using targeted molecular beacons [26].…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…Specificity: Unclear [309] Almost all variants 69del, 70del, Y144del, N501Y and A570D Compared to WGS, RT-PCR and sanger sequencing require shorter time and lower cost. LOD unclear [310] SNP genotyping Almost all variants Unreported >62% to be able to distinguish two variants of different genotypes [311] RT-LAMP P.1 (Gamma), P.2 (Zeta) N501Y, E484K/Q, K417N/T Sensitivity: 97%, Specificity: 100% (based on N/E targets) [312] B.1.1.7 (Alpha) 69del, 70del LOD: 39-10,000 RNA copies/reaction [313] CRISRP/Cas-based technique B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) N501Y LOD, sensitivity and specificity unclear [346] Almost all variants have D614G D614G LOD: 10 RNA copies/reaction [314] Other variants E174R/S542R/K548R, S254F LOD: 50-1000 RNA copies/reaction, specificity: 100% [315] Almost all variants have D614G D614G LOD: 82 RNA copies/reaction, % specificity unclear [155] Antigen test B.1.1.7 (Alpha), B.1.351 (Beta), Unclear LOD: 1.7×10 5 – 6.6×10 7 RNA copies/mL [316] Sensitivity and specificity: Unclear Antigen and antibody test B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) Unclear Detection rate of antigen/antibody tests to detect these three variants were reported to be >90% [317] …”

“…Other than RT-PCR and nucleic acid sequencing, the RT-LAMP-based approach was also being reported to be used in the detection of different SARS-CoV-2 variants [312] , [313] . This assay was used to identify various variants like Alpha (B.1.1.7) and Gamma (P.1) by detecting mutations like 69del, 70del, and N501Y [312] , [313] .…”

“…Other than RT-PCR and nucleic acid sequencing, the RT-LAMP-based approach was also being reported to be used in the detection of different SARS-CoV-2 variants [312] , [313] . This assay was used to identify various variants like Alpha (B.1.1.7) and Gamma (P.1) by detecting mutations like 69del, 70del, and N501Y [312] , [313] . The LOD of this test approach was reported to be ranging from 39 to 10,000 RNA copies in a reaction [313] , and the test sensitivity and specificity were shown to be around 97, and 100%, respectively [312] .…”

“…This assay was used to identify various variants like Alpha (B.1.1.7) and Gamma (P.1) by detecting mutations like 69del, 70del, and N501Y [312] , [313] . The LOD of this test approach was reported to be ranging from 39 to 10,000 RNA copies in a reaction [313] , and the test sensitivity and specificity were shown to be around 97, and 100%, respectively [312] . On the other side, a molecular diagnostic approach that is based on the CRISPR/Cas-based detection technique was also being employed in identifying different SARS-CoV-2 variants and the LOD of this test was reported to vary from 10 to 1000 RNA copies in a reaction [155] , [314] , [315] .…”