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

Abstract

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3–99.5%] sensitivity and 100% (95% CI = 94.5–100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non–SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction–free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

“…Detailed conditions of RT-LAMP reaction, including primer sequences, are described in our previous work. (4) Briefly, reactions were performed at 65ºC, during 30 min, using WarmStart ® Colorimetric LAMP 2× Master Mix (New England Biolabs #M1804). To validate the visual colorimetric output, samples resolved in 2% agarose gel stained with DNA intercalator GelRed ® (Biotium #41003), confirming the specific amplification when compared to the positive control (RNA of inactivated parental lineage B of SARS-CoV-2 extracted from the supernatant infected Vero E6 cells).…”

“…(1,2,3) As an alternative to RT-qPCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used with success to detect SARS-CoV-2 RNA from nasopharyngeal swabs and saliva samples. (4) During RT-LAMP reaction, the amplification of genetic material occurs at constant temperature (65ºC) without the need for sophisticated thermal cyclers as in RT-qPCR. (5) Furthermore, the colorimetric reaction allows the result to be interpreted faster than RT-qPCR, since the amplified products can be visually detected due to pH-dependent sensors that changes reaction color from pink to yellow (positive result).…”

“…(6) Previous results obtained by our group, showed that RT-LAMP reaction targeting SARS-CoV-2 N and E genes, can be equivalent to the gold standard RT-qPCR using nasopharyngeal swabs samples. (4) Given the fact that genomic mutations in SARS-CoV-2 omicron VOC are also present in N and E coding sequences, the main goal of this work was to verify whether RT-LAMP reaction targeting N and E genes (in a multiplex or singleplex manner), would works to detect the virus reinforcing the use of it as an affordable and robust COVID-19 diagnostic tool.…”

ViralFlow: A Versatile Automated Workflow for SARS-CoV-2 Genome Assembly, Lineage Assignment, Mutations and Intrahost Variant Detection

“…A range of molecular biology methods have been developed to diagnose SARS-CoV-2 infections, such as RT-qPCRs, RT-LAMP, immunoassays, and Sanger sequencing [5][6][7][8]. However, only whole-genome sequencing can provide enough genetic information (genome-wide mutation patterns) for the reliable lineage discrimination that is…”

Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation

“…The loop-mediated isothermal amplification (LAMP) method is cost-effective (1.50 USD/test), simple (the isothermal reaction requires a simple heating device), fast (results within 60 min) [10], and visually detected [11]. Because of this, LAMP has been used to detect parasites, [12][13][14][15], bacterias, [16,17], sexually transmitted diseases [18][19][20] , and viruses including SARS-CoV-2 [21][22][23].…”