Detection of SARS-CoV-2 in Saliva by RT-LAMP During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

Abstract

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers

“…Other techniques, such as Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP), can potentially simplify the execution of the test. The RT-LAMP has been proposed as a reliable diagnostic tool for detecting SARS-CoV-2 20 – 23 . This method can detect the target genome with high specificity and sensibility in < 40 min, without the need for a real-time thermocycler.…”

Saliva-based point-of-care testing techniques for COVID-19 detection

“…Each technique has its own merits and limitations. Colorimetric biosensors are portable, easy to use and rapid in response, with potential for large-scale POCT in remote locations ( Li et al, 2020 ; Alafeef et al, 2021 ; dos Santos et al, 2021 ; Ferreira et al, 2021 ). As a typical example, the lateral flow assay (LFA) is a type of these biosensors that uses conjugated gold nanoparticles, fluorescent molecules, or quantum dots for visual sensing of color changes.…”

“… LEAD ( de Lima et al, 2021 ) Nasopharyngeal/oropharyngeal and saliva SARS-CoV-2 spike protein The sensor enables on-site SARS-CoV-2 detection within 6.5 min, the sensitivity of which is comparable to the gold-standard methods (LOD = 229 fg/mL), and displays an excellent performance profile in saliva (100.0% sensitivity, 100.0% specificity, 100.0% accuracy) and nasopharyngeal/oropharyngeal (88.7% sensitivity, 86.0% specificity, 87.4% accuracy) samples. Colorimetric RT-LAMP assay ( dos Santos et al, 2021 ) Saliva RNA The sensor had a LOD of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. LFIA immunosensor ( Roda et al, 2021 ) Serum, saliva IgA The LFIA immunosensor coupled with the smartphone reading enabled the one-step affordable determination of IgA levels in saliva.…”