Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva

Abstract

Background The coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has killed millions of people worldwide. The current crisis has created an unprecedented demand for rapid test of SARS-CoV-2 infection. Methods Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. However, the sensitivity and specificity of RT-LAMP were generally regarded as inferior when compared with the gold standard RT-qPCR. To address this issue, we combined bioinformatic and experimental analyses to improve the assay performance for COVID-19 diagnosis. Findings First, by experimental screening as well as high-throughput sequencing studies, we discovered new primer features that impacted LAMP sensitivity and specificity. These features were then used to build an improved bioinformatics algorithm to design LAMP primers targeting SARS-CoV-2. We further rigorously validated these new assays for their efficacy and specificity. We demonstrated that multiplexed RT-LAMP assay could directly detect as low as 1.5 copies/µL of SARS-CoV-2 particles in saliva, without the need of RNA isolation. We further tested this ultra-sensitive and specific RT-LAMP assay using saliva samples from COVID-19 patients. Clinical validation results indicated that the new RT-LAMP assay was comparable to standard RT-qPCR in overall assay sensitivity and specificity. Interpretation In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases. Funding National Institutes of Health.

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance

“…While several saliva-based approaches have been demonstrated for the detection of SARS-CoV-2 using LAMP [ 8 , 14 , 30 , 31 , 35 , 41 – 45 ], including extraction-free or direct protocols [ 13 , 30 , 35 , 41 , 42 , 44 , 46 , 47 ], no gold standard method exists. We focused on the development of a simple and improved sample preparation protocol compatible with this complex sample and downstream LAMP reactions.…”

Development and Implementation of a Simple and Rapid Extraction-Free Saliva SARS-CoV-2 RT-LAMP Workflow for Workplace Surveillance

“…While several saliva-based approaches have been demonstrated for the detection of SARS-CoV-2 using LAMP [8,14,30,31,35,[37][38][39][40][41], including extraction-free or direct protocols [13,30,35,37,38,40,42,43], no gold standard method exists. We focused on the development of a simple and improved sample preparation protocol compatible with this complex sample and downstream LAMP reactions.…”

“…Only samples with very late Cq values (greater than 34) in RT-qPCR, failed to amplify in extraction-free RT-LAMP. High Cq values (>34) in RT-qPCR have been shown to correlate with low viral loads (<100-1,000 copies/μL) and these individuals are rarely infectious or not infectious [41].…”

A Review of SARS-CoV-2 Disease (COVID-19): Pandemic in Our Time

“…However, we must bear in mind that the CRISPR technology has its own limitations too since it is pretty much new technology that is still being worked on to understand more of it. The advancement in the technological world has made almost all of these methods achievable within 24 h or even less [ 75 , 76 , 77 , 78 , 79 , 80 , 81 ].…”