Extraction-Free Colorimetric RT-LAMP Detection of SARS-CoV-2 in Saliva

Abstract

The pandemic situation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need of fast, simple, and cost-effective tests for the diagnosis of emerging pathogens. RT-qPCR has been established as the reference technique for the diagnosis of SARS-CoV-2 infections. This method requires a time-consuming protocol for the extraction of the nucleic acids present in the sample. A colorimetric reverse transcription loop-mediated isothermal amplification using the calcein molecule combined with a simple extraction-free method for saliva samples (calcein RT-LAMP) has been developed. Samples are heated 95 °C for 10 min before amplification at 63 °C for 40 min. The results can be observed by fluorescence or by the naked eye with a color change from orange to green. The method was compared with commercialized available colorimetric and fluorescent RT-LAMP kits. The developed method shows better sensitivity and specificity than the colorimetric commercial RT-LAMP and the same as the fluorescent RT-LAMP, without the need of a fluorescent reader. Moreover, the calcein RT-LAMP has, compared to RT-qPCR, a sensitivity of 90% and a specificity of 100% for saliva samples with a Ct ≤ 34, without the need for expensive RT-qPCR instruments, demonstrating the potential of this method for population screening.

Self-Diagnosis of SARS-CoV-2 from Saliva Samples at Home: Isothermal Amplification Enabled by Do-It-Yourself Portable Incubators and Laminated Poly-ethyl Sulfonate Membranes

“…Recently, several groups have shown that accurate discrimination between COVID-19(+) and COVID-19(−) samples is possible using extraction-free RT-qPCR (i.e., average sensitivity and selectivity above 91% and 99%, respectively) [27,41]. High sensitivity and selectivity values have also been reported in extraction-free colorimetric LAMP implementations conducted using saliva samples (i.e., average sensitivity and selectivity values higher than 85% and 95%, respectively) [14,20,42]. In these protocols, inactivation and extraction are frequently mediated by heat treatment.…”

Clinical Performance of the Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for the Diagnosis of COVID-19 in a Thai Community Hospital at the Thailand-Myanmar Border

“…Silva et al reported the clinical evaluation of the RT-LAMP in a hospital setting with 95.83% accuracy, 88.89% sensitivity, and 98.55% specificity [ 23 ]. It was noted that other factors, such as specimens and specimen processing, could affect the clinical sensitivity of RT-LAMP [ 14 - 15 , 24 ]. For sample types, the RT-LAMP assay shared many considerations like rRT-PCR [ 8 ].…”

“…[23]. It was noted that other factors, such as specimens and specimen processing, could affect the clinical sensitivity of RT-LAMP [14][15]24]. For sample types, the RT-LAMP assay shared many considerations like rRT-PCR [8].…”

Evaluation of a Commercial Point-of-Care RT-LAMP Assay for Rapid Detection of SARS-CoV-2

“…Recently, researchers have been exploring the utility of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the point-of-care detection of SARS-CoV-2 [ 8 , 9 , 10 ]. For instance, Chow and co-workers developed a small van-sized mobile COVID-19-LAMP diagnostic unit for providing an on-site SARS-CoV-2 screening service [ 8 ].…”

“…In another study by Yip and co-workers, a nsp8 one-tube RT-LAMP-CRISPR (clustered regularly interspaced short palindromic repeat) assay was developed, showing a sensitivity of 98.6%, and a specificity of 100%, for SARS-CoV-2 detection [ 9 ]. Rivas-Macho and coworkers have evaluated an extraction-free colorimetric RT-LAMP method for the detection of SARS-CoV-2 in saliva [ 10 ]. They reported that the sensitivity and specificity of the assay were 90% and 100%, respectively, for saliva specimens with a Ct value equal to or less than 34.…”