Development of a Saliva-Optimized RT-LAMP Assay for SARS-CoV-2

Abstract

Conventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a 10-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads. Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva.

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

“…Our internal target was to achieve an analytical LoD (95% detection rate) for synthetic RNA targets of ≤15 copies/reaction. Our desired level of sensitivity would allow the assay to completely bypass any type of laborious RNA extraction/concentration and still be able to detect low viral titers 69 , 70 (<50 viral RNA copies/µl sample) in clinical specimens even with modest sample input volumes (~10% of reaction volume). In search of additional sensitivity, some groups have attempted to implement crude purification and concentration protocols, for example by using silica mesh/gel 55 , carboxylated nucleic acid capture beads, or dramatically increasing the reaction volume along with reagent usage and cost 71 .…”

Rapid and convenient detection of SARS-CoV-2 using a colorimetric triple-target reverse transcription loop-mediated isothermal amplification method

“…First, the specificity of these six sets of LAMP primers had been well studied in the previous studies reported by the different laboratories ( Broughton et al, 2020 ; Dong et al, 2021 ; Jamwal et al, 2021 ; Jiang et al, 2020 ; Nawattanapaiboon et al, 2021 ; Park et al, 2020 ; Yu et al, 2021 ; Zhang & Tanner, 2021 ). Second, the sequence of the LAMP primers was compared to aligned sequences of some other coronaviruses (including MERS-CoV, SARS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E), all of which had some nucleotides mismatching with our LAMP primers, supporting the specificity of the developed RT-LAMP assay.…”

“…In this study, the LAMP primer sets targeting the ORF1ab, N, and E genes were constructed based on RT-LAMP assays previously reported by the different laboratories, respectively ( Broughton et al, 2020 ; Dong et al, 2021 ; Jamwal et al, 2021 ; Jiang et al, 2020 ; Nawattanapaiboon et al, 2021 ; Park et al, 2020 ; Yu et al, 2021 ; Zhang & Tanner, 2021 ). A set of LAMP primers consisting of six primers (F3, B3, FIP, BIP, LF, LB) targeted eight distinct regions of the templates, and all primers were ordered from Sangon Biotech (Shanghai), LAMP primer mixtures (F3/B3 2 µM each; FIP/BIP 16 µM each; LF/LB 4 µM each) were prepared and used for the further RT-LAMP reactions.…”

Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay

“…RT-LAMP for COVID-19 diagnosis was also improved using more amenable samples, such as saliva. Rabe and Cepko, 15 and Yu et al 30 used saliva samples with a previous RNA extraction, while Lalli et al 22 and Kobayashi et al 31 reported an assay preserving RNA by the addition of proteinase K or guanidine/DTT to saliva samples, respectively. Several modifications to this test have been discussed.…”