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.
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 ten-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.
A protocol for the detection of SARS-CoV-2 from saliva samples featuring a rapid purification step and a highcontrast colorimetric readout. Saliva is first inactivated using a 100x inactivation reagent consisting of 2.5M TCEP, 100 mM EDTA, 1.2N NaOH solution diluted to approximately 1x final concentration and heated to 95C for 5 minutes. RNA is rapidly purified and concentrated with magnetic beads in a PEG/NaCl-based buffer using a 3Dprinted magnetic stick that enables selective separation of beads without carryover of saliva contaminants. Beads are eluted directly into an RT-LAMP reaction mix, which uses a novel high contrast dye that turns from purple to clear when acidified by nucleic acid amplification products that enables unambiguous identification of successful amplification. This protocol is sensitive down to 1 copy/µl of SARS-CoV-2 in 300 µl of saliva. This degree of sensitivity enables faithful detection of SARS-CoV-2 even in pooled samples.
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