Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Background
Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.
Results
Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.
Conclusions
LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
Article activity feed
-
-
-
-
-
-
SciScore for 10.1101/2020.08.13.20173757: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources RT-LAMP reaction mixtures: RT-LAMP reactions were prepared by mixing 7.5 μl commercial 2x LAMP master mix (NEB E1700L) or our own LAMP mix (40 mM TrisHCl, pH 8.5, 20 mM (NH4)2SO4, 100 mM KCl, 16 mM MgSO4, 0.2% Tween-20, 2.8 mM each dNTP, 16 µg/ml polA LF, and 2.6-7.7 µg/ml RT) with 1.5 μl of 10x primer/beacon master mix (final concentration: 1.6 μM FIP/BIP, 0.2 μM F3/B3, 0.4 μM LF/LB, LAMPsuggested: (LAMP, RRID:SCR_001740)The assay was performed using the ThermoFisher … SciScore for 10.1101/2020.08.13.20173757: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources RT-LAMP reaction mixtures: RT-LAMP reactions were prepared by mixing 7.5 μl commercial 2x LAMP master mix (NEB E1700L) or our own LAMP mix (40 mM TrisHCl, pH 8.5, 20 mM (NH4)2SO4, 100 mM KCl, 16 mM MgSO4, 0.2% Tween-20, 2.8 mM each dNTP, 16 µg/ml polA LF, and 2.6-7.7 µg/ml RT) with 1.5 μl of 10x primer/beacon master mix (final concentration: 1.6 μM FIP/BIP, 0.2 μM F3/B3, 0.4 μM LF/LB, LAMPsuggested: (LAMP, RRID:SCR_001740)The assay was performed using the ThermoFisher QuantStudio 5. ThermoFisher QuantStudiosuggested: (Primer Express Software, RRID:SCR_017376)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-