A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing
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Abstract
RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689 ), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.
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SciScore for 10.1101/2020.06.23.166397: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Informed consent was obtained from all patients. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Direct sample lysis buffer test: HEK293 cells were trypsinized and counted to make the appropriate dilutions in HBSS. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)As sample input for the proof-of-concept experiment shown in Figure S5D-F, sample pools containing different numbers of Covid-19 positive patient gargle sample in QuickExtract were mixed with HeLa cell lysate in … SciScore for 10.1101/2020.06.23.166397: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Informed consent was obtained from all patients. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Direct sample lysis buffer test: HEK293 cells were trypsinized and counted to make the appropriate dilutions in HBSS. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)As sample input for the proof-of-concept experiment shown in Figure S5D-F, sample pools containing different numbers of Covid-19 positive patient gargle sample in QuickExtract were mixed with HeLa cell lysate in QuickExtract. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)Software and Algorithms Sentences Resources 40 µl (matching the smallest pooled sample volume) of a Covid-19 positive or negative patient gargle sample were used as positive (qPCR positive) or negative (qPCR negative) controls, and also filled up to 100 µl with HBSS:QuickExtract (1:1) before LAMP. LAMPsuggested: (LAMP, RRID:SCR_001740)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.
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SciScore for 10.1101/2020.06.23.166397: (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. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Shown are percentages of positive ( detected in RT-LAMP and RT-qPCR , blue bars ) and negative ( not detected in either RT-LAMP or RTqPCR , black bars ) predictive agreement for sample groups ( defined by RT-qPCR-derived Cq values ) between RT-LAMP ( using E- and/or N-gene primers ) and 1-step RT-qPCR . D ) Cartoon indicating the workflow for testing different crude sample preparation methods using HEK293 cells as … SciScore for 10.1101/2020.06.23.166397: (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. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Shown are percentages of positive ( detected in RT-LAMP and RT-qPCR , blue bars ) and negative ( not detected in either RT-LAMP or RTqPCR , black bars ) predictive agreement for sample groups ( defined by RT-qPCR-derived Cq values ) between RT-LAMP ( using E- and/or N-gene primers ) and 1-step RT-qPCR . D ) Cartoon indicating the workflow for testing different crude sample preparation methods using HEK293 cells as input . HEK293suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045The image ( left ) shows HNB end-point colorimetric readout and the heatmap ( right ) shows co-measured end-point relative fluorescence units ( RFUs ) of RT-LAMP on serially diluted patient samples in QuickExtract-prepared HeLa cell lysate , with or without prior bead enrichment . HeLasuggested: CLS Cat# 300194/p772_HeLa, CVCL_0030Software and Algorithms Sentences Resources Considering robustness , a rarely discussed problem is that performing RT-LAMP outside certified diagnostic laboratories can easily lead to carryover crosscontamination , resulting in potentially large numbers of false positives ( Hsieh , Mage , Magesuggested: (MAGE, SCR_002313)D ) Performance ( measured as ‘time to threshold’ ) of different Bst DNA polymerase variants for LAMP in combination with NEB’s RTx Reverse Transcriptase on synthetic SARS-CoV-2 RNA standard. LAMPsuggested: (LAMP, SCR_001740)Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
- . The most significant limitation for SARS-CoV-2 testing in developing countries, however, is reagent availability.
- Our data indicate that Bst 2.0, in combination with RTx Reverse Transcriptase, shows superior performance over the original Bst LF in terms of reaction speed, assay sensitivity and dUTP incorporation efficiency.
- Bst 2.0 and RTx enzymes are engineered and proprietary enzymes.
- The open access sharing of expression plasmids for on-site production is therefore not possible.
- Efforts from research laboratories and the business sector are required to overcome socio-economic barriers preventing open reagent access by enabling the local production and distribution of RT-LAMP reagents, especially in developing countries.
- Combating the Covid-19 pandemic will require access to diagnostic tests in all countries ("The COVID-19 testing debacle," 2020).
- Our improvements of the RT-LAMP workflow provide a clear path forward to moving towards testing for everyone.
Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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