Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow
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Abstract
The ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65°C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.
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SciScore for 10.1101/2020.06.03.131474: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization The FRA is a barcoded transposome complex, which can cleave randomly DNA and add barcoded transposase adapters to the cleavage sites. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources All primers were aligned to the published SARS-CoV-2 sequences and others respiratory using BLAST. BLASTsuggested: (BLASTX, RRID:SCR_001653)All the dilution gradient and pure water (negative control) were amplified by LAMP. LAMPsuggested: (LAMP, RRID:SCR_001740)To test the limit of detection, the amplification products of dilution gradient 3.25×10^4, 3.25×10^3, … SciScore for 10.1101/2020.06.03.131474: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization The FRA is a barcoded transposome complex, which can cleave randomly DNA and add barcoded transposase adapters to the cleavage sites. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources All primers were aligned to the published SARS-CoV-2 sequences and others respiratory using BLAST. BLASTsuggested: (BLASTX, RRID:SCR_001653)All the dilution gradient and pure water (negative control) were amplified by LAMP. LAMPsuggested: (LAMP, RRID:SCR_001740)To test the limit of detection, the amplification products of dilution gradient 3.25×10^4, 3.25×10^3, 1.1 × 10^3, 6.5 × 10^2, 3.25×10^2 copies/mL and negative control total 12 samples were constructed another barcoding library (Oxford Nanopore, SQK-RBK004) as described above and sequenced using a PromethION flowcell to achieve more data. PromethIONsuggested: (PromethION, RRID:SCR_017987)Quality control was processed by MinKNOW (version 3.6.5) software pipeline in the machine with reads quality value score cutoff 7. MinKNOWsuggested: NoneWe align nanopore long reads to virus genome database with minimap2 (version 2.1) and use samtools (version 1.9) to demonstrate the coverage and depth. samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)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.
- No funding statement was detected.
- No protocol registration statement was detected.
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