Homebrew reagents for low-cost RT-LAMP

  1. Tamara Matute
  2. Isaac Nuñez
  3. Maira Rivera
  4. Javiera Reyes
  5. Paula Blázquez-Sánchez
  6. Aníbal Arce
  7. Alexander J. Brown
  8. Chiara Gandini
  9. Jennifer Molloy
  10. César A. Ramírez-Sarmiento
  11. Fernán Federici

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Abstract

No abstract available

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  1. Version published to 10.7171/jbt.21-3203-006
  2. ScreenIT

    SciScore for 10.1101/2021.05.08.21256891: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    Expression and purification of M-MLV and BstLF: The M-MLV gene was synthesized as a Gblock from IDT and cloned into at pET-19b vector with N-terminal 10x His-tag.
    pET-19b
    suggested: RRID:Addgene_153312)
    BstLF DNA sequence from the OpenEnzyme collection was cloned into pET15b with a 6x His-tag at its N-terminus.
    pET15b
    suggested: RRID:Addgene_129689)
    Software and Algorithms
    SentencesResources
    Both DNAs can be obtained from the Open Bioeconomy Lab and/or FreeGenes collection32.
    FreeGenes
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A potential limitation of our proof of concept method is that the use of intercalating dyes can make target-specific real-time detection challenging44, and could pose as a barrier for use in resource limited areas. Future lower resource implementations of our method could substitute EvaGreen for 100-200 µM halochromic dyes, like Cresol Red, in pH 8.8 buffered reaction mix to cause detectable colorimetric change when amplification products have been produced47. Alternatively more advanced detection methods utilizing sequence specific methods based on the use of probes (e.g. LAMP-BEAC23, OSD48, DARQ49 and QUASR50) could also be utilized as end-point detection methods, and remain to be tested in future work. Such methods would be advantageous as they could be multiplexed for different targets in the same reaction. QUASR, for instance, has been shown to allow single step and close-tube multiplexing50. The versions of both M-MLV and BstLF enzymes used in this paper are in the public domain in Chile but some of the M-MLV mutations are still subject to protection in other jurisdictions e.g. in the US (patent no. US7056716B251) with anticipated expiration date of 15 March 2021. Future work will, therefore, explore the use of off-patent enzymes Open-MMLV or HIV RT15 from the E. coli expression toolkit from ReClone initiative52 in order to have freedom-to-operate in any country. The use of public domain enzymes would also permit commercial partnerships seeking to scale-up production lo...

    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.

    Results from scite Reference Check: We found no unreliable references.

    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 checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.05.08.21256891v1 on medRxiv