Portable and Label-Free Quantitative Loop-Mediated Isothermal Amplification (LF-qLamp) for Reliable COVID-19 Diagnostics in Three Minutes of Reaction Time: Arduino-Based Detection System Assisted by a pH Microelectrode

  1. Mario Moisés Alvarez
  2. Sergio Bravo-González
  3. Everardo González-González
  4. Grissel Trujillo-de Santiago

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.

Article activity feed

  1. Version published to 10.3390/bios11100386
  2. ScreenIT

    SciScore for 10.1101/2021.05.23.21256350: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Saliva samples were also collected from a COVID-19(+) patient after obtaining written consent and in full compliance with the principles stated in the Helsinki Declaration.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    These LAMP primers were designed to target a region of the sequence of the SARS-CoV-2 N gene; specifically, they were based on the analysis of alignments of the SARS-CoV-2 N gene sequences using the Geneious software (Auckland, New Zealand).
    Geneious
    suggested: (Geneious, RRID:SCR_010519)

    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: We detected the following sentences addressing limitations in the study:
    One of the limitations of colorimetric LAMP-based methods based on the phenol red color shift is that some degree of color change exists in samples containing no template (see also ref [27]). This has been associated to the occurrence of non-specific amplification induced by the interaction among the LAMP primers [23,32], which often serve as templates for off-target polymerizations. In general, non-specific amplification is believed to be the main reason for false positives in LAMP colorimetric reactions. Figure 4 A shows the typical profile of the progression of the electric potential in negative samples (with primers, reaction mix, but no SARS-CoV-2 template). Four representative replicates are shown. The shape of the progression of the electric potential in the negative samples is highly consistent. Figure 4B presents a normalization of the four representative curves shown in Figure 4A. The curves were normalized by subtracting the initial potential value for each curve. In doing so, all curves practically collapse into an invariant shape. The black line in Figure 4B depicts the average of the four curves corresponding to independently run negative samples. This profile, characteristic of negative samples, is distinguishable from that inherent to the specific amplification observed in samples that do contain SARS-CoV-2 genetic material. Figure 4C shows the trajectories of the electric potential associated with positive samples containing different quantities of SARS-CoV-2...

    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 10.1101/2021.05.23.21256350v1 on medRxiv