A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings

  1. Arunkumar Arumugam
  2. Matthew L. Faron
  3. Peter Yu
  4. Cole Markham
  5. Season Wong

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Abstract

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. RT-qPCR requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers, which are not available in many low resource settings and developing countries. As a pandemic, COVID-19 has quickly spread to the rest of the world. Many underdeveloped and developing counties do not have the means for fast and accurate COVID-19 detection to control this outbreak. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 minutes using untreated samples, heat-inactivated samples, or extracted RNA templates. Rapid RT-PCR was achieved using thin-walled PCR tubes and a setup including sous vide immersion heaters/circulators. Our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in situations where sophisticated laboratory instruments are not available.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.29.069591: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Alternatively, software such as ImageJ can be utilized.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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:
    The only limitation we expect for this set up is when using it in high altitude locations where boiling temperature is lower than 95°C. Lower denaturation temperature setting would be needed, along with longer denaturation time. RT-qPCR analysis of ten COVID-19 positive clinical samples: To determine the relative viral load of the clinical samples, 60 μL of the specimen was extracted using an automated system (Promega Maxwell) and run on a commercial real-time thermal cycler. Three μL of the templates were used in each RT-qPCR reaction. The qPCR threshold cycle (Ct) values of these samples are listed in Table 1. It took the commercial cycler 1 hour and 22 minutes to complete the reaction. Furthermore, to test the need for RNA extraction, 3 μLs of each media was spiked in 17 μL of the master mix. The results show that untreated media samples can produce qualitative PCR results matching those performed using extracted RNA templates. Rapid RT-PCR detection of SARS-CoV-2 with N1, N2, and RNase P reactions using water baths: To test for SAR-CoV-2 using water baths, we prepared three singleplex PCR reactions, each targeting N1, N2, and RNase P. Extracted templates from COVID-19 positive clinical specimens and contrived negative samples were tested using water bath-based RT-PCR. The RT-PCR run was completed in 12 minutes. The picture of the PCR tubes after 40 cycles shows that we can use a blue LED gel box and cell phone camera to determine the test results (Figure 2). In a COVID-19...

    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.

    About SciScore

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  2. Version published to 10.1101/2020.04.29.069591v1 on bioRxiv