Point of care testing for detection of coronaviruses including SARS-CoV-2 from saliva without treating RNA in advance

  1. Masaaki Muraoka
  2. Osamu Kawaguchi
  3. Mikio Mizukoshi
  4. Shunsuke Sejima

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Abstract

Coronavirus disease 2019 (COVID-19) outbreak was reported to the WHO (World Health Organization) as an outbreak on end of 2019, afterwards pandemic on the worldwide in 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has be reported it to cause COVID-19, and highly transmissible. Therefore, it is important that it is rapidly and continuously detected and monitored on site so that the infection is prevented. Namely, POCT (point of care testing) may be important to control cross infection of SARS-CoV-2. At present, the routine confirmation of SARS-CoV-2 is based on detection of sequence unique in the virus RNA by nucleic acid amplification tests (NAATs) such as rRT-PCR. Taking POCT into account, it is clear that it takes time and labour very much or much. Thus, it was our purpose this time to contribute to develop POCT for microbes such as SARS-CoV-2, and thus needed to improve NAATs method.

First, combining the mobile real-time RT-PCR (rRT-PCR) device PCR1100 with the appropriate rRT-PCR reagent, we found that it is possible to detect RNA of SARS-CoV-2 for less 14 minutes with equivalent accuracy to conventional devices. Next, we found that the above method made it possible for us to detect coronaviruses by direct rRT-PCR without pre-treatments. Furthermore, it also made clear that coronaviruses in saliva could be detected by the similar direct rRT-PCR method.

Hence, it was concluded that this method made it possible to detect virus in saliva without treating in advance (extraction, purification, concentration, etc.), and moreover, samples would be able to be collected with non-invasive. For this reason, we suggest that this method is useful for POCT of coronaviruses including SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.01.21256441: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: ETHICS STATEMENT: The study was approved by the Ethics Committee of Certified Non-Profit Organization Biomedical Science Association, Japan (BMSA2020-12-01) for the evaluation of detecting HCoV-229E in saliva.
    Consent: Spoken informed consent was obtained from each volunteer participated to offer saliva.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    HCoV-229E was purchased (VR-740™, American Type Culture Collection [ATCC]®, USA), followed by infection with MRC-5 cells (CCL-171™, ATCC®) and proliferating with EMEM medium (30-2003™, ATCC®) included 2 % FBS (30-2020™, ATCC®) for 5 to 8 days in 5 % CO2 at 35 °C.
    MRC-5
    suggested: None
    Primer and Probe: Based on previously reported protocols, for SARS-CoV-2 by the NIID protocol [10, 11] and the CDC protocol [9, 13], and moreover, for HCoV-229E [15], respectively, oligonucleotide primers and probes for rRT-PCR detection were selected to target each N gene and synthesised (Nihon Gene Research Laboratories Inc., Japan) (Table 1).
    HCoV-229E
    suggested: None
    Software and Algorithms
    SentencesResources
    Primer and Probe: Based on previously reported protocols, for SARS-CoV-2 by the NIID protocol [10, 11] and the CDC protocol [9, 13], and moreover, for HCoV-229E [15], respectively, oligonucleotide primers and probes for rRT-PCR detection were selected to target each N gene and synthesised (Nihon Gene Research Laboratories Inc., Japan) (Table 1).
    Nihon Gene Research Laboratories
    suggested: (University of Southern California; Los Angeles; USA, RRID:SCR_008093)

    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.

    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.

  2. Version published to 10.1101/2021.05.01.21256441 on medRxiv