Dry loop–mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens

  1. Yuki Higashimoto
  2. Masaru Ihira
  3. Yoshiki Kawamura
  4. Masato Inaba
  5. Kazuya Shirato
  6. Tadaki Suzuki
  7. Hideki Hasegawa
  8. Tsutomu Kageyama
  9. Yohei Doi
  10. Tadayoshi Hata
  11. Tetsushi Yoshikawa

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Abstract

Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop–mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

Author summary

Coronavirus disease 2019 (COVID-19) is a major public health problem around the world. A reliable point-of-care (POC) test for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is urgently needed, especially in developing countries. The loop-mediated isothermal amplification (LAMP) method amplifies template nucleotides under isothermal conditions with high efficiency and specificity, both of which are major advantages for a POC test. In addition, because dry LAMP reagents can be stored at 4°C, it is suitable for use in developing countries.

We evaluated the specificity and sensitivity of the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan), a dry LAMP method for amplifying viral RNA. The initial validation study revealed that the method was highly specific and sensitive (lower detection limit: 10 copies/reaction). We then analyzed 24 nasopharyngeal swab specimens from patients suspected to have COVID-19. Using the Loopamp SARS-CoV-2 Detection kit, SARS-CoV-2 RNA was detected in 15 (62.5%) of the 24 samples. Compared with the standard real-time reverse transcription PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp SARS-CoV-2 Detection kit were 78.9%, 100%, 100%, and 55.6%, respectively.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.29.20204297: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was approved by the institutional review board of Fujita Health University (No. HM19-493).
    Consent: Written informed consent was obtained from each patient.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    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.

    About SciScore

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  2. Version published to 10.1101/2020.09.29.20204297v1 on medRxiv