Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

  1. Soon Keong Wee
  2. Suppiah Paramalingam Sivalingam
  3. Eric Peng Huat Yap

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Abstract

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.17.042366: (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
    The slopes and intercepts of the linear regression lines were tested for statistical significance using Analysis of Covariance (ANCOVA) (GraphPad Prism 6).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One caveat however for fast DIRECT-PCR of samples with low viral load is that of sampling error, since only 1 μL of sample is used. Nucleic acid extractions, on the other hand, serve to concentrate RNA from typical sample volumes of 150-300 μL, although their yield can also be low and variable. Hence, where samples are expected to have low counts near to the limit of detection, DIRECT-PCR with larger volume reactions (25 μL) to include higher template volume may be necessary to reduce risk of false negatives. Reduction in amplification efficiency is a common concern in DIRECT-PCR from crude samples (e.g. respiratory samples, blood and serum). The presence of PCR inhibitors can decrease the sensitivity and accuracy of pathogen detection through interfering with polymerase activity, degradation of nucleic acids and efficient cell lysis (23). A variety of methods have been developed to overcome such inhibition, including inhibitor tolerant polymerases, additives and buffer modification. In our study, we used one commercially available formulation that tolerated PCR inhibition in sputum and nasal exudates as well as blood/serum/urine (data not shown), and it is not unlikely that other formulations could be used as well in our study. While amplification directly from sputum and nasal exudate reduced the efficiency of PCR compared to water controls, there was no net effect on the threshold of detection. Nasopharyngeal swabs and sputum have been suggested to be effective clinical sa...

    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 10.1101/2020.04.17.042366v1 on bioRxiv