Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection

  1. yanxia liu
  2. zhengan cao
  3. mei chen
  4. Yan zhong
  5. yuhao luo
  6. gang shi
  7. Hongxia Xiang
  8. Jianping Luo
  9. Hongwei Zhou

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Abstract

Background: Real-time reverse transcription PCR (rRT-PCR) is commonly used to diagnose SARS-CoV-2 infection. Heat inactivation prior to nucleic acid isolation may allow safe testing, while the effects of heat inactivation on SARS-CoV-2 rRT-PCR detection result need to be determined. Methods: 14 positive nasopharyngeal swab specimens were inactivated at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min, and another 2 positive nasopharyngeal swab specimens were also inactivated at 100°C for 10min, 100°C for 30min, 100°C for 60min, after which the samples were isolated and detected by rRT-PCR. Results: All 14 heat treated samples remained positive. The range of threshold cycle (Ct) values observed when detecting ORF1a/b was 27.228-34.011 in heat-treated samples, while 25.281-34.861 in unheated samples, and the range of threshold cycle (Ct) values observed at the time of detecting N was 25.777-33.351 in heat-treated samples, while 24.1615-35.433 in unheated samples, on basis of which it showed no statistical difference otherwise a good correlation of Ct values between the heat-inactivated samples and the untreated samples. However, the 2 samples inactivated at 100°C 30min, 100°C 60min turned into negative. Conclusions: Heat inactivation at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min shall not affect the detection results of Real-Time Reverse Transcription PCR of the SARS-COV2. Furthermore, it is recommended to inactive nasopharyngeal swab specimens 10min at 100°C before RNA extraction in consideration of efficiency and reliable results. Key Words: SARS-CoV-2, Heat Inactivation, rRT-PCR, Comparison

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  1. ScreenIT

    SciScore for 10.1101/2020.05.19.20101469: (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
    Graphpad Prism software (Version8.0) was used for statistical analysis.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    Our research has some limitations. First, only nasopharyngeal swab samples were used for testing; which may make our results not applicable to those obtained using sputum, fecal specimen or blood samples. Second, we tested a relatively small number of samples under limit test condition, affecting the representative significance. Third, the thermostability of SARS-CoV-2 RNA in nasopharyngeal swab specimens at 100 °C for 10 min need to be determined. In the light of which, our findings need to be further verified by other large-scale researches and virus infectivity experiments using cell culture.

    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

    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 10.1101/2020.05.19.20101469v2 on medRxiv
  3. Version published to 10.1101/2020.05.19.20101469v1 on medRxiv