Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

  1. Shovon Lal Sarkar
  2. A. S. M. Rubayet Ul Alam
  3. Prosanto Kumar Das
  4. Md. Hasan Ali Pramanik
  5. Hassan M. Al-Emran
  6. Iqbal Kabir Jahid
  7. M. Anwar Hossain

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Abstract

TaqMan probe-based commercial real-time (RT) PCR kits are expensive but most frequently used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections needs to meet the challenge of testing more persons at a reasonable cost. This study developed a simple and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay targeting two virus-specific genes along with a host-specific internal control. A total of 180 randomly selected samples portioning into two subsets based on crude and high-quality RNA extraction were used to compare this assay with a nationwide available commercial kit (Sansure Biotech Inc., (Hunan, China)), so that we could analyze the variation and validity of this in-house developed method. Our customized-designed primers can specifically detect the viral RNA likewise Sansure. We separately optimized SYBR Green RT-PCR reaction of N, E, S, and RdRp genes based on singleplex melting curve analysis at the initial stage. After several rounds of optimization on multiplex assays of different primer combinations, the optimized method finally targeted N and E genes of the SARS-CoV-2 virus, together with the β-actin gene of the host as an internal control. Comparing with the Sansure commercial kit, our proposed assay provided up to 97% specificity and 93% sensitivity. The cost of each sample processing ranged between ~2 and ~6 USD depending on the purification level of extracted RNA template. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in low-income countries.

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  1. Version published to 10.1038/s41598-022-10413-7
  2. ScreenIT

    SciScore for 10.1101/2021.05.06.21256629: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The study was approved by the ethical review committee (ERC) of Jashore University of Science and Technology, Bangladesh (Reference: ERC/FBS/JUST/2020-45, Date: 06/10/2020).
    Sex as a biological variablenot detected.
    RandomizationClinical sample selection and ethical consideration: The sample size was calculated based on the prevalence of positive samples using the following equation

    Randomly (random number generator using Microsoft Excel inc.) selected 6 positive and 4 negative samples were selected from left-over samples once in a week and tested by our proposed method besides the routine TaqMan based method.

    Blindingnot detected.
    Power AnalysisClinical sample selection and ethical consideration: The sample size was calculated based on the prevalence of positive samples using the following equation

    Randomly (random number generator using Microsoft Excel inc.) selected 6 positive and 4 negative samples were selected from left-over samples once in a week and tested by our proposed method besides the routine TaqMan based method.

    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    , HCoV-229E, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV, and finally main respiratory and opportunistic viruses and pathogens in PrimerBLAST (supplementary table S1).
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    Software and Algorithms
    SentencesResources
    Clinical sample selection and ethical consideration: The sample size was calculated based on the prevalence of positive samples using the following equation

    Randomly (random number generator using Microsoft Excel inc.) selected 6 positive and 4 negative samples were selected from left-over samples once in a week and tested by our proposed method besides the routine TaqMan based method.

    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Validation by Gel Electrophoresis and Targeted Amplification: We performed gel electrophoresis in 3% agarose gel having ethidium bromide at 60V and 100mA for 100 minutes to ensure amplification of the correct RT-qPCR products and analyzed in an automated Gel Doc Imager (Molecular Imager® Gel Doc™ XR+ System with Image Lab™ Software by Bio-Rad (Catalog # 170-8195) and the software Image Lab™ Software version 5.2.1.
    Image Lab™
    suggested: (Image Lab Software, RRID:SCR_014210)
    The four representative amplicons were then subjected to Sanger sequencing with BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) in Applied Biosystems SeqStudio genetic analyzer as per the optimized protocol of Islam et al. (2021).
    BigDye™
    suggested: (UH Manoa Sequencing Facility, RRID:SCR_010114)
    NCBI BLAST was performed initially and the alignment to SARS-CoV-2 spike gene was also checked in MEGA7 (https://www.megasoftware.net/).
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    MEGA7
    suggested: None

    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:
    Nevertheless, this study has some limitations. We could not be consistent with the gold standard outcomes obtained by commercial kits with respect to Ct value measured for each gene. Discrepant results wherein one gene was detected in SYBR green assay were observed in more than half of the samples for both SYBR positive-Sansure kit positive (27/49) and SYBR positive-Sansurel kit negative groups (17/27). Other researchers however reported the similar trend despite with a very small percentage of the samples 20. Using a lower amount of RNA template, the less reliable results than TaqMan assay, multiplexing of three genes in SYBR Green, and arises of possible mutations during the study in highly dynamic nucleocapsid protein20,21 might reduce the chance to amplify N gene specific region. Moreover, the sensitivity and the specificity were not completely determined due to not validating all the studied samples, though an identical sensitivity and specificity was determined while comparing the 33 deviated samples between SYBR Green and TaqMan assay (Table 2). The reasons behind a low R2 value (0.938 for JUST_N1 and 0.917 for JUST_E1) might be the presence of non-specific RNA due to a quick RNA extraction system, possible presence of polymerase inhibitors, and formation of primer-dimer. Another subtle limitation of our technology is that only expert personnel will be able to analyze the results since it is based on melting curve analysis. SYBR Green technique was suitable for detecti...

    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.
    • Thank you for including a protocol registration statement.

    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.

  3. Version published to 10.1101/2021.05.06.21256629 on medRxiv