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. 2020 Jul 24;15(7):e0236564.
doi: 10.1371/journal.pone.0236564. eCollection 2020.

Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

Mohammad Rubayet Hasan  1   2 , Faheem Mirza  1 , Hamad Al-Hail  1 , Sathyavathi Sundararaju  1 , Thabisile Xaba  1 , Muhammad Iqbal  1 , Hashim Alhussain  3 , Hadi Mohamad Yassine  3 , Andres Perez-Lopez  1   2 , Patrick Tang  1   2
Affiliations

Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

Mohammad Rubayet Hasan et al. PLoS One. .

Erratum in

Abstract

To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x103 copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR CT values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Linearity of SARS-CoV-2 RT-qPCR by direct approach.
A tittered SARS-CoV-2 positive nasopharyngeal specimen was serially diluted using a negative specimen, pre-heated at 65°C for 10 min and assessed by SARS-CoV-2 RT-qPCR as described in the Supplemental methods. A) Amplification curves from SARS-CoV-2 RT-qPCR assay on the serially diluted sample. B) RT-qPCR CT values were plotted against estimated copy number of SARS-CoV-2 RNA in each dilution. Each data point represents an average of data obtained from 8 replicates.
Fig 2
Fig 2. Correlation of RT = qPCR CT values obtained by direct versus standard approach.
See this image and copyright information in PMC

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References

    1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coronavirus from Patients with Pneumonia in China, N Engl J Med 2019;382(8):727–733. - PMC - PubMed
    1. Sohrabi C, Alsafi Z, O’Neill N, Khan M, Kerwan A, Al-Jabir A, et al. World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg 2020;76:71–76. - PMC - PubMed
    1. Steffens I. A hundred days into the coronavirus disease (COVID-19) pandemic, Eurosurveillance. https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.14.... Accessed 18 April 2020. - DOI - PMC - PubMed
    1. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020;25(3). - PMC - PubMed
    1. Ranney ML, Griffeth V, Jha AK. Critical Supply Shortages—The Need for Ventilators and Personal Protective Equipment during the Covid-19 Pandemic. N Engl J Med 2020; 10.1056/NEJMp2006141 - DOI - PubMed

Grants and funding

The authors received no specific funding for this work.