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. 2020 Oct 30;10(1):18764.
doi: 10.1038/s41598-020-75958-x.

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Michela Deiana  1 , Antonio Mori  1   2 , Chiara Piubelli  1 , Salvatore Scarso  1 , Mosè Favarato  3 , Elena Pomari  4
Affiliations

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Michela Deiana et al. Sci Rep. .

Abstract

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Results of the limit of detection analysis on UTM.
Figure 2
Figure 2
Results of the limit of detection analysis on ESwab.
Figure 3
Figure 3
Flow chart of the study.
See this image and copyright information in PMC

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References

    1. Astuti I, Ysrafil Y. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an overview of viral structure and host response. Diabetes Metab. Syndr. Clin. Res. Rev. 2020;14(4):407–412. doi: 10.1016/j.dsx.2020.04.020. - DOI - PMC - PubMed
    1. Venter M, Richter K. Towards effective diagnostic assays for COVID-19: a review. J. Clin. Pathol. 2020;73(7):370–377. doi: 10.1136/jclinpath-2020-206685. - DOI - PubMed
    1. Corman VM, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance. 2020;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045. - DOI - PMC - PubMed
    1. Monleau M, et al. Evaluation of different RNA extraction methods and storage conditions of dried plasma or blood spots for human immunodeficiency virus type 1 RNA quantification and PCR amplification for drug resistance testing ∇. J. Clin. Microbiol. 2009;47(4):1107–1118. doi: 10.1128/JCM.02255-08. - DOI - PMC - PubMed
    1. Lim NYN, Roco CA, Frostegård Å. Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses. Front. Microbiol. 2016;7:1588. doi: 10.3389/fmicb.2016.01588. - DOI - PMC - PubMed

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