Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs.
Journal
Disease markers
ISSN: 1875-8630
Titre abrégé: Dis Markers
Pays: United States
ID NLM: 8604127
Informations de publication
Date de publication:
2021
2021
Historique:
received:
28
07
2020
revised:
15
01
2021
accepted:
23
02
2021
entrez:
22
3
2021
pubmed:
23
3
2021
medline:
14
4
2021
Statut:
epublish
Résumé
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.
Sections du résumé
BACKGROUND
BACKGROUND
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples.
METHODS
METHODS
The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy).
RESULTS
RESULTS
The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2
CONCLUSION
CONCLUSIONS
During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.
Identifiants
pubmed: 33747257
doi: 10.1155/2021/8890221
pmc: PMC7934789
doi:
Substances chimiques
RNA, Viral
0
Types de publication
Journal Article
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
8890221Informations de copyright
Copyright © 2021 Catia Mio et al.
Déclaration de conflit d'intérêts
The authors declare no conflicts of interest.
Références
Clin Biochem Rev. 2008 Aug;29 Suppl 1:S49-52
pubmed: 18852857
Eur J Nucl Med Mol Imaging. 2020 May;47(5):1275-1280
pubmed: 32107577
Infez Med. 2020 Ahead of print Jun 1;28(2):174-184
pubmed: 32275259
Euro Surveill. 2020 Jan;25(3):
pubmed: 31992387
Talanta. 2021 Mar 1;224:121726
pubmed: 33379001
Int J Antimicrob Agents. 2020 Mar;55(3):105924
pubmed: 32081636
Anal Chem. 2021 Jan 19;93(2):715-721
pubmed: 33289545
Dis Markers. 2020 Dec 9;2020:8869424
pubmed: 33343767
Emerg Microbes Infect. 2020 Dec;9(1):1259-1268
pubmed: 32438868
Mol Med Rep. 2017 Aug;16(2):1157-1166
pubmed: 29067441
Ann Hematol. 2018 Dec;97(12):2299-2308
pubmed: 30056580
PLoS Negl Trop Dis. 2018 Dec 26;12(12):e0007063
pubmed: 30586355
PLoS One. 2020 Sep 8;15(9):e0236311
pubmed: 32898153
Biomol Detect Quantif. 2017 Apr 29;12:1-6
pubmed: 28702366
Biomol Detect Quantif. 2016 Aug 30;9:29-39
pubmed: 27617230
Mol Oncol. 2018 Apr;12(4):545-560
pubmed: 29465788
Clin Exp Pediatr. 2020 Apr;63(4):119-124
pubmed: 32252141
Anal Bioanal Chem. 2014 Jan;406(3):661-7
pubmed: 24276251
Aging (Albany NY). 2020 Nov 1;12(21):20997-21003
pubmed: 33136068
Clin Chem. 2020 Apr 1;66(4):549-555
pubmed: 32031583
Ann Hematol. 2016 Apr;95(5):739-44
pubmed: 26931113
J Int AIDS Soc. 2018 Sep;21(9):e25185
pubmed: 30375818