Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
The SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.
Article activity feed
-
SciScore for 10.1101/2020.04.26.20081307: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical statement: The study was conducted at the University of the Basque Country (UPV/EHU, Leioa, Spain) in collaboration with the Cruces University Hospital after approval by the UPV/EHU Ethics Committee (project 2020/059, CEIAB Randomization not detected. Blinding In a second stage of blind validation, we used 105 additional NPS samples (Set #2), unaware of the diagnosis assessment made by HUC. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources As a negative control, RNA extracted from non-infected HeLa cells was included (30 ng/well). HeLaSciScore for 10.1101/2020.04.26.20081307: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical statement: The study was conducted at the University of the Basque Country (UPV/EHU, Leioa, Spain) in collaboration with the Cruces University Hospital after approval by the UPV/EHU Ethics Committee (project 2020/059, CEIAB Randomization not detected. Blinding In a second stage of blind validation, we used 105 additional NPS samples (Set #2), unaware of the diagnosis assessment made by HUC. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources As a negative control, RNA extracted from non-infected HeLa cells was included (30 ng/well). HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)Software and Algorithms Sentences Resources RT-qPCR was performed using the NZYTech Speedy One-step RT-qPCR Probe Master Mix (NZYTech, Lisbon, Portugal), following the manufacturer’s instructions. NZYTechsuggested: (NZYTech, RRID:SCR_016772)The primers for the E gene, which encodes for the envelope, were discarded because of their low specificity and hybridization in other viral targets based on BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). BLASTsuggested: (BLASTX, RRID:SCR_001653)https://blast.ncbi.nlm.nih.gov/Blast.cgisuggested: (TBLASTX, RRID:SCR_011823)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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.
-
