A simple, safe and sensitive method for SARS‐CoV‐2 inactivation and RNA extraction for RT‐qPCR
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Abstract
The SARS‐CoV‐2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS‐CoV‐2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two‐step RT‐qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one‐step RT‐qPCR against SARS‐CoV‐2 using NP and OP samples. We furthermore tested a SARS‐CoV‐2 dilution series to determine the detection threshold. The method enables downstream detection of SARS‐CoV‐2 by RT‐qPCR with high sensitivity (~4 viral RNA copies per RT‐qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT‐qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR‐ready viral RNA and circumvents the need for commercial RNA purification kits.
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SciScore for 10.1101/2020.06.29.179176: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. 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 Negative controls were reactions without the RT enzyme and without sample, and the positive control was RNA isolated from HeLa cells. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)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: An explicit section about the limitations of the …SciScore for 10.1101/2020.06.29.179176: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. 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 Negative controls were reactions without the RT enzyme and without sample, and the positive control was RNA isolated from HeLa cells. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)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: 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.
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