A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR.
APMIS
; 129(7): 393-400, 2021 Jul.
Article
in English
| MEDLINE | ID: covidwho-1388189
Preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
See preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
See preprint
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.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Specimen Handling
/
RNA, Viral
/
COVID-19 Nucleic Acid Testing
/
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
APMIS
Journal subject:
Allergy and Immunology
/
Microbiology
/
Pathology
Year:
2021
Document Type:
Article
Affiliation country:
APM.13123
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