One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection.
J Infect Dev Ctries
; 14(7): 679-684, 2020 07 31.
Article
in English
| MEDLINE | ID: covidwho-721535
ABSTRACT
INTRODUCTION:
Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR.METHODOLOGY:
The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR.RESULTS:
Amplification was achieved for the positive control and specific regions in both techniques.CONCLUSIONS:
This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Pneumonia, Viral
/
RNA, Viral
/
Coronavirus Infections
/
Real-Time Polymerase Chain Reaction
/
Betacoronavirus
Type of study:
Diagnostic study
/
Prognostic study
Topics:
Vaccines
Limits:
Humans
Language:
English
Journal:
J Infect Dev Ctries
Journal subject:
Communicable Diseases
Year:
2020
Document Type:
Article
Affiliation country:
Jidc.12726
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