Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis.
Int J Infect Dis
; 102: 437-439, 2021 Jan.
Article
in English
| MEDLINE | ID: covidwho-997013
ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers-probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Real-Time Polymerase Chain Reaction
/
SARS-CoV-2
/
COVID-19
Type of study:
Case report
/
Diagnostic study
/
Observational study
/
Prognostic study
Topics:
Long Covid
Limits:
Humans
Language:
English
Journal:
Int J Infect Dis
Journal subject:
Communicable Diseases
Year:
2021
Document Type:
Article
Affiliation country:
J.ijid.2020.10.079
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