RT-qPCR half reaction optimization for the detection of SARS-CoV-2
Abstract
BACKGROUND
The main laboratory test for the diagnosis of COVID-19 is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, the RT-qPCR is an expensive method due to the number of tests required.
OBJECTIVES
To evaluate an alternative RT-qPCR approach for the detection of SARS-CoV-2 sing half of the total volume currently recommended by the US Centers for Disease Control and Prevention.
METHODS
The analytical limit of detection (LoD) and the reaction efficiency using half volumes of RT-qPCR assay were evaluated for both the N1 and N2 regions by using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples were evaluated to establish the clinical sensitivity and specificity.
FINDINGS
The RT-qPCR assay efficiency was 105% for both the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. Clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds when compared with the standard RT-qPCR.
CONCLUSIONS
The use of RT-qPCR half reaction proved to be a feasible and economic strategy for detection of SARS-CoV-2 RNA.
Sponsorship
This work was supported by FAPERGS (20/2551-0000265-9) and by Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) (Project no. 2020-0163).
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