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RT-qPCR half-reaction optimization for the detection of SARS-CoV-2
Priscila Lamb Wink1,2,3, Fabiana Volpato1,2,4, Daiana de Lima-Morales1,2
1Hospital de Clínicas de Porto Alegre, Centro de Pesquisa Experimental, Laboratório de Pesquisa em Resistência Bacteriana, Porto Alegre, RS, Brasil.
Introduction:
The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is 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 the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity.
Results:
The RT-qPCR assay efficiency was 105% for 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. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR.
Conclusions:
The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.
