RT-qPCR half reaction optimization for the detection of SARS-CoV-2

Wink, P. L.; Volpato, F.; Lima-Morales, D.; Paiva, R. M.; Willig, J. B.; Bock, H.; de-Paris, F.; Barth, A. L.

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/uL, 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.