Context.—: The gold standard test to identify the presence of SARS-CoV-2 in COVID-19 patients is the real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), but inconclusive data and false-positive diagnosis remain the major problem of this approach.
Objective.—: To compare the fitness of 2 primer sets to the SARS-CoV-2 nucleocapsid phosphoprotein gene (NP) in the molecular diagnosis of COVID-19, we verified the inconclusive data and confidence of high cycle threshold (Ct) values in SARS-CoV-2 detection.
Design.—: The 970 patient samples were tested by using United States Centers for Disease Control and Prevention protocol. We compared the fitness of 2 primer sets to 2 different regions of the NP gene. In addition, we checked the consistency of positive samples with high Ct values by retesting extracted SARS-CoV-2 RNA or by second testing of patients.
Results.—: N1 and N2 displayed similar fitness during testing, with no differences between Ct values. Then, we verified security range Ct values related to positive diagnostics, with Ct values above 34 failing in 21 of 32 cases (65.6%) after retesting of samples. The patient samples with Ct values above 34.89 that were doubly positive revealed a low sensitivity (52.4%) and specificity (63.6%) of the test in samples with Ct values above 34.
Conclusions.—: It is safe to use 1 primer set for the NP gene to identify SARS-CoV-2 in samples. However, samples with high Ct values may be considered inconclusive and retested to avoid false-positive diagnosis.