Preliminary evaluation of qualitative RT-PCR and indirect immunofluorescence for SARS-CoV-2 detection in nasopharyngeal swabs samples

Introduction: The COVID-19 pandemic made it essential to expand SARS-CoV-2 diagnostic capacity. Two laboratory techniques—qualitative RT-PCR and indirect immunofluorescence (IIF)—were preliminarily evaluated for virus detection in nasopharyngeal samples. Methods: RNA extracted from nasopharyngeal swabs of patients with infection confirmed by RT-qPCR was used to establish the amplification conditions for a 128-bp fragment of the SARS-CoV-2 N gene by RT-PCR. Under the selected conditions, 65 samples from patients with clinical manifestations compatible with COVID-19 were analyzed. For IIF, a purified preparation of anti-SARS-CoV-2 IgG antibodies obtained from positive serum samples previously analyzed with a commercial ELISA was used. The antibodies bound to viral antigens in cells from nasopharyngeal swabs and were detected with a fluorescein isothiocyanate-conjugated anti-human IgG antibody. Results: The selected conditions for amplification of the 128-bp fragment were 1.5 mM MgCl₂, 0.6 µM of each primer, 0.2 mM dNTPs, 5.0 U of Taq polymerase and 5.0 U of reverse transcriptase, and an annealing temperature of 57 °C. Under these conditions, 64 of 65 samples (98.46%) yielded the expected amplicon. IIF revealed fluorescence-positive cells and showed no reactivity against the evaluated panel of other viruses. Conclusion: Qualitative RT-PCR and IIF showed favorable preliminary results for SARS-CoV-2 detection in nasopharyngeal samples. A complete analytical and clinical evaluation is required before either technique can be considered for diagnostic use.