Exploring the use of nested RT-PCR for detecting a nuclear gene of SARS-CoV-2 confirming spike gene detection

Background: Laboratories used different diagnostic methods to detect SARS-CoV-2 at a time when the world was unprepared for a pandemic like COVID-19. Although real-time PCR is a gold standard diagnostic method, its use in diagnosing SARS-CoV-2 has decreased significantly due to its high costs, and thus it cannot be used for a large number of samples. Aim: The current study aimed to compare a commercial kit targeting S and N genes using reverse transcriptase real-time PCR (rRT-PCR) with a conventional nested RT-PCR approach for the N gene marker, thereby improving the true positive results obtained from other tests. Methods: This study included 80 COVID-19 patients from Baghdad Teaching Hospital with moderate (50) and severe (30) infection. The positive samples in fast rRT-PCR for SARS-CoV-2 species were selected to implement a conventional one-step RT-PCR, with nested PCR as the initial run. Results: Findings showed significant differences in detection (p = 0.002) of the amplification nested RT-PCR of a nuclear region in 48/80 (60%) SARS-CoV-2-positive samples in the first run and all 80 samples in the second run. The nested RT-PCR provided powerful detection for clinical cases with high Ct values, indicating low viral loads. Conclusion: nested RT-PCR confirmed ERIDA's reactivity to the E, RdRp, and S genes, but not the N gene. This suggests that a nuclear gene profile with a recently evolved Spike gene can detect SARS-CoV-2 more effectively in the early stages of the pandemic, particularly among variants and mutations. Nested PCR offers five multiplications over Simplex PCR, making it promising for detecting low-abundance spike and nuclear genes.