Unmasked G-quadruplexes for real-time fluorescence detection of viral genomic RNA

Real-time (RT) PCR is currently one of the most powerful molecular approaches and has gained popularity as a key laboratory technique for diagnosing infectious diseases because of its sensitivity, specificity, accuracy and low contamination risk. Many real-time PCR systems use a fluorescent reporter or a probe for detection and quantification. During the COVID-19 pandemic, the real-time PCR assay has emerged as the gold standard. Here, we present a novel molecular beacon-based real-time RT-PCR assay, termed TQ_unm-based RT-PCR, that utilizes unmasked G-quadruplexes and the fluorogenic dye thioflavin T (ThT) as the fluorescent reporter for detecting the SARS-CoV-2 virus. The formation and recognition of G-quadruplexes by ThT allows the real-time detection of amplification products. Primers and the molecular beacon, i.e., the probe of the assay, were designed to detect a viral nucleocapsid protein N gene. The green channel of the Rotor-Gene Q instrument was selected to record the fluorescence signal resulting from the binding of ThT to the GQ as the reporter dye of the N gene. The clinical performance and sensitivity of the assay were tested using RNA samples from SARS-CoV-2-positive patients and compared with those of a commercial TaqMan kit. The clinical sensitivity of the assay was 100% in detecting positive cases. Furthermore, the limit of detection (LOD) value was determined to be equivalent to a 6.76*10² copy number. A strong correlation (r = 0.9602) was observed between the Ct values of samples tested using one-step and two-step TQ_unm. The combination of a non-fluorescent GQ-generating probe and ThT in a PCR provides promising nucleic acid amplification tests (NAATs) for the detection of the genomic materials of viruses. Also, compared to the TaqMan probe-based RT-PCR method, the TQ_unm-RT-PCR method could be a suitable method for low-cost viral genomic RNA detection.