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Unmasked G-quadruplexes for real-time fluorescence detection of viral genomic RNA
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DOI：10.1039/D6AY00654J.png)
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Abstract
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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 TQunm-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 TQunm. 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 TQunm-RT-PCR method could be a suitable method for low-cost viral genomic RNA detection.
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