State Key Laboratory of Analytical Chemistry for Life Science, School of Life Sciences, Nanjing University, Nanjing 210023, P. R. China.
The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing 210003, P. R. China.
Anal Chem. 2023 Feb 14;95(6):3358-3362. doi: 10.1021/acs.analchem.2c04572. Epub 2023 Feb 1.
The development of sensitive, accurate, and conveniently operated methods for the simultaneous assay of two nucleic acids is promising while still challenging. In this work, by using two genes (the N gene and RdRp gene) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we have designed an ingenious dual-gene-controlled rolling circle amplification (RCA) strategy to propose an accurate and sensitive electrochemical method. Specifically, the coexistence of the two target genes can trigger the RCA reaction to generate a number of repeated G-quadruplex (G4)-forming sequences. These sequences then switch into G4/hemin complexes with redox activity after the incubation of hemin, which can catalyze the TMB/HO substrates to produce significantly enhanced current responses. Experimental results reveal that the proposed method exhibits satisfying feasibility and analytical performance, enabling the sensitive detection of SARS-CoV-2 in the range of 0.1-5000 pM, with the detection limit of 57 fM. Meanwhile, because only the simultaneous existence of the two target genes can effectively trigger the downstream amplification reaction, this method can effectively avoid false-positives and ensure specificity as well as accuracy. Furthermore, our method can distinguish the COVID-19 samples from healthy people, and the outcomes show a satisfying agreement with the results of RT-PCR, manifesting that our label-free dual-gene-controlled RCA strategy exhibits great possibility in clinical application.
开发用于同时测定两种核酸的灵敏、准确且操作简便的方法具有广阔的前景，但仍然具有挑战性。在这项工作中，我们以严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的两个基因（N 基因和 RdRp 基因）为例，设计了一种巧妙的双基因控制滚环扩增（RCA）策略，提出了一种准确灵敏的电化学方法。具体而言，两种目标基因的共存可以触发 RCA 反应，产生大量重复的 G-四链体（G4）形成序列。这些序列在孵育过的血红素存在下，与具有氧化还原活性的血红素结合，然后切换成 G4/血红素复合物，能够催化 TMB/HO 底物产生明显增强的电流响应。实验结果表明，该方法具有令人满意的可行性和分析性能，能够在 0.1-5000 pM 的范围内灵敏检测 SARS-CoV-2，检测限为 57 fM。同时，由于只有两种目标基因的同时存在才能有效地触发下游扩增反应，因此该方法可以有效地避免假阳性，确保特异性和准确性。此外，我们的方法可以区分 COVID-19 样本和健康人群，结果与 RT-PCR 的结果非常吻合，表明我们的无标记双基因控制 RCA 策略在临床应用中具有很大的可能性。
