Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several step-by-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly. The proposed homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.

环到环扩增（C2CA）是一种特定且精确的级联核酸扩增方法，由多于一轮的挂锁探针连接和滚环扩增（RCA）组成。尽管C2CA提供了高扩增效率，而假阳性风险的增加可以忽略不计，但它包含几个分步操作过程。我们在本文中展示了基于C2CA和磁性纳米粒子（MNP）组件的光磁分析的均质和等温核酸定量策略。拟议的均相环间扩增无需在第一轮RCA之后进行额外的单体化和连接步骤，并在一个反应​​罐中合并了两个扩增轮。第二轮RCA产生扩增子线圈，该扩增子与嫁接到MNP上的检测探针退火，从而可以使用光电传感器实时检测MNP组件。拟议的方法应用于SARS-CoV-2（严重急性呼吸系统综合症冠状病毒2，也称为2019-nCoV）RdRp（RNA依赖性RNA聚合酶）编码序列的合成互补DNA检测，检测限为动态检测范围为3个数量级，总检测时间为0.4 fMca. 100分钟 建立了数学模型并进行了验证，以预测化验性能。此外，所提出的方法是专门用于区分具有高度相似性的SARS-CoV和SARS-CoV-2序列的。