Rapid virus detection by primer-independent linear rolling circle amplification combined with DNA-templated silver nanoclusters.

BACKGROUND

DNA and RNA virus detections commonly rely on qPCR (quantitative polymerase chain reaction) -based methods that need dyes or Taqman probes as signaling probes and expertized instruments. Isothermal amplification such as linear rolling-circle amplification (LRCA) has attracted large interest in its role to act as a supplement to the traditional qPCR. However, the current LRCA-based signaling probes suffered from long time-consumption and complex procedures for nucleic acid detections. Novel systems are urgently needed to integrate the simplicity of LRCA with rapid signal amplification of label-free probes to spread the advantages of isothermal amplification for virus detections.

RESULTS

A primer-independent LRCA system utilizing AgNCs/DNA as signaling probes was developed for rapid virus detection. LRCA was optimized into a one-step reaction by integrating target-triggered ligation reaction of padlock probes (PlPs) with DNA amplification. After an easy H2O2 treatment to minimize dithiothreitol's interference, LRCA product with tandem repeats of G-rich sequence was hybridized with AgNCs/DNA, promptly amplifying fluorescence signals through the G-proximity enhancement effect. The system precisely discriminated single-base mismatch at ligation junctions of PlPs. The formation of 3'-hairpin structure in PlPs was found to be prioritized over 5'-exposed linear sequences to maximize the fluorescence signals. It requires only 1 μL sample and costs 40 min at 25-30 °C, facilitating a sensitive and broad linear range of detection for E (10 -9-10 μM) gene in RNA virus (SARS-CoV-2) and VP2/3 gene (10 -9 - 1 μM) in DNA virus (BK human polyomavirus, BKV), and successful application in urine samples.

SIGNIFICANCE: AgNCs/DNA lighting up LRCA products with rapid signal amplifications alongside the extension of single strand DNA enables the direct use of LRCA for nucleic acids detections. The modular design of PlPs, universal signal probes, near-room temperature reaction conditions, small sample requirements and rapid reaction make it adapted for various DNA detections, and give it great promise for future point-of-care tests.