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Updated: May 16, 2026
Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
Published on: December 23, 2022
Exponential signal amplification through coupled rolling circle transcription and CRISPR/Cas13a cleavage for
Yongkun Guo1, Zaobing Zhu2, Can Zhu3
1Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Hainan Research Institute, Shanghai Jiao Tong University, Shanghai 200240, PR China; Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China.
Abstract:
Rapid, sensitive nucleic acid detection is crucial for clinical diagnostics, food safety, and environmental monitoring. We present the Sensitive Padlock Rolling-circle INtegrated Transcription with clustered regularly interspaced short palindromic repeats (CRISPR)/LwCas13a detector (SPRINT-C), a transformative one-pot isothermal platform capable of detecting single molecules within 10 min. By rationally designing padlock probes, we enable seamless coupling of three enzymatic reactions: template-mediated padlock ligation, rolling circle transcription (RCT), and CRISPR/LwCas13a cleavage. Our mechanistic studies reveal that the substantially higher transcription rate of T7 RNA polymerase (5.97 cycles/s) relative to LwCas13a cis-cleavage (<0.019 s-1) results in incomplete cis-cleavage of RCT products, generating short RNA fragments that re-initiate the amplification cascade. This creates exponential amplification, transforming linear RCT into a self-perpetuating chain reaction. SPRINT-C achieves ultrasensitivity (1-2 copies/reaction) with single-nucleotide specificity, successfully detecting severe acute respiratory syndrome coronavirus 2 variants in clinical samples, including 'gray-zone' specimens (Ct 37-40). Integration with lateral flow enables an equipment-free visual detection method, demonstrating versatility across pathogen detection, genetically modified organism identification, and microRNA analysis.
