Ultrasensitive fluorescent biosensor for detecting CaMV 35S promoter with proximity extension mediated multiple cascade strand displacement amplification and CRISPR/Cpf 1

• Published in: Analytica chimica acta 2022-07, Vol.1215, p.339973-339973, Article 339973
• Main Authors: Liu, Yin, Zhou, Shiying, Sun, Human, Dong, Jiangbo, Deng, Liyuan, Qi, Na, Wang, Yongzhong, Huo, Danqun, Hou, Changjun
• Format: Article
• Language: English
• Subjects: CaMV 35S; CRISPR/Cpf 1; DNA detection; Fluorescence; Strand displacement amplification (SDA)
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2022.339973

A novel fluorescent biosensor was proposed for detecting the CaMV 35S promoter in genetically modified organisms (GMOs). It was based on a proximity extension mediated multiple cascade strand displacement amplification connected with CRISPR/Cpf 1 (termed PE-MC/SDA-CRISPR/Cpf1). In this protocol, the CaMV 35S was recognized by proximity reaction in the presence of two adjacent primer probes. The proximity extension further triggered the multiple cascade strand displacement amplification (MC/SDA), generating a mass of ssDNA. The products compelled the trans-cleavage activity of CRISPR/Cpf 1, so as to cleave nearby ssDNA-FQ reporters and generate a strong fluorescent signal. The ingenious three-link combination design allowed the CaMV 35S a low background interference. And the MC/SDA combined with CRISPR/Cpf 1 dramatically improved the detection sensitivity. Under optimized conditions, the detection linear range of ultrasensitive fluorescent biosensor for CaMV 35S was from 50 fM to10 pM and 10 pM–500 pM, along with the limit of detection (LOD) down to 14.4 fM. The sensing platform also had excellent performance in the assay of selectivity and real samples. Therefore, the method earned great application potential for transgenic crops. [Display omitted] •A novel sensing strategy (PE-MC/SDA-CRISPR/Cpf1) was constructed for the detection of genetically modified organisms.•The combination of multiple cascade SDA and CRISPR/Cas12a achieved effective signal amplification.•Three-way junction, bases mismatch and terminal modification greatly reduced the background noise.•The high sensitivity was realized with the limit of detection down to 14.4 fM.•This work held great application potential in food safety and provided a universal platform for other nucleic acid assays.