Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification with CRISPR/Cpf1 for RNA virus assay

• Published in: Process biochemistry (1991) 2023-07, Vol.130, p.358-365
• Main Authors: Xiong, Yifan, Cao, Gaihua, Nie, Fuping, Shi, Meimei, Yang, Jun, Li, Yingguo, Huo, Danqun, Hou, Changjun
• Format: Article
• Language: English
• Subjects: CRISPR/Cpf1; Foot-and-mouth disease; Lateral flow assay (LFA); On-situ detection; Reverse Transcriptase-Enzyme-assisted recombinant isothermal amplification technique (RT-ERA)
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2023.04.030

Foot-and-mouth disease virus (FMDV) causes Foot-and-mouth disease, which was listed as a reported disease by Office international des épizooties. It has caused great economic losses to the meat industry. Here, we developed a sensitive, rapid, and visible method based Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification technique (RT-ERA) and CRISPR/Cpf1 for detecting FMDV. RT-ERA achieved simultaneous reverse transcription and amplification in a short time at 37 ℃. Moreover, CRISPR/Cpf1 specifically recognized the products and amplified the signals through the trans-cleavage activity, which converted the DNA signals into fluorescence signals and lateral flow assay strip. Within 30 min, the method detected FMDV as low as 10 copies·μL−1, making it 103 times more sensitive than gel electrophoresis and 10 times more sensitive than reverse transcription quantitative real-time PCR. Moreover, the method had good performance in actual samples. Therefore, this method realized visual detection without the large instruments and showed significant potential for FMDV assay. [Display omitted] •RT-ERA fulfills reverse transcription and amplification in 20 min to reduce time.•The role of the primers and crRNA improves the specificity of the method.•The method has high sensitivity by the trans-cleavage activity of Cpf1 and RT-ERA.•The method achieves visual detected by LFA without large equipment.