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

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. [ABSTRACT FROM AUTHOR]