Figure 1 Effects of essential components of CRISPR/Cas12a detection system on the fluorescence intensity of the reaction (A) The schematic diagram indicates the formation process of crRNA-Cas12a-DNA ternary complex, and shows principle of fluorescence signal generation in CRISPR/Cas12a detection system (F represents the 6-FAM fluorescence group and Q represents the BHQ1 quenched group); Once crRNA and Cas12a forming a binary complex, the binary complex searches for target sites on the DNA sequence; If there is a target site, the Ruvc site in Cas12a performs its enzyme activity (orange), and then transects the single-strand fluorescent probe; If there is no target site, the Ruvc site is not activated (gray) and the fluorescent probe is not cut by the Cas12a; (B) The absence of components in different treatment groups under blue light, normal light and UV (+ represents the addition of this component, - represents the absence of this component, and a-g represents the each treatment group); (C) Real-time fluorescence signal of the absence components in different treatment groups were recorded in qPCR instrument

Figure 2 Effect of crRNA at different sites on fluorescence intensity of CRISPR/Cas12a detection system (A) The schematic diagram shows the position of crRNA binding locations; the forward and reverse primer binding locations in RPA amplification were highlighted in red and orange, and the selected PAM sites were highlighted in blue; the base sequence after the PAM sites (shown in blue or box) are the crRNA binding sites; (B) Image illuminated of CRISPR/Cas12a reaction of crRNA at different sites under blue light, normal light and UV, respectively; (C) Real-time fluorescence signal recording diagram of different crRNAs-mediated reaction systems in qPCR instrument, in which NTC is the negative control without adding target sequence in CRISPR/Cas12a detection system.

Figure 3 Comparison of minimum concentration limits of target sequences detected by RPA-CRISPR/Cas12a as well as PCR and qPCR methods (A) Image illuminated by blue light after using RPA-CRISPR/Cas12a to detect different concentrations target sequences, the minimum detectable level was 3×102 copies∙μL-1 (red box); (B) Real-time fluorescence signal recording of different concentrations target sequences in RPA-CRISPR/Cas12a detection; (C) Gel electrophoresis map after PCR; the minimum detectable level was 3×105 copies∙μL-1 (red box); (D) The detection results of qPCR method are expressed in the form of standard curve, the initial concentration corresponding to ct value that was closest to and less than 30 was regarded as the minimum of detection; the abscissa shows the initial concentration of each template diluted by step by step gradient (expressed in logarithm), and the ordinate represents ct value, the units of concentration were copies∙μL-1. (A)-(C) NTC: Negative control

Figure 4 Flow chart of RPA-CRISPR/Cas12a system for plant virus detection The sample was added into the nucleic acid crude extraction buffer and grinded at room temperature. The grinding solution was added into the reverse transcription reaction system to synthesize cDNA (37°C, 20 min). The cDNA was placed in the RPA reaction tube for constant temperature amplification (37°C, 20 min). A small amounts of RPA reaction products were placed into CRISPR/Cas12a for incubation (37°C, 20 min), and the reaction results were observed under blue light/UV/normal light.

Figure 5 Detection results of 15 potato plantlets by RPA-CRISPR/Cas12a, PCR and qPCR methods (A) Image illuminated under blue light after 15 potato plantlets detection by RPA-CRISPR/Cas12a; 1-15 are the number of the sample to be tested; (B) Gel electrophoresis map after PCR amplification (35 cycles); (C) Fluorescence curve detected by qPCR