图1 CRISPR/Cas12a检测体系必需组分对反应荧光强度的影响 (A) CRISPR/Cas12a检测体系内DNA、crRNA及Cas12a三元复合物形成及荧光信号产生示意图(F表示6-FAM荧光基团, Q表示BHQ1淬灭基团); 图示过程为crRNA与Cas12a先形成二元复合物, 复合体在DNA链上寻找靶标位点, 若存在靶标位点, 则Cas12a中的Ruvc位点发挥其酶活(橙色), 继而反式酶切单链的荧光探针; 若不存在靶标位点, 则Ruvc位点不发挥酶活(灰色), 荧光探针未被酶切; (B) 在不同组分缺失的情况下, 分别在蓝光、普通光和紫外光下的观察结果(+表示添加该组分, -表示缺失该组分, a-g表示各处理组); (C) 不同组分缺失情况下的实时荧光信号记录图

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

图2 不同位点crRNA对CRISPR/Cas12a反应体系荧光强度的影响 (A) crRNA设计靶点位置示意图, 其中红色、橙色序列为RPA扩增时的上、下游引物结合位置, 蓝色背景突出序列为选择的PAM位点; PAM位点后的碱基序列(蓝色或方框所示)为crRNA结合位置; (B) 不同位点crRNA经CRISPR/Cas12a反应后分别在蓝光、普通光与紫外光下的观察结果; (C) 不同位点crRNA在CRISPR/Cas12a反应中的实时荧光信号记录图, 其中NTC为阴性对照, 即CRISPR/Cas12a反应体系内不添加靶标序列。

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.

图3 RPA-CRISPR/Cas12a与PCR、qPCR法检测靶序列最低浓度限度比较 (A) 不同浓度靶序列经RPA-CRISPR/Cas12a反应后蓝光照射的荧光照片, 图示能检测到的最低限度为3×102 copies∙μL-1 (红框所示); (B) 不同浓度靶序列在RPA-CRISPR/Cas12a反应中的实时荧光信号记录图; (C) PCR扩增后的凝胶电泳检测图; 图示能检测到的最低限度为3×105 copies∙μL-1 (红框所示); (D) qPCR检测结果, 以标准曲线形式表示, 取ct值最接近且小于30对应的初始浓度即为检测的最低限度; 横坐标为模板经逐级浓度梯度稀释后的各初始浓度(取对数表示), 纵坐标表示ct值, 浓度单位为copies∙μL-1。(A)-(C) NTC: 阴性对照

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

图4 植物病毒RPA-CRISPR/Cas12a检测流程 将样品加入核酸粗提缓冲液中, 于室温下研磨。取研磨液加入逆转录反应体系中合成cDNA (37°C, 20分钟)。将cDNA置于RPA反应管中进行恒温扩增(37°C, 20分钟)。取少量RPA反应产物进行CRISPR/Cas12a反应(37°C, 20分钟), 反应结束后在蓝光/紫外光/普通光下观察反应结果。

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.

图5 用RPA-CRISPR/Cas12a、PCR与qPCR三种方法对15株马铃薯试管苗的检测结果 (A) 用RPA-CRISPR/Cas12a法对15株马铃薯试管苗在蓝光下的检测结果, 1-15为待检测样本的序号; (B) PCR检测结果, 经35个循环扩增后的凝胶电泳检测图; (C) qPCR检测结果荧光曲线

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