2024-09-06
《Development of a novel Cas13a/Cas12a-mediated “one-pot”dual detection assay for genetically modified crops》
Genetically modified crops have been widely planted around the world, and many countries have gradually improved their regulatory measures to ensure the safety of genetically modified crops. The development of rapid and accurate on-site genetic detection methods is very important for agriculture and biosafety, and can alleviate public concerns about genetically modified products.
In this study, the research group developed a new one-pot reaction ——MIRA dual detection combined with dual CRISPR (MR-DCA method) for the simultaneous detection of CaMV35S and NOS genetic targets in genetically modified crops, providing a simple and efficient detection method for the detection of genetically modified crops.
The MIRA dual product is analyzed by Cas13a and Cas12a dual system to generate two independent signals that do not interfere with each other, and then the fluorescent color is read by an illumination instrument or by a test strip with two T lines to achieve dual-channel readout. The entire process is completed in less than 35 minutes, with a detection limit as low as 20 copies.
（Principle of MR-DCA detection of CaMV35S and NOS）
The entire experiment is divided into three steps: MIRA amplification, CRISPR reaction, and two methods of result interpretation.
The CaMV35S gene is used as the target of Cas13a, and the NOS gene is used as the target of Cas12a. Two pairs of MIRA primers were designed respectively. The Cas13a system only recognizes RNA, so the forward primer region is designed, which contains a T7 promoter for subsequent transcription and activation of Cas13a. A PAM is added to the NOS gene. The above additions ensure that both Cas13a and Cas12a proteins can bind to their respective crRNAs, and there is no interference or cross-reaction between the two systems.
The sample was diluted in a gradient to test the fluorescence intensity of the MIRA reaction. It was found that the fluorescence intensity could still be detected at a sample concentration of 20 μL. The concentration of 20 μL was finally selected for the experiment.
（Different sample concentrations and fluorescence intensity tests）
（Different sample concentrations and test strips are used for color development and intensity quantification calculation）
To further evaluate the specificity of MR-DCA, the research team tested mixed samples of non-transgenic animals and non-transgenic plants. The results showed that only transgenic samples containing CaMV35S and NOS had high fluorescence signals, while the fluorescence intensity of other samples was close to that of the negative control. MR-DCA can be used as a specific dual-target nucleic acid detection platform.
24 crop samples were tested by MR-DCA. Among them, 1, 2, 15, 16, 21, and 22 are transgenic corn containing CaMV35S promoter. 7, 8, 23, and 24 are disease-resistant transgenic rice containing NOS terminator. 3, 4, 11, 12, 13, 14, 17, and 18 are transgenic soybeans containing CaMV35S promoter and NOS terminator.
（Test results of 24 samples）
Reaction time is an important factor in on-site detection. The research team observed that the fluorescence signals of FAM and HEX could be detected by a fluorescence instrument when the reaction time was 10 minutes. MIRA combined with CRISPR reaction also observed yellow fluorescence after 15 minutes.
（Fluorescence signal at different MIRA reaction times）
（The color development of the test strips at different MIRA reaction times and the intensity quantification calculation）
The experimental results showed that the dual MIRA has good specificity and can accurately obtain the amplification products of CaMV35S and NOS. The Cas13a and Cas12a systems react independently without interfering with each other. The MR-DCA test can accurately detect 20 copies at a low temperature of 35 minutes, which has great value in detecting genetically modified crops in the field.
Article Information
Journal Name：《Journal of Advanced Research》
Article Title：《Development of a novel Cas13a/Cas12a-mediated “one-pot”dual detection assay for genetically modified crops》
Impact Factor：11.4
Research team: Wang Xiaofu, Zhejiang Academy of Agricultural Sciences
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