Abstract：Currently, the detection of gene editing products mostly relies on methods such as genome sequencing analysis and PCR technology, which are time-consuming, costly, and require large instruments and equipment. In order to establish a rapid, sensitive, visual and suitable method for the detection of gene edited crops, primers and single guide RNA (sgRNA) were designed according to the rice OsSWEET14 gene editing material. Recombinase polymerase amplification (RPA) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 12b technology combined with lateral flow strips (LFS) strip were used to detect gene edited rice (Oryza sativa). The results showed that RPA-CRISPR/Cas12b combined with LFS could make the detection results visualized through the test strips within 33 min at most, and the detection sensitivity was 100 fg/μL. The RPA-CRISPR/Cas12b-LFS detection method developed in this study was simple and rapid, did not need large instruments and equipment. This study provides technical support for the field rapid detection with limited experimental conditions.
[1] 罗亮, 牛蒙亮, 乾义柯, 等. 2025. 基因编辑水稻RPA-CRISPR/Cas12b快速检测方法的建立[J]. 西北农业学报, 34(07): 1201-1208. (Luo L, Niu M L, Qian Y K, et al.2025. Establishment of a rapid detection method for gene-edited rice using RPA-CRISPR/Cas12b[J]. Acta Agriculturae Boreali-occidentalis Sinica, 34(07): 1201-1208.) [2] 庞建虎, 乔龙亮, 黄海龙, 等. 2018. 重组酶聚合酶扩增技术快速检测鳗利斯顿氏菌[J]. 农业生物技术学报, 26(06): 1056-1063. (Pang J H, Qiao L L, Huang H L, et al.2018. Rapid detection of Listonella anguillarum by recombinase polymerase amplification techniqu[J]. Journal of Agricultural Biotechnology, 26(06): 1056-1063.) [3] 王梦雨, 王颢潜, 王旭静, 等. 2021. 基因编辑产品检测技术研究进展[J]. 生物技术进展, 11(4): 438-445. (Wang M Y, Wang H Q, Wang X J, et al.2021. Research progress of gene editing products detection technology[J]. Current Biotechnology, 11(4): 438-445.) [4] 袁俊杰, 龙阳, 渭婷玉, 等. 2020. 添加扩增内标的逆转录重组酶聚合酶扩增技术检测三种大豆病毒[J]. 农业生物技术学报, 28(12): 2261-2269. (Yuan J J, Long Y, Wei T Y, et al.2020. RT-RPA technology included with IAC for detection of three soybean (Glycine max) viruses[J]. Journal of Agricultural Biotechnology, 28(12): 2261-2269.) [5] 张笑天, 王智, 朱鹏宇, 等. 2023. 一种基于定量PCR的CRISPR/Cas9基因编辑作物快速检测方法的研究[J]. 生物技术进展, 13(06): 907-912. (Zhang X T, Wang Z, Zhu P Y, et al.2023. A rapid detection method based on qPCR for CRISPR/Cas9 edited crops[J]. Current Biotechnology, 13(06): 907-912.) [6] Abudayyeh O O, Gootenberg J S, Konermann S, et al.2016. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 353(6299): aaf5573. [7] Alcock B P, Raphenya A R, Lau T T Y, et al.2020. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database[J]. Nucleic Acids Research, 48(D1): D517-D525. [8] Aman R, Marsic T, Sivakrishna Rao G, et al.2022. iSCAN-V2: A one-pot RT-RPA-CRISPR/Cas12b assay for point-of-care SARS-CoV-2 detection[J]. Frontiers in Bioengineering and Biotechnology, 9: 800104. [9] Chen J S, Ma E, Harrington L B, et al.2018. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 360(6387): 436-439. [10] Hijjawi N, Zahedi A,Ryan U.2023. Point of care diagnostics for Cryptosporidium: New and emerging technologies[J]. Current Opinion in Gastroenterology, 39(1): 3-8. [11] Huang Y, Gu D, Xue H, et al.2021. Rapid and accurate Campylobacter jejuni detection with CRISPR-Cas12b based on newly identified Campylobacter jejuni-specific and -conserved genomic signatures[J]. Frontiers in Microbiology, 12: 649010. [12] Li L, Li S, Wu N, et al.2019. HOLMESv2: A CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation[J]. ACS Synthetic Biology, 8(10): 2228-2237. [13] Li S Y, Cheng Q X, Wang J M, et al.2018. CRISPR-Cas12a-assisted nucleic acid detection[J]. Cell Discovery, 4(1): 20. [14] Li Y, Li S, Wang J, et al.2019. CRISPR/Cas systems towards next-generation biosensing[J]. Trends in Biotechnology, 37(7): 730-743. [15] Luan H, Wang S, Ju L, et al.2024. KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus[J]. Frontiers in Immunology, 15: 1358960. [16] Pinchon E, Henry S, Leon F, et al.2024.Rapid detection of Measles virus using reverse transcriptase/recombinase polymerase amplification coupled with CRISPR/Cas12a and a lateral flow detection: A proof-of-concept study[J]. Diagnostics (Basel), 14(5): 517. [17] Teng F, Guo L, Cui T, et al.2019. CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity[J]. Genome Biology, 20(1): 132. [18] Varshney R K, Barmukh R, Roorkiwal M, et al.2021. Breeding custom-designed crops for improved drought adaptation[J]. Advanced Genetics (Hoboken), 2(3): e202100017. [19] Wang X, Chen Y, Cheng X, et al.2023. CDetection.v2: One-pot assay for the detection of SARS-CoV-2[J]. Frontiers in Microbiology, 14: 1158163. [20] Wang Z, Wang Y, Zhang Y, et al.2024. On-site detection and differentiation of African swine fever virus variants using an orthogonal CRISPR-Cas12b/Cas13a-based assay[J]. iScience, 27(4): 109050. [21] Yamano T, Nishimasu H, Zetsche B, et al.2016. Crystal structure of Cpf1 in complex with guide RNA and target DNA[J]. Cell, 165(4): 949-962. [22] Yang L, Chen G, Wu J, et al.2024. A PAM-free one-step asymmetric RPA and CRISPR/Cas12b combined assay (OAR-CRISPR) for rapid and ultrasensitive DNA detection[J]. Analytical Chemistry, 96(14): 5471-5477. [23] Yang X, Huang J, Chen Y, et al.2023. Development of CRISPR/Cas12b-based multiple cross displacement amplification technique for the detection of mycobacterium tuberculosis complex in clinical settings[J]. Microbiology Spectrum, 11(2): e0347522. [24] Zetsche B, Gootenberg J S, Abudayyeh O O, et al.2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 163(3): 759-771.
