基于LAMP-CRISPR/Cas12a技术快速检测转基因玉米DBN9501转化体特异性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0974

生物技术通报 ›› 2026, Vol. 42 ›› Issue (2): 149-157.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0974

基于LAMP-CRISPR/Cas12a技术快速检测转基因玉米DBN9501转化体特异性

王晶¹(), 刘卓艳², 张晓磊¹, 刘宝海¹, 关海涛¹(), 温洪涛¹()

^1.黑龙江省农业科学院农产品质量安全研究所农业农村部谷物及制品质量检验测试中心（哈尔滨）国家市场监督管理总局重点实验室（谷物及制品质量与安全），哈尔滨 150086
^2.哈尔滨师范大学生命科学与技术学院，哈尔滨 150500

收稿日期:2025-09-10 出版日期:2026-02-26 发布日期:2026-03-17
通讯作者: 温洪涛，男，博士，副研究员，研究方向：转基因农作物产品成分检测技术和安全性评价；E-mail: wen0891@163.com
关海涛，男，博士，副研究员，研究方向：农作物品种真实性和纯度检测技术；E-mail: ght20080808@126.com
作者简介:王晶，女，博士，助理研究员，研究方向：生物技术产品检测技术；E-mail: buyijingjing@163.com
第一联系人：（王晶、刘卓艳并列第一作者）
基金资助:
国家市场监督管理总局重点实验室（谷物及制品质量与安全）自立课题(GWZL2025-008);科技创新2030—重大项目子课题(2022ZD040190803);黑龙江省农业科技创新跨越工程农业科技基础创新优青项目(CX22YQ08)

Rapid Detection of Transgenic Maize DBN9501 Event-specificity Based on LAMP-CRISPR-Cas12a Technology

WANG Jing¹(), LIU Zhuo-yan², ZHANG Xiao-lei¹, LIU Bao-hai¹, GUAN Hai-tao¹(), WEN Hong-tao¹()

^1.Quality and Safety Institute of Agricultural Products, Heilongjiang Academy of Agricultural Sciences, Quality Testing Center of Cereals and Their Products (Harbin), Ministry of Agriculture and Rural Affairs, Key Laboratory of Quality and Safety of Cereals and Their Products, State Administration for Market Regulation, Harbin 150086
^2.College of Life Science and Technology, Harbin Normal University, Harbin 150500

Received:2025-09-10 Published:2026-02-26 Online:2026-03-17

摘要/Abstract

摘要：

目的转化体特异性检测是转基因生物安全监管中识别转化体合法性的重要技术手段，目前转化体特异性检测以实验室检测为主，急需开发能够现场快速检测的方法，满足转基因生物安全监管需求。方法以抗虫耐除草剂转基因玉米DBN9501为材料，利用环介导等温扩增（loop-mediated isothermal amplification, LAMP）技术结合CRISPR/Cas12a（clustered regularly interspaced short palindromic repeats,CRISPR）技术（LAMP-CRISPR/Cas12a），辅助试纸条显示，开发转化体特异性快速检测方法。结果本方法具有检测特异性，检出限为20拷贝，转化体含量为0.1%，检测时间<60 min。结论 LAMP-CRISPR/Cas技术能够实现快速检测转基因玉米DBN9501转化体特异性，该方法将为转基因生物安全监管快速检测技术研究提供重要技术支撑。

关键词: 转基因玉米, 转化体特异性, 环介导等温扩增, CRISPR/Cas, 快速检测

Abstract:

Objective The event specificity is a key focus of transgenic regulation. Currently, laboratory testing remains the primary method for detecting event specificity. Therefore, it is an urgent need to develop a method that can be quickly detected on-site, thus meeting the regulatory needs for genetically modified organisms. Method This study used insect-resistant and herbicide-tolerant transgenic maize DBN9501 as the material, and utilized loop-mediated isothermal amplification (LAMP) combined with CRISPR/Cas12a technology, assisted by a lateral flow test strip display, to develop a rapid detection method suitable for on-site event-specificity. Result This detection method has a detection specificity. The limit of detection is about 20 copies, that is, 0.1% event content, time of detection<60 min. Conclusion The LAMP-CRISPR/Cas method enables the rapid detection of maize DBN9501 event-specificity, providing technical support in supervising the safety of genetically modified organisms in China.

Key words: transgenic maize, event-specificity, LAMP, CRISPR/Cas12a, rapid detection

王晶, 刘卓艳, 张晓磊, 刘宝海, 关海涛, 温洪涛. 基于LAMP-CRISPR/Cas12a技术快速检测转基因玉米DBN9501转化体特异性[J]. 生物技术通报, 2026, 42(2): 149-157.

WANG Jing, LIU Zhuo-yan, ZHANG Xiao-lei, LIU Bao-hai, GUAN Hai-tao, WEN Hong-tao. Rapid Detection of Transgenic Maize DBN9501 Event-specificity Based on LAMP-CRISPR-Cas12a Technology[J]. Biotechnology Bulletin, 2026, 42(2): 149-157.

图/表 7

表1 引物、报告探针、crRNA序列信息

Table 1 Sequence information of primers, probe reporters, and CrRNA

名称 Name	序列 Sequence （5′-3′）
DBN9501-LB-F3	CCAGCCACTATTAAGTTTCATG
DBN9501-LB-B3	AATCTGTACCTAGTTTAGCTAGT
DBN9501-LB-FIP	TGGCAAAAAGGCATCCTGTTTTAAAAAATCCAAAGAAACCATACG
DBN9501-LB-BIP	TTCAGGAAAAACAGGGGAAAAGTAGGACGTTAACAGGGACG
DBN9501-RB-F3	TTAATACAAATCCACCTGCG
DBN9501-RB-B3	GAACATTTCTTTATTCGGAAACA
DBN9501-RB-FIP	GAGTCACGTTATGACCCCCGTTTTGCGGTTCTGTCAGTTC
DBN9501-RB-BIP	AATTCTCCGCTCATGATCAGATTGTAATCCGGACTACTATACCATT
crRNA-LB	AAUUUCUACUGUUGUAGAUAAAAAAUCCAAAGAAACCAUACG
crRNA-RB	AAUUUCUACUGUUGUAGAUGCGGUUCUGUCAGUUCCAAACG
FB-reporter	6FAM-TTTTTTTTTT-Biotin

图1 DBN9501转化体特异性的快速检测流程A：LAMP-F3/B3扩增DBN9501转化体LB和RB序列的PCR产物（1：无模板对照；2：LB扩增产物；3：RB扩增产物）；B：DBN9501-LB和DBN9501-RB质粒测序结果示意图；C：LAMP扩增结果；D：顺式酶切条件验证实验；E：顺式酶切实验完全切割结果；F：反式酶切试纸条检测结果。C-F图中，1：NTC；2：非转基因玉米；3：DBN9501 转化体；4：DBN9501-LB质粒；5：DBN9501-RB质粒

Fig. 1 Rapid detection workflow of DBN9501 event-specificityA: PCR products of LB and RB sequences of DBN9501 event amplified by LAMP-F3/B3 (1: No template control (NTC) ; 2: LB PCR product; 3:RB PCR product); B: Illustration of DBN9501-LB plasmid sequencing results; C: LAMP amplified results. D: Verification of cis-enzyme digestion condition. E: Trans-enzyme digestion results. F: Strip test assay. In Fig.C-F, 1: NTC; 2: Non-transgenic maize. 3: DBN9501 event. 4: DBN9501-LB plasmid. 5: DBN9501-RB plasmid

图2 LAMP-CRISPR/Cas12a反应条件优化结果A：LAMP引物FIP/BIP∶F3/B3浓度比例优化；B：LAMP反应温度优化；C：Cas12a/crRNA体积比例优化；D：FB reporter优化

Fig. 2 Optimized results of reaction conditions for the LAMP-CRISPR/Cas12aA: LAMP primer FIP/BIP∶F3/B3 Concentration optimization (1: Positive control (2∶1): 2: NTC; 3: 6∶1; 4: 8∶1; 5: 10∶1). B: Optimization of LAMP reaction temperature (1: 62 ℃; 2: 63 ℃; 3: 64 ℃; 4: 65 ℃; 5: 66 ℃; 6: NTC). C: Optimization of Cas12a/crRNA ratio (1: H2O; 2: 0.1∶0.5; 3: 0.2∶0.4; 4: 0.3∶0.3; 5: 0.4∶0.2; 6: 0.5∶0.1). D: Optimization of FB reporter (1: H2O; 2: 62.5; 3: 50.0; 4: 37.5; 5: 25.0; 6: 12.5 nmol/L)

图3 LAMP-CRISPR/Cas12a方法特异性测试A：LAMP扩增结果；B：试纸条检测结果；C：内标准基因PCR扩增结果。1：空白对照；2：非转基因玉米；3：DBN9501转化体；4：转基因玉米（不含DBN9501）；5：转基因油菜；6：转基因大豆；7：转基因水稻；8：转基因棉花

Fig. 3 Specificity detection for LAMP-CRISPR/Cas12a methodA: LAMP amplified results; B: strip test assay results; C: internal standard gene PCR amplified results. 1: Blank control; 2: non-transgenic maize; 3: DBN9501 event; 4: transgenic maize (excluding DBN9501); 5:transgenic rape; 6: transgenic soybean; 7: transgenic rice; 8: transgenic cotton

图4 LAMP-CRISPR/Cas12a方法灵敏度和检出限测试A：灵敏度测试，左图为LAMP扩增结果，右图为试纸条检测；B：检出限测试，左图为质粒检出限测试，右图为DBN9501转化体试纸条测试

Fig. 4 Assay of sensitivity and limit of detection for LAMP-CRISPR/Cas12a methodA: Sensitivity assay, the left picture is LAMP amplified results; the right picture is strip detection assay (1: 8.0×103; 2: 8.0×102; 3: 8.0×10; 4: 4.0×10; 5: 2.0×10; 7: 1.0×10; 7: 1.0; 8: NTC). B: Assay of detection limit, left picture is the assay of detection limit for plasmid, and right picture is DBN9501 event by strip test assay (1: NTC; 2-11: 20 copies DBN9501-LB plasmid)

图5 盲样检测和方法应用A：盲样测试（1：DBN9501-LB；2：非转基因玉米；3：NTC；4：盲样（DBN9501）；5：盲样（非转基因玉米）；6：盲样（Bt11）；7：盲样（DBN9936）；8：盲样（MON810））；B：检测方法应用，左图为LAMP扩增结果，右图为试纸条测试（M：marker；1：非转基因玉米；2：NTC；3：DBN9501-LB；4：DBN9501叶子；5：DBN9501种子，试纸条试验3次重复）

Fig. 5 Blind sample testing and method applicationA: Blind sample test (1: DBN9501-LB; 2: non-transgenic maize; 3: NTC; 4: blind sample (DBN9501). 5: Blind samples (non-transgenic maize); 6: blind sample (Bt11); 7: blind sample (DBN9936); 8: blind sample (MON810)). B: Application of assay method, the left is LAMP amplification results, the right is a test strip test (M: marker; 1: non-transgenic maize; 2: NTC; 3: DBN9501-LB; 4: DBN9501 leaves; 5: DBN9501 seeds, 3 replicates for strip test assay)

表2 基于CRISPR/Cas技术的转基因检测时间比较

Table 2 Comparison of transgenic detecting time based on CRISPR/Cas technology

方法 Method	操作步骤 Procedure	检测对象 Test object	灵敏度 Sensitivity	检测时间 Testing time	参考文献 References
RPA-Cas12a-FS	（1） RPA （2） CRISPR/Cas12a （3）荧光/试纸条显示	P-CaMV35S，T-nos	10拷贝	~45 min	［25］
MR-DCA	（1）多重RPA （2） CRISPR/Cas13a或Cas12a+荧光/试纸条显示	CaMV35S，NOS	20拷贝	~35 min	［7］
RPA-CRISPR/Cas12a	（1） RPA （2） CRISPR/Cas （3） G-四链体比色测定	CaMV35	10 amol/L和0.01%	~45 min	［26］
RPA-CRISPR/Cas12a	（1） RPA （2） CRISPR/Cas12a （3）荧光/试纸条显示	CP4-EPSPS，Cry1Ab/Ac	10 拷贝，0.5%	~50 min	［27］
RAA-CRISPR-Cas12a	（1）重组酶辅助扩增（RAA）；（2） CRISPR/Cas12a （3）裸眼观察金纳米棒颜色变化	NOS	1 amol/L和0.1%	~60 min	［18］
LAMP-CRISPR/Cas12b	（1） LAMP （2） CRISPR/Cas12b （3）荧光显示	CaMV35S	10拷贝，0.05%	~40 min	［4］
LAMP-CRISPR/Cas12a	（1） LAMP （2） CRISPR/Cas 12a （3）试纸条显示	DBN9501	20拷贝	~60 min	本方法

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基于LAMP-CRISPR/Cas12a技术快速检测转基因玉米DBN9501转化体特异性

Rapid Detection of Transgenic Maize DBN9501 Event-specificity Based on LAMP-CRISPR-Cas12a Technology

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