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

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

Abstract

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

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.

Figures/Tables 7

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

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

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)

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

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)

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)

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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