Establishment of Specific Qualitative PCR Detection Method of Transgenic Maize Bt11

doi:10.19586/j.2095-2341.2025.0058

Current Biotechnology ›› 2025, Vol. 15 ›› Issue (6): 1031-1039.DOI: 10.19586/j.2095-2341.2025.0058

• Articles • Previous Articles Next Articles

Establishment of Specific Qualitative PCR Detection Method of Transgenic Maize Bt11

Jin TIAN¹^,²(), Fangrui GAO¹, Ling LI², Ziyan CHEN¹, Yifan CHEN³, Hua ZHANG¹, Hong CHEN¹(), Haoqian WANG¹, Jingang LIANG¹()

^1.Development Center of Science and Technology，Ministry of Agriculture and Rural Affairs，Beijing 100176，China
^2.College of Food and Bioengineering，Beijing Vocational College of Agriculture，Beijing 102442，China
^3.Biotechnology Research Institute，Shanghai Academy of Agricultural Sciences，Shanghai 201106，China

Received:2025-04-30 Accepted:2025-08-13 Online:2025-11-25 Published:2026-01-04
Contact: Hong CHEN,Jingang LIANG

Abstract

Abstract:

This study aims to establish a specific qualitative PCR detection method for the precise identification of the Bt11 transgenic insect-resistant maize， in order to meet the requirements of biosafety management of genetically modified （GM） organisms and ensure the safety and authenticity of GM maize products. The genomic DNA of Bt11 was extracted using the QIAGEN DNeasy^? Plant Maxi kit， and specific primers were designed and screened based on the flanking sequences of the inserted foreign gene segment in Bt11. The detection conditions were optimized by adjusting the annealing temperature and primer concentration， specificity， detection limit， and reproducibility of the method were tested. The primer pair Bt11-RB-F3/R3 was ultimately selected， with an annealing temperature of 60 ℃ and a primer concentration of 0.4 μmol·L^-1. The selected primer pair Bt11-RB-F3/R3 specifically amplified a 260 bp target fragment without any non-specific amplification. The method was able to stably detect the Bt11 transgenic event in samples with a copy number ratio of 0.1%， establishing a detection limit of 0.1%. The reproducibility test demonstrated that the detection results across different operators， time periods， and PCR instruments are consistent and meet the expected outcomes. This study successfully established a scientific and reliable detection method for Bt11 transgenic maize， which is capable of precisely and efficiently identifying the Bt11 transgenic event. This method provides strong technical support for biosafety management institutions and holds significant importance for strengthening the regulatory system of GM biosafety.

Key words: genetically modified maize, Bt11, qualitative PCR, specific detection

摘要：

研究旨在建立一套用于精准检测转基因抗虫玉米Bt11的特异性定性PCR检测方法，以满足转基因生物安全管理的需求，确保转基因玉米产品的安全性和真实性。采用QIAGEN DNeasy^? Plant Maxi kit提取Bt11基因组DNA，基于Bt11外源基因插入片段侧翼序列设计并筛选特异性引物。通过调整退火温度和引物浓度，进行特异性、检出限和再现性测试，优化检测条件。最终选择Bt11-RB-F3/R3引物组合，确定退火温度为60 ℃，引物浓度为0.4 μmol·L^-1。该引物组合能特异性扩增260 bp的目标片段，且无非特异性扩增。在拷贝数比值为0.1%的样品中，该方法可稳定检出Bt11转化体，检出限为0.1%。再现性测试表明，不同操作人员、不同时间段和不同PCR仪的检测结果一致，符合预期。研究成功建立了一种科学、可靠的Bt11转基因玉米检测方法，能够精准、高效地检测Bt11转化体，为转基因生物安全管理机构提供了强有力的技术支持，对加强转基因生物安全监管体系具有重要意义。

关键词: 转基因玉米, Bt11, 定性PCR技术, 特异性检测

CLC Number:

Jin TIAN, Fangrui GAO, Ling LI, Ziyan CHEN, Yifan CHEN, Hua ZHANG, Hong CHEN, Haoqian WANG, Jingang LIANG. Establishment of Specific Qualitative PCR Detection Method of Transgenic Maize Bt11[J]. Current Biotechnology, 2025, 15(6): 1031-1039.

田锦, 高芳瑞, 李玲, 陈子言, 陈一帆, 张华, 陈红, 王颢潜, 梁晋刚. 转基因玉米Bt11特异性定性PCR检测方法的建立[J]. 生物技术进展, 2025, 15(6): 1031-1039.

Figures/Tables 10

References 18

[1]	中华人民共和国农业农村部.农业农村部公告第732号[Z].2023-12-07.
[2]	中华人民共和国农业农村部.农业农村部公告第830号[Z].2024-10-08.
[3]	温洪涛,杨洋,丁一佳,等.抗虫玉米CM8101定性检测方法的建立[J].生物技术进展,2022,12(2):248-255.
	WEN H T, YANG Y, DING Y J, et al.. Establishment of qualitative PCR detection of transgenic insect-resistant maize CM8101[J]. Curr. Biotechnol., 2022, 12(2): 248-255.
[4]	傅凯,黄文胜,邓婷婷,等.多重PCR-液相芯片技术检测13个品系转基因玉米[J].中国食品学报,2015,15(1):188-197.
	FU K, HUANG W S, DENG T T, et al.. Multiplex PCR assay and liquid bead array for detection of 13 lines genetically modified maize[J]. J. Chin. Inst. Food Sci. Technol., 2015, 15(1): 188-197.
[5]	郑莹,刘家益,左璇,等.基于文献计量和内容挖掘的转基因玉米科研态势研究[J].生物技术通报,2016,32(12):203-213.
	ZHENG Y, LIU J Y, ZUO X, et al.. Research situation of transgenic corn based on bibliometrics and content mining[J]. Biotechnol. Bull., 2016, 32(12): 203-213.
[6]	农业部农业转基因生物安全管理办公室.2004—2011年进口用作加工原料的农业转基因生物审批情况[EB/OL]. [2025-08-21]. .
[7]	覃文,曹际娟,朱水芳.加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测[J].生物技术通报,2003,19(6):46-50.
	QIN W, CAO J J, ZHU S F. Quantitative/Identified detection of the genetically modified maize Bt11 components in processed products[J]. Biotechnol. Bull., 2003, 19(6): 46-50.
[8]	兰青阔,王永,赵新,等.转基因玉米LAMP检测体系的建立及应用[J].华北农学报,2010,25(4):49-52.
	LAN Q K, WANG Y, ZHAO X, et al.. Development and application of loop-mediated isothermal amplification for detection of GM maize Bt11[J]. Acta Agric. Boreali Sin., 2010, 25(4): 49-52.
[9]	马文良,刘峰,刘恩凯,等.用于转基因玉米品系检测的PCR芯片的应用研究[J].植物检疫,2016,30(6):44-48.
	MA W L, LIU F, LIU E K, et al.. Research on PCR chip for detection of genetically modified maize[J]. Plant Quar., 2016, 30(6): 44-48.
[10]	李凯,金芜军,李亮,等.转基因玉米Bt11品系特异性荧光RPA检测[J].分子植物育种,2017,15(11):4741-4745.
	LI K, JIN W J, LI L, et al.. Specific detection of genetically modified maize Bt11 with novel realtime RPA assay[J]. Mol. Plant Breed., 2017, 15(11): 4741-4745.
[11]	李葱葱,闫伟,夏蔚,等.应用简并PCR方法检测转cry1A基因作物[J].食品科学,2018,39(14):317-322.
	LI C C, YAN W, XIA W, et al.. Detection of genetically modified crops with cry1A gene by PCR with degenerate primers[J]. Food Sci., 2018, 39(14): 317-322.
[12]	田锦,张月秋,张华,等.转基因玉米浙大瑞丰8特异性定性PCR检测方法研究[J].生物技术通报,2024,40(12):45-52.
	TIAN J, ZHANG Y Q, ZHANG H, et al.. Specific qualitative PCR detection method of transgenic maize zheda ruifeng 8[J]. Biotechnol. Bull., 2024, 40(12): 45-52.
[13]	GRYSON N. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review[J]. Anal. Bioanal. Chem., 2010, 396(6): 2003-2022.
[14]	中华人民共和国农业部. 转基因植物及其产品成分检测玉米内标准基因定性PCR方法:农业部1861号公告-3—2012 [S].北京:中国农业出版社,2013.
[15]	ENGL-GMFF. Definition of minimum performance requirements for analytical methods of GMO testing, European Network of GMO Laboratories 2015.
[16]	转基因植物及其产品成分检测抗虫和耐除草剂玉米Bt11及其衍生品种定性PCR方法:农业部2122号公告-14—2014 [S]. 2014.
[17]	转基因植物及其产品成分检测抗虫和耐除草剂玉米Bt11及其衍生品种定性PCR方法:农业部869号公告-3—2007 [S]. 2007.
[18]	MANO J, YANAKA Y, AKIYAMA H, et al.. Improvement of polymerase chain reaction-based Bt11 maize detection method by reduction of non-specific amplification[J]. Food Hyg. Safe. Sci., 2010, 51(1): 32-36.

引物名称	序列(5´→3´)	引物名称	序列(5´→3´)
Bt11-LB-F1	5´-ATGGCTATTTTCAAGGTCGCT-3´	Bt11-RB-F2	5´-ACTTTTCGGGGAAATGTGCG-3´
Bt11-LB-F2	5´-AATCCGTAGGTGTAGCCTCTAGT-3´	Bt11-RB-F3	5´-ACTAGATCTGGGCCTCGTGA-3´
Bt11-LB-F3	5´-CATCGACTTCCATGACCAAA-3´	Bt11-RB-F4	5´-TACTAGATCTGGGCCTCGT-3´
Bt11-LB-R1	5´-TTTCTACGGGGTCTGACGCT-3´	Bt11-RB-R1	5´-CGAGCCTCTGGTCGATGATA-3´
Bt11-LB-R2	5´-TGGTAGCTCTTGATCCGGCA-3´	Bt11-RB-R2	5´-ATGTGGGGTGAGGTGTACGA-3´
Bt11-LB-R3	5´-CGCTCAGTGGAACGAAAACTC-3´	Bt11-RB-R3	5´-TTTTGATGAAAATAGCCATGAG-3´
Bt11-LB-R4	5´-AACAAACCACCGCTGGTAGC-3´	Bt11-RB-R4	5´-GAGGCTAACACCTACAGATTTTAGA-3´
Bt11-LB-R5	5´-GTATCTGCGCTCTGCTGAAG-3´	Bt11-RB-R5	5´-CTCCACCAAAAATCCAAGAATC-3´
(S1)Bt11-F	5´-GCCTCGTGATACGCCTATTTTT-3´	(S2)Bt11-F	5´-GCGGAACCCCTATTTGTTTA-3´
(S1)Bt11-R	5´-CAAAAATCCAAGAATCCCTCCA-3´	(S2)Bt11-R	5´-CAAGAAATGGTCTCCACCAAA-3´
Bt11-RB-F1	5´-AGACGTCAGGTGGCACTTTT-3´

引物名称	序列(5´→3´)	引物名称	序列(5´→3´)
Bt11-LB-F1	5´-ATGGCTATTTTCAAGGTCGCT-3´	Bt11-RB-F2	5´-ACTTTTCGGGGAAATGTGCG-3´
Bt11-LB-F2	5´-AATCCGTAGGTGTAGCCTCTAGT-3´	Bt11-RB-F3	5´-ACTAGATCTGGGCCTCGTGA-3´
Bt11-LB-F3	5´-CATCGACTTCCATGACCAAA-3´	Bt11-RB-F4	5´-TACTAGATCTGGGCCTCGT-3´
Bt11-LB-R1	5´-TTTCTACGGGGTCTGACGCT-3´	Bt11-RB-R1	5´-CGAGCCTCTGGTCGATGATA-3´
Bt11-LB-R2	5´-TGGTAGCTCTTGATCCGGCA-3´	Bt11-RB-R2	5´-ATGTGGGGTGAGGTGTACGA-3´
Bt11-LB-R3	5´-CGCTCAGTGGAACGAAAACTC-3´	Bt11-RB-R3	5´-TTTTGATGAAAATAGCCATGAG-3´
Bt11-LB-R4	5´-AACAAACCACCGCTGGTAGC-3´	Bt11-RB-R4	5´-GAGGCTAACACCTACAGATTTTAGA-3´
Bt11-LB-R5	5´-GTATCTGCGCTCTGCTGAAG-3´	Bt11-RB-R5	5´-CTCCACCAAAAATCCAAGAATC-3´
(S1)Bt11-F	5´-GCCTCGTGATACGCCTATTTTT-3´	(S2)Bt11-F	5´-GCGGAACCCCTATTTGTTTA-3´
(S1)Bt11-R	5´-CAAAAATCCAAGAATCCCTCCA-3´	(S2)Bt11-R	5´-CAAGAAATGGTCTCCACCAAA-3´
Bt11-RB-F1	5´-AGACGTCAGGTGGCACTTTT-3´

序号	组合	产物大小/bp	序号	组合	产物大小/bp	序号	组合	产物大小/bp
1	Bt11-LB-F1/R1	133	15	Bt11-RB-F1/R4	152	29	Bt11-RB-F4/R5	181
2	Bt11-LB-F1/R2	243	16	Bt11-RB-F1/(S2)Bt11-R	126	30	Bt11-RB-F4/(S2)Bt11-R	192
3	Bt11-LB-F1/R4	223	17	Bt11-RB-F2/R1	221	31	Bt11-RB-F4/(S1)Bt11-R	175
4	Bt11-LB-F1/R5	286	18	Bt11-RB-F2/R2	238	32	(S2)Bt11-F/Bt11-RB-R1	202
5	Bt11-LB-F2/R1	176	19	Bt11-RB-F2/R3	181	33	(S2)Bt11-F/Bt11-RB-R2	219
6	Bt11-LB-F2/R2	286	20	Bt11-RB-F2/R4	138	34	(S2)Bt11-F/Bt11-RB-R3	162
7	Bt11-LB-F2/R3	160	21	Bt11-RB-F3/R1	300	35	(S1)Bt11-R/Bt11-RB-R1	289
8	Bt11-LB-F2/R4	266	22	Bt11-RB-F3/R3	260	36	(S1)Bt11-R/Bt11-RB-R3	249
9	Bt11-LB-F3/R2	179	23	Bt11-RB-F3/R4	217	37	(S1)Bt11-R/Bt11-RB-R4	206
10	Bt11-LB-F3/R4	159	24	Bt11-RB-F3/R5	180	38	(S1)Bt11-R/Bt11-RB-R5	169
11	Bt11-LB-F3/R5	222	25	Bt11-RB-F3/(S2)Bt11-R	191	39	(S1)Bt11-R/(S2)Bt11-R	180
12	Bt11-RB-F1/R1	235	26	Bt11-RB-F3/(S1)Bt11-R	174	40	(S1)Bt11-R/(S1)Bt11-F	163
13	Bt11-RB-F1/R2	252	27	Bt11-RB-F4/R3	261
14	Bt11-RB-F1/R3	195	28	Bt11-RB-F4/R4	218

序号	组合	产物大小/bp	序号	组合	产物大小/bp	序号	组合	产物大小/bp
1	Bt11-LB-F1/R1	133	15	Bt11-RB-F1/R4	152	29	Bt11-RB-F4/R5	181
2	Bt11-LB-F1/R2	243	16	Bt11-RB-F1/(S2)Bt11-R	126	30	Bt11-RB-F4/(S2)Bt11-R	192
3	Bt11-LB-F1/R4	223	17	Bt11-RB-F2/R1	221	31	Bt11-RB-F4/(S1)Bt11-R	175
4	Bt11-LB-F1/R5	286	18	Bt11-RB-F2/R2	238	32	(S2)Bt11-F/Bt11-RB-R1	202
5	Bt11-LB-F2/R1	176	19	Bt11-RB-F2/R3	181	33	(S2)Bt11-F/Bt11-RB-R2	219
6	Bt11-LB-F2/R2	286	20	Bt11-RB-F2/R4	138	34	(S2)Bt11-F/Bt11-RB-R3	162
7	Bt11-LB-F2/R3	160	21	Bt11-RB-F3/R1	300	35	(S1)Bt11-R/Bt11-RB-R1	289
8	Bt11-LB-F2/R4	266	22	Bt11-RB-F3/R3	260	36	(S1)Bt11-R/Bt11-RB-R3	249
9	Bt11-LB-F3/R2	179	23	Bt11-RB-F3/R4	217	37	(S1)Bt11-R/Bt11-RB-R4	206
10	Bt11-LB-F3/R4	159	24	Bt11-RB-F3/R5	180	38	(S1)Bt11-R/Bt11-RB-R5	169
11	Bt11-LB-F3/R5	222	25	Bt11-RB-F3/(S2)Bt11-R	191	39	(S1)Bt11-R/(S2)Bt11-R	180
12	Bt11-RB-F1/R1	235	26	Bt11-RB-F3/(S1)Bt11-R	174	40	(S1)Bt11-R/(S1)Bt11-F	163
13	Bt11-RB-F1/R2	252	27	Bt11-RB-F4/R3	261
14	Bt11-RB-F1/R3	195	28	Bt11-RB-F4/R4	218

序号	样品名称	样品组成	样品状态
1	空白对照	水	—
2	阴性对照	抗虫玉米Bt11非转基因对照玉米基因组DNA	植株叶片
3	阳性对照	抗虫玉米Bt11转化体基因组DNA，拷贝数分数为1%	植株叶片
4	其他转基因玉米混合样	本实验室制备^[12]	粉末
5	转基因大豆混合样	本实验室制备^[12]	粉末
6	转基因水稻混合样	本实验室制备^[12]	粉末
7	转基因油菜混合样	本实验室制备^[12]	粉末
8	转基因棉花混合样	本实验室制备^[12]	粉末

Establishment of Specific Qualitative PCR Detection Method of Transgenic Maize Bt11

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 18

Related Articles 8

Recommended Articles

Metrics

Comments

[1]	Xin QI, Xinran LI, Yaning GUO, Dan WANG, Kai LI, Qiong WU, Liang LI. Comparison of Endogenous Genes in Maize Based on Digital PCR [J]. Current Biotechnology, 2025, 15(1): 78-85.
[2]	Haoqian WANG, Yan HUANG, Yuting SHI, Pengyu ZHU, Yuzhou XIE, Chunmeng HUANG, Xiaoyu LU, Wei FU. Construction and Application of Reference Plasmids for Detection of Transgenic Insect-resistant Crops [J]. Current Biotechnology, 2023, 13(1): 83-89.
[3]	Xing DANG, Binwei ZHI, Kehao CAO, Tingting LIU, Biao CHEN, Yuanjie DING. Patent Analysis on Genetically Modified Maize Biological Breeding Technology and Development Suggestions [J]. Current Biotechnology, 2022, 12(4): 614-622.
[4]	Hongtao WEN, Yang YANG, Yijia DING, Ran YUAN, Ruiying ZHANG, Zhiyuan XU, Jingang LIANG. Establishment of Qualitative PCR Detection of Transgenic Insect⁃resistant Maize CM8101 [J]. Current Biotechnology, 2022, 12(2): 248-255.
[5]	REN Wen1, YANG Haixia2, CHEN Lizhu2, LI Yufeng2, LIU Ya1*. Establishment and Application of Nucleic Acid Chromatography for Rapid Detection of Transgenic Plants [J]. Curr. Biotech., 2020, 10(6): 680-687.
[6]	WEN Hongtao1§,YANG Yang1§,DING Yijia1,YUAN Ran1,ZHANG Xiujie2,ZHANG Ruiying1*. Qualitative PCR Methods for the Detection of Transgenic Herbicide-tolerant Maize G1105E-823C [J]. Curr. Biotech., 2020, 10(6): 688-695.
[7]	WU Wenyan^§, LIU Xinxiang^§, ZHOU Miaoyi^*, LIU Jinxiang, LIU Ya. Establishment of Event-specific PCR Detection Method of Transgenic Maize Line 2A-5 [J]. Curr. Biotech., 2020, 10(4): 363-370.
[8]	ZHANG Zhi-fang1, LIU Ya2, REN Wen2, DAI Lu-yuan3. Identifying the Drought Resistance of TsVP* Transgenic Maize [J]. Curr. Biotech., 2013, 3(3): 206-210.