转基因玉米MON87411实时荧光PCR定性检测方法的建立及其标准化

doi:10.19586/j.2095-2341.2023.0158

生物技术进展 ›› 2024, Vol. 14 ›› Issue (2): 257-262.DOI: 10.19586/j.2095-2341.2023.0158

• 研究论文 • 上一篇

转基因玉米MON87411实时荧光PCR定性检测方法的建立及其标准化

李凌燕¹^,²(), 肖冰³(), 张旭冬¹, 张华¹, 陈子言¹, 王颢潜¹, 张秀杰¹, 陈红¹(), 梁晋刚¹()

^1.农业农村部科技发展中心，北京 100176
^2.北京农业职业学院食品与生物工程学院，北京 102442
^3.中国农业科学院农业资源与农业区划研究所，北京 100081

收稿日期:2023-12-08 接受日期:2024-01-24 出版日期:2024-03-25 发布日期:2024-04-17
通讯作者: 陈红,梁晋刚
作者简介:李凌燕 E-mail: 80200@bvca.edu.cn
肖冰E-mail: 17319374552@163.com第一联系人：李凌燕和肖冰为本文并列第一作者
基金资助:
科技创新2030-重大专项(2022ZD0401909);农业农村部农业科研杰出人才培养计划项目(农办科〔2021〕27号)

Establishment and Standardization of Real-time PCR Method for Qualitative Detection of Genetically Modified Maize MON87411

Lingyan LI¹^,²(), Bing XIAO³(), Xudong ZHANG¹, Hua ZHANG¹, Ziyan CHEN¹, Haoqian WANG¹, Xiujie ZHANG¹, Hong CHEN¹(), Jingang LIANG¹()

^1.Development Center of Science and Technology，Ministry of Agriculture and Rural Affairs，Beijing 100176，China
^2.Department of Food and Bioengineering，Beijing Vocational College of Agriculture，Beijing 102442，China
^3.Institute of Agricultural Resources and Regional Planning，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2023-12-08 Accepted:2024-01-24 Online:2024-03-25 Published:2024-04-17
Contact: Hong CHEN,Jingang LIANG

摘要/Abstract

摘要：

转基因玉米MON87411是孟山都远东有限公司研发的抗虫耐除草剂玉米转化体，该转化体已获得进口加工原料的农业转基因生物安全证书。以转基因玉米MON87411品系特异性序列为靶标设计引物和探针，经特异性测试、体系优化、灵敏度测试及检出限测试，建立了转基因玉米MON87411的实时荧光PCR定性检测方法。结果表明，该方法能检测出转基因玉米MON87411的转化体成分，检出限（limit of detection，LOD）可达0.05%，具有稳定性好、特异性强、灵敏度高的特点。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试，循环验证报告显示该方法符合国家标准方法的各项要求，可在检测行业推广应用。研究建立的方法可为我国对转基因玉米MON87411品系的安全监管提供有效的技术支撑。

关键词: 抗虫耐除草剂玉米, MON87411, 实时荧光PCR, 安全监管, 标准化

Abstract:

Genetically modified （GM） maize MON87411 is an event developed by Monsanto， and it has obtained the biological safety certificate imported as raw materials for processing. In this paper， the primers and probes were designed with the specific sequences as targets. Through specificity test， system optimization， sensitivity test and limit of detection （LOD） test， a real-time PCR method for the qualitative detection of GM maize MON87411 was established. The results showed that the method could detect the components of GM maize MON87411， and had the characteristics of good stability， strong specificity and high sensitivity and the LOD could reach 0.05%. The specific test， LOD test and reproducibility test of the method were carried out by 8 domestic GMO safety testing institutions. The cyclic verification report showed that the method met the requirements of the national standard method and could be promoted and applied in the testing industry. The establishment of this method could provide effective technical support for the safety supervision of GM maize MON87411 strain in China.

Key words: insect-resistant and herbicide-tolerant maize, MON87411, real-time PCR, safety supervision, standardization

中图分类号:

Q-33

李凌燕, 肖冰, 张旭冬, 张华, 陈子言, 王颢潜, 张秀杰, 陈红, 梁晋刚. 转基因玉米MON87411实时荧光PCR定性检测方法的建立及其标准化[J]. 生物技术进展, 2024, 14(2): 257-262.

Lingyan LI, Bing XIAO, Xudong ZHANG, Hua ZHANG, Ziyan CHEN, Haoqian WANG, Xiujie ZHANG, Hong CHEN, Jingang LIANG. Establishment and Standardization of Real-time PCR Method for Qualitative Detection of Genetically Modified Maize MON87411[J]. Current Biotechnology, 2024, 14(2): 257-262.

图/表 9

参考文献 23

1	ISAAA. Global status of commercialized Biotech/GM crops in 2019: Biotech crops drive socio-economic development and sustainable environment in the new frontier[R/OL]. (2020-11-30)[2024-01-20]. .
2	甄贞,高学军,张明辉.主要农作物种子转基因检测方法及应用[J].中国种业,2018(4):8-11.
	ZHEN Z, GAO X J, ZHANG M H. Method and application of genetically modified organism detection for major crop seeds[J]. China Seed Ind., 2018(4): 8-11.
3	董文凤,梁晋刚,李夏莹,等.转基因耐除草剂玉米C0010.2.2定性PCR检测方法的建立[J].玉米科学,2021,29(5):57-64.
	DONG W F, LIANG J G, LI X Y, et al.. Development of detection method of genetically modified herbicide-tolerant maize C0010.2.2[J]. J. Maize Sci., 2021, 29(5): 57-64.
4	龙丽坤,赵宁,李葱葱,等.转基因玉米CM8101实时荧光定量PCR检测方法的建立[J].农业生物技术学报,2021,29(5):1007-1015.
	LONG L K, ZHAO N, LI C C, et al.. Establishment of qRT-PCR method of detection transgenic maize (Zea mays) CM8101[J]. J. Agric. Biotechnol.,2021, 29(5): 1007-1015.
5	王永,兰青阔,赵新,等.数字PCR在转基因水稻拷贝数鉴定中的应用[J].生物技术通报,2018,34(3):53-58.
	WANG Y, LAN Q K, ZHAO X, et al.. Estimation of the copy number of exogenous genes in genetically modified rice by droplet digital PCR[J]. Biotechnol. Bull., 2018, 34(3): 53-58.
6	OLICEIRA C A, KOMMERS C M, LEHMANN F K, et al.. Detection of genetically modified maize in processed products,dry grains,and corn ears intended for fresh consumption in South Brazil[J]. Genet. Mol. Res., 2016, 15(4): 8818-8827.
7	ALASAAD N, ALZUBI H, KADER A A. Data in support of the detection of genetically modified organisms (GMOs) in food and feed samples[J]. Data Brief, 2016, 7: 243-252.
8	尹全,李忆,王东,等.转基因抗除草剂玉米DAS-40278-9品系特异实时荧光定量PCR检测方法的建立[J]. 中国食品学报,2016,16(5):147-154.
	YIN Q, LI Y, WANG D, et al.. Establishment of event-specific quantitative PCR of event DAS-40278-9 herbicide-tolerant maize[J]. J. Chin. Inst. Food Sci. Technol., 2016, 16(5): 147-154.
9	石盼盼,谢文佳,刘燕,等.多重实时荧光PCR快速检测转基因大豆[J].江苏农业科学,2020,48(18):77-81.
	SHI P P, XIE W J, LIU Y, et al.. Rapid detection of genetically modified soybeans by multiplex real-time fluorescence PCR[J]. Jiangsu Agric. Sci., 2020,48(18):77-81.
10	WANG X, TANG T, MIAO Q, et al.. Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR,and droplet digital PCR[J]. Food Contr., 2018, 98: 380-388.
11	中华人民共和国农业农村部. 转基因植物及其产品成分检测定性PCR方法制定指南:2259号公告-4-2015 [S].北京:中国农业出版社,2015.
12	ENGL. Definition of minimum performance requirements for analytical methods of GMO testing [S]. European Network of GMO Laboratories, 2015.
13	国家质量监督检验检疫总局. 转基因检测非标方法确认评价指南: [S].北京:中国标准出版社, 2017.
14	李葱葱,谢苹,董立明,等.抗虫耐除草剂玉米GH5112E-117C定性PCR检测方法[J].生物技术通报,2020,36(5):64-67.
	LI C C, XIE P, DONG L M, et al.. Qualitative PCR assay for the detection of GH5112E-117C transgenic maize resistant to insects and herbicides[J]. Biotechnol. Bull.,2020,36(5):64-67.
15	LIU M, ZHANG X J, GAO Y,et al.. Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance[J]. J. Zhejiang Univ. Sci. B, 2018, 19(8): 610-619.
16	CHU H W, TU R J, NIU F A, et al.. A new PCR/LDR-based multiplex functional molecular marker for marker-assisted breeding in rice[J]. Rice Sci., 2021, 28(1): 6-10.
17	SALISU I B, SHAHID A A, YAQOOB A, et al.. Molecular approaches for high throughput detection and quantification of genetically modified crops: a review[J/OL]. Front. Plant Sci., 2017, 8: 1670[2024-02-22]. .
18	RAO J, YANG L, GUO J, et al.. Development of event-specific qualitative and quantitative PCR detection methods for the transgenic maize BVLA430101[J].Eur. Food Res. Technol., 2016, 242(8): 1277-1284.
19	ZHANG F, SONG J, NIU B, et al.. An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples[J]. Food Contr., 2015, 57: 1-8.
20	琚鹏举,宁蕾,葛林豪,等.采用优化的数字PCR方法分析转基因小麦外源基因拷贝数[J].中国农业科学,2020,53(10):1931-1939.
	LU P J, NING L, GE L H, et al.. Analysis of foreign gene copy number in transgenic wheat by optimized digital PCR[J]. Sci. Agric. Sin., 2020, 53(10): 1931-1939.
21	刘晓,朱鹏宇,景小艳,等.双重数字PCR在转基因大豆检测中的应用[J].生物技术进展,2020,10(1):60-66.
	LIU X, ZHU P Y, JING X Y, et al.. Application of duplex droplet digital PCR for detection of genetically modified soybean[J]. Curr. Biotechnol., 2020, 10(1): 60-66.
22	温洪涛,杨洋,丁一佳,等. 耐除草剂玉米G1105E-823C定性检测方法[J].生物技术进展,2020,10(6):688-695.
	WEN H T, YANG Y, DING Y J, et al. Qualitative PCR methods for the detection of transgenic herbicide-tolerant maize G1105E-823C[J]. Curr. Biotechnol., 2020, 10(6): 688-695.
23	武文艳,刘新香,周秒依,等.转基因玉米2A-5特异性PCR方法的建立[J].生物技术进展,2020,10(4):363-370.
	WU W Y, LIU X X, ZHOU M Y, et al.. Establishment of event-specific PCR detection method of transgenic maize line 2A-5[J]. Curr. Biotechnol., 2020, 10(4): 363-370.

样品名称	含有的转化体
转基因玉米混合样品	Bt11、Bt176、MON810、MON863、GA21、NK603、T25、TC1507、MON89034、MON88017、59122、MIR604、3272、MON87460、MIR162、DAS40278-9、4114、MON87427、5307
转基因大豆混合物样品	GTS40-3-2、MON89788、A5547-127、A2704-12、356043、305423、CV127、MON87701、MON87708、MON87769、MON87705、FG72、DAS68416-4、SHZD32-1
转基因水稻混合物样品	TT51-1、KF-6、KMD-1、M12、KF-8、KF-2、G6H1、T1C-19
转基因油菜混合物样品	MS1、MS8、RF1、RF2、RF3、T45、Oxy-235、Topas19/2、MON88302、73496
转基因棉花混合物样品	MON1445、MON531、MON15985、LLCOTTON25、MON88913、GHB614、COT102

样品名称	含有的转化体
转基因玉米混合样品	Bt11、Bt176、MON810、MON863、GA21、NK603、T25、TC1507、MON89034、MON88017、59122、MIR604、3272、MON87460、MIR162、DAS40278-9、4114、MON87427、5307
转基因大豆混合物样品	GTS40-3-2、MON89788、A5547-127、A2704-12、356043、305423、CV127、MON87701、MON87708、MON87769、MON87705、FG72、DAS68416-4、SHZD32-1
转基因水稻混合物样品	TT51-1、KF-6、KMD-1、M12、KF-8、KF-2、G6H1、T1C-19
转基因油菜混合物样品	MS1、MS8、RF1、RF2、RF3、T45、Oxy-235、Topas19/2、MON88302、73496
转基因棉花混合物样品	MON1445、MON531、MON15985、LLCOTTON25、MON88913、GHB614、COT102

5′端引物和探针	序列（5′→3′）	3′端引物和探针	序列（5′→3′）
MON87411-5-Q	5′-CCGGGAAATCTACATGGATC-3′	MON87411-3-2Q	5′-CGGCCGCGTTTAAACTAT-3′
MON87411-5-1F-2	5′-GGCAAAACACTAATGAATAGTTAAG-3′	MON87411-3-1F	5′-TGTAACAGAAAACACCATC-3′
MON87411-5-1F-3	5′-GGCAAAACACTAATGAATAG-3′	MON87411-3-1R	5′-TGCAACAAAGTGAACACCAG-3′
MON87411-5-1R	5′-GCTGCGGACATCTACATTTT-3′	MON87411-3-2R	5′-AAGTGAACTAGTTCTAGGGTAG-3′
MON87411-5-2R	5′-AATAACGCTGCGGACATCT-3′
MON87411-5-3R	5′-CTCCATATTGACCATCATACTC-3′

5′端引物和探针	序列（5′→3′）	3′端引物和探针	序列（5′→3′）
MON87411-5-Q	5′-CCGGGAAATCTACATGGATC-3′	MON87411-3-2Q	5′-CGGCCGCGTTTAAACTAT-3′
MON87411-5-1F-2	5′-GGCAAAACACTAATGAATAGTTAAG-3′	MON87411-3-1F	5′-TGTAACAGAAAACACCATC-3′
MON87411-5-1F-3	5′-GGCAAAACACTAATGAATAG-3′	MON87411-3-1R	5′-TGCAACAAAGTGAACACCAG-3′
MON87411-5-1R	5′-GCTGCGGACATCTACATTTT-3′	MON87411-3-2R	5′-AAGTGAACTAGTTCTAGGGTAG-3′
MON87411-5-2R	5′-AATAACGCTGCGGACATCT-3′
MON87411-5-3R	5′-CTCCATATTGACCATCATACTC-3′

编号	上游引物	下游引物	引物探针组	片段长度/bp
5′-1	MON87411-5-1F-2	MON87411-5-1R	MON87411-5-Q	141
5′-2	MON87411-5-1F-2	MON87411-5-2R	MON87411-5-Q	146
5′-3	MON87411-5-1F-2	MON87411-5-3R	MON87411-5-Q	84
5′-4	MON87411-5-1F-3	MON87411-5-1R	MON87411-5-Q	141
5′-5	MON87411-5-1F-3	MON87411-5-2R	MON87411-5-Q	146
5′-6	MON87411-5-1F-3	MON87411-5-3R	MON87411-5-Q	84
3′-1	MON87411-3-1F	MON87411-3-1R	MON87411-3-2Q	146
3′-2	MON87411-3-1F	MON87411-3-2R	MON87411-3-2Q	103
3′-3	MON87411-QF	MON87411-QR	MON87411-QP	109

转基因玉米MON87411实时荧光PCR定性检测方法的建立及其标准化

Establishment and Standardization of Real-time PCR Method for Qualitative Detection of Genetically Modified Maize MON87411

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 23

相关文章 5

编辑推荐

Metrics

本文评价

样品	实时荧光定性PCR检测结果
阳性对照	+	+	+	+	+	+
阴性对照	-	-	-	-	-	-
1% MON87411转化体	+	+	+	+	+	+
其他转基因玉米混合样	-	-	-	-	-	-
转基因大豆混合样	-	-	-	-	-	-
转基因水稻混合样	-	-	-	-	-	-
转基因棉花混合样	-	-	-	-	-	-
转基因油菜混合样	-	-	-	-	-	-
非转基因玉米混合样	-	-	-	-	-	-
0.05% MON87411转化体	+	+	+	+	+	+

[1]	柯振华. 冷链食品中铜绿假单胞菌的检测方法研究及进化树构建[J]. 生物技术进展, 2023, 13(6): 940-944.
[2]	李晶晶, 赵海波, 翟睿, 梁亮, 范培蕾, 赵雨佳. 糖化血红蛋白（HbA_1c）检测技术与标准化研究进展[J]. 生物技术进展, 2022, 12(6): 837-846.
[3]	甄珍, 窦迎港, 刘晓兰. 玉米蛋白粉DNA提取方法比较研究[J]. 生物技术进展, 2022, 12(1): 99-104.
[4]	温洪涛,杨洋,丁一佳,苑冉,张秀杰,张瑞英. 耐除草剂玉米G1105E-823C定性检测方法[J]. 生物技术进展, 2020, 10(6): 688-695.
[5]	黄春蒙,,朱鹏宇,王智,王晨光,杜智欣,魏霜4,张永江,付伟. 基于RNAi技术的转基因植物监管现状及其面临的育种领域的挑战[J]. 生物技术进展, 2020, 10(1): 10-14.