CM8101 Transgenic Maize: Qualitative PCR Assay Detection of Specificity

doi:10.11924/j.issn.1000-6850.casb2020-0576

Abstract

Abstract:

To provide technical support for GM (genetically modified) safety supervision, a qualitative detection method for GM maize CM8101 was established. According to the insertion sequence information of CM8101, PCR primers were designed in the 3' end flanking sequence. Through the amplification system optimization, the specificity test, sensitivity and stability tests, a specific method of CM8101 was developed. The results showed that all test parameters of this method met the requirements of transgenic molecular detection standard. The method had good specificity, and the detection sensitivity reached 0.1%. CM8101 is a new transgenic maize line with industrial application prospect independently developed in China, so this method can provide a technical means for the identification of CM8101 and its derives.

Key words: transgenic maize, CM8101, qualitative method, molecular detection, PCR, specificity

CLC Number:

S188

Long Likun, Zhao Ning, Xia Wei, Li Congcong, Dong Liming, Yan Wei, Li Feiwu. CM8101 Transgenic Maize: Qualitative PCR Assay Detection of Specificity[J]. Chinese Agricultural Science Bulletin, 2021, 37(23): 23-28.

Figures/Tables 9

References 25

[1]	国际农业生物技术应用服务组织. 2018 年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 2019, 39(8):1-6.
[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.
[3]	沈泓, 李超, 李珏. 分子生物学技术在转基因食品检测领域中的研究进展[J]. 中国农业信息, 2017(15):57-59.
[4]	周琳华, 肖维威, 吴永彬, 等. 苏云金芽孢杆菌Cry1A(b)抗虫基因LAMP检测方法的建立与应用[J]. 生物技术通报, 2012(4):165-169.
[5]	徐君怡, 曹际娟, 郑秋月, 等. 基于Taqman微流控芯片技术高通量检测17种转基因玉米品系[J]. 分析化学, 2020, 48(11):1477-1488.
[6]	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.
[7]	缪青梅, 汪小福, 陈笑芸, 等. 基于双重微滴数字PCR精准定量转基因水稻G6H1的方法研究[J]. 农业生物技术学报, 2019, 27(1):159-169.
[8]	段星阳, 田晶晶, 张园, 等. 转基因成分功能核酸生物传感检测技术[J]. 农业生物技术学报, 2019, 27(12):2265-2271.
[9]	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]. Front Plant Science, 2017, 8:1670. doi: 10.3389/fpls.2017.01670 URL
[10]	Fraiture M A, Herman P, de Loose M, et al. How can we better detect unauthorized GMOs in food and feed chains?[J]. Trends Biotechnology, 2017, 35(6):508-517. doi: 10.1016/j.tibtech.2017.03.002 URL
[11]	汤婷, 谢实龙, 祝旋, 等. CaMV35S启动子及其在转基因作物中的应用和检测[J]. 浙江农业学报, 2019, 31(1):161-170.
[12]	郭文芳, 王楠, 李刚强, 等. CP4-EPSPS转基因棉花植株鉴定方法比较分析[J]. 生物技术通报, 2017, 33(4):114-118.
[13]	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.
[14]	李太强, 高亚男, 高居荣, 等. 转基因小麦中bar基因检测方法研究[J]. 麦类作物学报, 2015, 35(9):1190-1193.
[15]	Deisingh A K, Badrie N. Detection approaches for genetically modified organisms in foods[J]. Food Research International, 2005, 38(6):639-649. doi: 10.1016/j.foodres.2005.01.003 URL
[16]	Babekova R, Funk T, Pecoraro S, et al. Development of an eventspecific Real-time PCR detection method for the transgenic Bt rice line KMD1[J]. European Food Research Technology, 2009, 228(5):707-716. doi: 10.1007/s00217-008-0981-0 URL
[17]	Datukishvili N, Kutateladze T, Gabriadze I, et al. New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 2015, 6:757. doi: 10.3389/fmicb.2015.00757 pmid: 26257724
[18]	Fu W, Zhu P, Wang C, et al. A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment[J]. Scientific. Report, 2015, 53(5):12715.
[19]	Dobnik D, Spilsberg B, Kosir A B, et al. Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction[J]. Analytical Chemistry, 2015, 87(16):8218-8226. doi: 10.1021/acs.analchem.5b01208 pmid: 26169291
[20]	Demeke T, Dobnik D. Critical assessment of digital PCR for the detection and quantification of genetically modified organisms. Analytical and Bioanalytical Chemistry, 2018, 410:4039-4050.
[21]	王国义, 贺晓云, 许文涛, 等. 转基因植物食用安全性评估与监管研究进展[J]. 食品科学2019, 40(11):343-345.
[22]	Jae-HwanKim, Dabing Zhang, Hae-Yeong Kim Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems[J]. Food Control, 2014, 1(35):345-353
[23]	RAO J, Yang L T, Guo J C, et al. Development of event-specific qualitative and quantitative PCR detection methods for the transgenic maize BVLA430101[J]. European Food Research and Technology, 2016, 242(8):1277-1284. doi: 10.1007/s00217-015-2631-7 URL
[24]	Fu L Z, Song J, Niu B, et al. An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples[J]. Food Control, 2015(57):1-8
[25]	罗云波, 贺晓云. 中国转基因作物产业发展概述[J]. 中国食品学报, 2014, 14(8):10-15.

样品名称	包括的转化体
转基因玉米混合物	Bt11、Bt176、MON810、MON863、GA21、NK603、T25、TC1507、MON88017、59122、MIR604、3272、MON87460、MIR162、DAS40278-9、4114、MON87427、5307、 MON89034
转基因大豆混合物	GTS40-3-2、MON89788、CV127、A5547-127、A2704-12、305423、356043、MON87769、 MON87705、FG72、DAS68416-4
转基因棉花混合物	MON15985、LLCOTTON25、MON1445、MON531 、MON88913
转基因油菜混合物	GT73、MS1、MS8、RF1、RF2、RF3、T45、Oxy235、Topas19/2

样品名称	包括的转化体
转基因玉米混合物	Bt11、Bt176、MON810、MON863、GA21、NK603、T25、TC1507、MON88017、59122、MIR604、3272、MON87460、MIR162、DAS40278-9、4114、MON87427、5307、 MON89034
转基因大豆混合物	GTS40-3-2、MON89788、CV127、A5547-127、A2704-12、305423、356043、MON87769、 MON87705、FG72、DAS68416-4
转基因棉花混合物	MON15985、LLCOTTON25、MON1445、MON531 、MON88913
转基因油菜混合物	GT73、MS1、MS8、RF1、RF2、RF3、T45、Oxy235、Topas19/2

引物名称	引物序列(5’--3’)	引物长度/bp	产物大小/bp	位置
8101-F1	GTGCCAGAGTCAACCCA	17	132	5’端
8101-R1	ATTCGGCGTTAATTCAGTAC	20	132	5’端
8101-F2	ATCCCAACATGAGCTAGTCG	20	179	5’端
8101-R2	GCCTGAATGGCGAATGCTA	19	179	5’端
8101-F3	GAGTCAACCCATGTTAGTCT	20	150	5’端
8101-R3	GCCTGAATGGCGAATGCTA	19	150	5’端
8101-F4	GTGCCAGAGTCAACCCA	17	177	5’端
8101-R4	GCCCTTCCCAACAGTTGC	18	177	5’端
8101- F5	GGAGAGGCGGTTTACGAGTTT	21	100	3’端
8101- R5	GTCAAAACCTCACCCCACA	19	100	3’端
8101- F6	GGAGAGGCGGTTTACGAGTTT	21	197	3’端
8101- R6	GCCATTGATCGTCGACCAG	19	197	3’端
8101- F7	GGAAGCATAAAGTGTAAAGCC	21	242	3’端
8101- R7	CATTTCTTGGTCAAAACCTCAC	22	242	3’端

引物名称	引物序列(5’--3’)	引物长度/bp	产物大小/bp	位置
8101-F1	GTGCCAGAGTCAACCCA	17	132	5’端
8101-R1	ATTCGGCGTTAATTCAGTAC	20	132	5’端
8101-F2	ATCCCAACATGAGCTAGTCG	20	179	5’端
8101-R2	GCCTGAATGGCGAATGCTA	19	179	5’端
8101-F3	GAGTCAACCCATGTTAGTCT	20	150	5’端
8101-R3	GCCTGAATGGCGAATGCTA	19	150	5’端
8101-F4	GTGCCAGAGTCAACCCA	17	177	5’端
8101-R4	GCCCTTCCCAACAGTTGC	18	177	5’端
8101- F5	GGAGAGGCGGTTTACGAGTTT	21	100	3’端
8101- R5	GTCAAAACCTCACCCCACA	19	100	3’端
8101- F6	GGAGAGGCGGTTTACGAGTTT	21	197	3’端
8101- R6	GCCATTGATCGTCGACCAG	19	197	3’端
8101- F7	GGAAGCATAAAGTGTAAAGCC	21	242	3’端
8101- R7	CATTTCTTGGTCAAAACCTCAC	22	242	3’端