Specific Qualitative PCR Detection Method for Transgenic Maize Zheda Ruifeng 8

doi:10.13560/j.cnki.biotech.bull.1985.2024-0324

Abstract

Abstract:

【Objective】Zheda Ruifeng 8 is an insect-resistant transgenic maize developed by Hangzhou Ruifeng Biotechnology Co. Ltd., the safety certificate for production and application was obtained in December 2021. The specific PCR detection method for Zheda Ruifeng 8 was investigated to provide technical support for market regulation and industrialization. 【Method】In this study, the flanking sequence of the insertion end of exogenous gene of Zheda Ruifeng 8 event was used as the target sequence, specific primers were designed at the 5'-end（right flanking）and the 3'-end（left flanking）respectively. Twenty pairs of primers for each of the left flanking and the right flanking were screened by the standard reaction system and the reaction procedure that was consistent with that of the standard gene zSSIIb in maize, and the primers with high specificity and fine stability were obtained. A preliminary method for the detection of PCR of the Zheda Ruifeng 8 event was established by changing the two key parameters of the annealing temperature and the concentration of the primer. 【Result】The screened Zheda Ruifeng 8 PCR amplification primer RF 8-RB-R4/F1 may specifically amplify the target fragment of 265 bp, and a standard reaction system and reaction procedure consistent with the standard gene zSSIIb in maize was established, and it achieved high throughput for batch detection. 【Conclusion】The established specific qualitative PCR detection method demonstrated excellent stability and adaptability. It could consistently detect a copy number fraction as low as 0.1%, equivalent to approximately 20 copies. This method proved to be highly accurate and specific, effectively detecting the transformants of Zheda Ruifeng 8.

Key words: genetically modified maize, Zheda Ruifeng 8, qualitative PCR, specific detection

TIAN Jin, ZHANG Yue-qiu, ZHANG Hua, CHEN Zi-yan, TIAN Lu, WANG Hao-qian, GAO Fang-rui, LIANG Jin-gang, CHEN Hong. Specific Qualitative PCR Detection Method for Transgenic Maize Zheda Ruifeng 8[J]. Biotechnology Bulletin, 2024, 40(12): 45-52.

Figures/Tables 11

References 16

[1]	黎裕, 徐辰武, 秦峰, 等. 玉米生物育种:现状与展望[J]. 中国基础科学, 2022, 24(4): 18-28.
	Li Y, Xu CW, Qin F, et al. Maize breeding: advances and prospects[J]. China Basic Sci, 2022, 24(4): 18-28.
[2]	徐晓丽, 姜媛媛, 王鹏飞, 等. 表达Cry1Ab和Cry2Ab蛋白的转基因玉米GAB-3对四种主要鳞翅目害虫的抗性评价[J]. 中国农业科技导报, 2020, 22(12): 97-104. doi: 10.13304/j.nykjdb.2020.0388
	Xu XL, Jiang YY, Wang PF, et al. Resistance evaluation of genetically modified maize GAB-3 expressing Cry1Ab and Cry2Ab against four major lepidopteran pests[J]. J Agric Sci Technol, 2020, 22(12): 97-104.
[3]	雷展, 王建成, 张晨, 等. 转基因油菜NS-B50027-4定性定量检测方法的建立[J]. 农业生物技术学报, 2021, 29(9): 1825-1835.
	Lei Z, Wang JC, Zhang C, et al. Establishment of qualitative and quantitative detection method for transgenic Brassica napus NS-B50027-4[J]. J Agric Biotechnol, 2021, 29(9): 1825-1835.
[4]	斯能武, 李俊, 武玉花, 等. 数字PCR在转基因定量检测中的研究进展[J]. 中国油料作物学报, 2021, 43(1): 40-50.
	Si NW, Li J, Wu YH, et al. Research progress of digital PCR in quantitative detection of genetically modified organism[J]. Chin J Oil Crop Sci, 2021, 43(1): 40-50. doi: 10.19802/j.issn.1007-9084.2020265
[5]	刘欣, 祝长青, 王毅谦, 等. 大豆转基因检测中DNA提取方法的比较研究[J]. 食品科学, 2013, 34(4): 199-203.
	Liu X, Zhu CQ, Wang YQ, et al. Methodology comparison of DNA extraction from soybean for detection of genetically modified organism[J]. Food Sci, 2013, 34(4): 199-203.
[6]	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. doi: 10.1007/s00216-009-3343-2 pmid: 20012944
[7]	中华人民共和国农业部.转基因植物及其产品成分检测定性PCR方法制定指南: 农业部2259号公告-4—2015[S]. 北京: 中国农业出版社, 2015.
	Ministry of Agriculture of the People's Republic of China.Detection of Genetically Modified Plants and Derived Products Guidelines for Establishing qualitative PCR Method: Department of Agriculture Notice 2259-4-2015[S]. Beijing: China Agriculture Press, 2015.
[8]	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.
	Ministry of Agriculture of the People's Republic of China.Detection of genetically modified plants and derived products. Target-taxon-specifis qualitative PCR method for maize: Department of Agriculture Notice 1861-3-2012[S]. Beijing: China Agriculture Press, 2013.
[9]	李凌燕, 张旭冬, 陈子言, 等. 转基因抗虫耐除草剂玉米MON87411精准定量检测方法的建立[J]. 生物安全学报, 2023, 32(1): 38-45.
	Li LY, Zhang XD, Chen ZY, et al. Establishment of accurate quantitative detection method for insect-resistant and herbicide-tolerant maize MON87411[J]. J Biosaf, 2023, 32(1): 38-45.
[10]	Gatto F, Savini C, Baillie C, et al. Definition of minimum performance requirements for analytical methods of GMO testing[J]. Luxembourg: Publications Office, 2023.
[11]	阳丽, 操志林. 转基因玉米种子的PCR检测[J]. 江西农业学报, 2014, 26(4): 98-99, 106.
	Yang L, Cao ZL. PCR detection of transgenic maize seeds[J]. Acta Agric Jiangxi, 2014, 26(4): 98-99, 106.
[12]	尹全, 李忆, 宋君, 等. 多重PCR筛查检测进口转基因玉米[J]. 核农学报, 2016, 30(6): 1045-1053. doi: 10.11869/j.issn.100-8551.2016.06.1045
	Yin Q, Li Y, Song J, et al. Multiplex PCR detection of exogenous gene in the imported genetically modified maize[J]. J Nucl Agric Sci, 2016, 30(6): 1045-1053. doi: 10.11869/j.issn.100-8551.2016.06.1045
[13]	李葱葱, 王青山, 李飞武, 等. 用实时荧光PCR方法定量检测Bt176转基因玉米[J]. 吉林农业科学, 2007, 32(5): 24-27.
	Li CC, Wang QS, Li FW, et al. Quantitative detection of the genetically modified Bt176 maize using real-time fluorescent PCR method[J]. J Jilin Agric Sci, 2007, 32(5): 24-27.
[14]	金永梅, 马瑞, 于志晶, 等. 转基因水稻吉生粳2号的外源基因旁侧序列分离及事件特异性PCR检测方法[J]. 东北农业科学, 2016, 41(1): 14-19.
	Jin YM, Ma R, Yu ZJ, et al. Identification of the T-DNA flanking sequences and event-specific PCR detection of transgenic rice‘jishengjing 2’[J]. J Northeast Agric Sci, 2016, 41(1): 14-19.
[15]	郑子繁, 王颢潜, 高佳奇, 等. 耐除草剂转基因大豆SHZD32-1数字PCR绝对定量方法的建立[J]. 计量学报, 2023, 44(3): 415-421.
	Zheng ZF, Wang HQ, Gao JQ, et al. Development of digital PCR absolute quantification method for herbicide-tolerant genetically modified soybean SHZD32-1[J]. Acta Metrol Sin, 2023, 44(3): 415-421.
[16]	林萍萍, 史云鹏, 安鹏天, 等. 影响实时荧光PCR检测转基因作物的因素分析[J]. 植物检疫, 2022, 36(5): 50-53.
	Lin PP, Shi YP, An PT, et al. Analysis of influence factors on detection of transgenic crops by real-time PCR[J]. Plant Quar, 2022, 36(5): 50-53.

引物Primer	序列 Sequence（5'-3'）	引物Primer	序列Sequence（5'-3'）
RF 8-LB-F1	GGGTTTCGCTCATGTGTTGAG	RF 8-RB-F1	AGTTGGCGTCTCTCTGTTCG
RF 8-LB-F2	CGTCCGCAATGTGTTATTAAGTTGTCTA	RF 8-RB-F2	GTCGTTACTGAAACCGGTCC
RF 8-LB-F3	AGGAGCTCGAGTTTCTCCAT	RF 8-RB-F3	TCGTTACTGAAACCGGTCCAT
RF 8-LB-F4	AGCGTCAATTTGTTTACACCACA	RF 8-RB-F4	ATCCCAACCATTGCCGTCTT
RF 8-LB-F5	ACTAAAATCCAGATCCCCCGAA	RF 8-RB-F5	CCGTCTTGATGAGTTGGCGT
RF 8-LB-F6	GGTTTCGCTCATGTGTTGAG	RF 8-RB-F6	TGCCGTCTTGATGAGTTGGC
RF 8-LB-R1	GTGTCGACGGCTGAGATGAA	RF 8-RB-R1	AAAAACGTCCGCAATGTGTT
RF 8-LB-R2	AGTGCCAATACATACGCAACTGTTGCAG	RF 8-RB-R2	CTGAGGCCACCGCCTAATA
RF 8-LB-R3	ATACGATCGGATTCGTGGGA	RF 8-RB-R3	CTGAGGCCACCGCCTAAT
RF 8-LB-R4	GAACCAGAGCACTCTCACCA	RF 8-RB-R4	CGTCCGCAATGTGTTATTAAGT
RF 8-LB-R5	GGCCCATGAGTCCAAGAACC	RF 8-RB-R5	CATTAAAAACGTCCGCAATGTGT
RF 8-LB-R6	CAACTGGCGACACAGGGT	RF 8-RB-R6	TTAAAAACGTCCGCAATGTGTT

引物Primer	序列 Sequence（5'-3'）	引物Primer	序列Sequence（5'-3'）
RF 8-LB-F1	GGGTTTCGCTCATGTGTTGAG	RF 8-RB-F1	AGTTGGCGTCTCTCTGTTCG
RF 8-LB-F2	CGTCCGCAATGTGTTATTAAGTTGTCTA	RF 8-RB-F2	GTCGTTACTGAAACCGGTCC
RF 8-LB-F3	AGGAGCTCGAGTTTCTCCAT	RF 8-RB-F3	TCGTTACTGAAACCGGTCCAT
RF 8-LB-F4	AGCGTCAATTTGTTTACACCACA	RF 8-RB-F4	ATCCCAACCATTGCCGTCTT
RF 8-LB-F5	ACTAAAATCCAGATCCCCCGAA	RF 8-RB-F5	CCGTCTTGATGAGTTGGCGT
RF 8-LB-F6	GGTTTCGCTCATGTGTTGAG	RF 8-RB-F6	TGCCGTCTTGATGAGTTGGC
RF 8-LB-R1	GTGTCGACGGCTGAGATGAA	RF 8-RB-R1	AAAAACGTCCGCAATGTGTT
RF 8-LB-R2	AGTGCCAATACATACGCAACTGTTGCAG	RF 8-RB-R2	CTGAGGCCACCGCCTAATA
RF 8-LB-R3	ATACGATCGGATTCGTGGGA	RF 8-RB-R3	CTGAGGCCACCGCCTAAT
RF 8-LB-R4	GAACCAGAGCACTCTCACCA	RF 8-RB-R4	CGTCCGCAATGTGTTATTAAGT
RF 8-LB-R5	GGCCCATGAGTCCAAGAACC	RF 8-RB-R5	CATTAAAAACGTCCGCAATGTGT
RF 8-LB-R6	CAACTGGCGACACAGGGT	RF 8-RB-R6	TTAAAAACGTCCGCAATGTGTT

序号 No.	组合 Group	产物大小 Amplication size/bp	序号 No.	组合 Group	产物大小 Amplication size/bp	序号 No.	组合 Group	产物大小 Amplication size/bp
1	RF 8-RB-R1/F1	270	15	RF 8-RB-R5/F5	285	28	RF 8-LB-F2/R6	282
2	RF 8-RB-R1/F4	294	16	RF 8-RB-R5/F6	287	29	RF 8-LB-F3/R3	299
3	RF 8-RB-R1/F5	281	17	RF 8-RB-R6/F1	272	30	RF 8-LB-F4/R4	141
4	RF 8-RB-R1/F6	283	18	RF 8-RB-R6/F4	296	31	RF 8-LB-F5/R1	149
5	RF 8-RB-R2/F2	295	19	RF 8-RB-R6/F5	283	32	RF 8-LB-F5/R2	179
6	RF 8-RB-R2/F3	294	20	RF 8-RB-R6/F6	285	33	RF 8-LB-F5/R3	128
7	RF 8-RB-R3/F2	295	21	RF 8-LB-F1/R1	254	34	RF 8-LB-F5/R4	222
8	RF 8-RB-R3/F3	294	22	RF 8-LB-F1/R2	284	35	RF 8-LB-F5/R5	237
9	RF 8-RB-R4/F1	265	23	RF 8-LB-F1/R3	233	36	RF 8-LB-F5/R6	165
10	RF 8-RB-R4/F4	289	24	RF 8-LB-F1/R6	270	37	RF 8-LB-F6/R1	253
11	RF 8-RB-R4/F5	276	25	RF 8-LB-F2/R1	266	38	RF 8-LB-F6/R2	283
12	RF 8-RB-R4/F6	278	26	RF 8-LB-F2/R2	290	39	RF 8-LB-F6/R3	232
13	RF 8-RB-R5/F1	274	27	RF 8-LB-F2/R3	245	40	RF 8-LB-F6/R6	269
14	RF 8-RB-R5/F4	298

序号 No.	组合 Group	产物大小 Amplication size/bp	序号 No.	组合 Group	产物大小 Amplication size/bp	序号 No.	组合 Group	产物大小 Amplication size/bp
1	RF 8-RB-R1/F1	270	15	RF 8-RB-R5/F5	285	28	RF 8-LB-F2/R6	282
2	RF 8-RB-R1/F4	294	16	RF 8-RB-R5/F6	287	29	RF 8-LB-F3/R3	299
3	RF 8-RB-R1/F5	281	17	RF 8-RB-R6/F1	272	30	RF 8-LB-F4/R4	141
4	RF 8-RB-R1/F6	283	18	RF 8-RB-R6/F4	296	31	RF 8-LB-F5/R1	149
5	RF 8-RB-R2/F2	295	19	RF 8-RB-R6/F5	283	32	RF 8-LB-F5/R2	179
6	RF 8-RB-R2/F3	294	20	RF 8-RB-R6/F6	285	33	RF 8-LB-F5/R3	128
7	RF 8-RB-R3/F2	295	21	RF 8-LB-F1/R1	254	34	RF 8-LB-F5/R4	222
8	RF 8-RB-R3/F3	294	22	RF 8-LB-F1/R2	284	35	RF 8-LB-F5/R5	237
9	RF 8-RB-R4/F1	265	23	RF 8-LB-F1/R3	233	36	RF 8-LB-F5/R6	165
10	RF 8-RB-R4/F4	289	24	RF 8-LB-F1/R6	270	37	RF 8-LB-F6/R1	253
11	RF 8-RB-R4/F5	276	25	RF 8-LB-F2/R1	266	38	RF 8-LB-F6/R2	283
12	RF 8-RB-R4/F6	278	26	RF 8-LB-F2/R2	290	39	RF 8-LB-F6/R3	232
13	RF 8-RB-R5/F1	274	27	RF 8-LB-F2/R3	245	40	RF 8-LB-F6/R6	269
14	RF 8-RB-R5/F4	298

Lab	样品名称/编号 Sample name/No.	1	2	3	4	5	6
Lab1 - Lab8	阳性对照 Positive control	+	+	+	+	+	+
	阴性对照 Negative control	-	-	-	-	-	-
	1%浙大瑞丰8 1% Zheda Ruifeng 8	+	+	+	+	+	+
	其他转基因玉米混样 Transgenic corn mixture samples	-	-	-	-	-	-
	转基因大豆混样 Transgenic soybean mixture samples	-	-	-	-	-	-
	转基因水稻混样 Transgenic rice mixture samples	-	-	-	-	-	-
	转基因棉花混样 Transgenic cotton mixture samples	-	-	-	-	-	-
	转基因油菜混样 Transgenic rape mixture samples	-	-	-	-	-	-
	非转基因玉米混样 Non-transgenic maize mixture samples	-	-	-	-	-	-
	0.1%浙大瑞丰8 0.1% Zheda Ruifeng 8	+	+	+	+	+	+