转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立

doi:10.16288/j.yczz.21-110

遗传 ›› 2021, Vol. 43 ›› Issue (8): 802-812.doi: 10.16288/j.yczz.21-110

• 技术与方法 • 上一篇

转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立

陈欲^1,²(), 陈笑芸¹, 彭城¹, 徐俊锋¹, 沈洁³, 李玥莹²(), 汪小福¹()

^1. 浙江省农业科学院,农产品质量安全危害因子与风险防控国家重点实验室,杭州 310021
^2. 沈阳师范大学生命科学学院,沈阳 110034
^3. 宁夏医科大学临床医学院,银川 750004

收稿日期:2021-03-24 修回日期:2021-07-06 出版日期:2021-08-20 发布日期:2021-07-29
通讯作者: 李玥莹,汪小福 E-mail:yuchen_115@126.com;yueyinglicn@163.com;yywxf1981@163.com
作者简介:陈欲,在读硕士研究生,专业方向：植物基因工程。E-mail: yuchen_115@126.com
基金资助:
国家自然科学基金项目资助编号(31772098)

Establishment of a field visual detection method for genetically modified maize ‘Shuangkang’12-5 by fluorescence RPA

Yu Chen^1,²(), Xiaoyun Chen¹, Cheng Peng¹, Junfeng Xu¹, Jie Shen³, Yueying Li²(), Xiaofu Wang¹()

^1. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
^2. College of Life Science, Shenyang Normal University, Shenyang 110034, China
^3. School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China

Received:2021-03-24 Revised:2021-07-06 Online:2021-08-20 Published:2021-07-29
Contact: Li Yueying,Wang Xiaofu E-mail:yuchen_115@126.com;yueyinglicn@163.com;yywxf1981@163.com
Supported by:
Supported by the National Natural Science Foundation of China No(31772098)

摘要/Abstract

摘要：

转基因玉米双抗12-5具有良好的抗虫性和除草剂耐受性,是我国第一批获得安全证书的转基因玉米之一,具有广阔的应用前景。本研究利用重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)建立转基因玉米双抗12-5的现场快速检测方法。针对转基因玉米双抗12-5的转化体特异性序列片段,设计引物和探针,通过引物筛选实验得到最佳引物与探针组合。荧光RPA扩增结果可以在蓝光下直接进行可视化分析。结果表明,建立的转基因玉米双抗12-5可视化检测体系特异性强,检测灵敏度达到10拷贝。进一步研究发现RPA的扩增体系对温度有很强的适应性,样品在34℃~46℃之间都能得到扩增,据此,本研究利用市面上常见的自发热暖贴代替常规的加热仪器来激发RPA。结果表明,自发热暖贴满足RPA扩增体系对温度的需要。最终,本研究将自发热暖贴加热法与RPA可视化检测体系结合,对转基因玉米双抗12-5进行现场检测,并与qPCR方法检测结果作比较,检测结果表明,本研究建立的现场可视化检测方法与qPCR方法检测结果一致,并且可视化检测方法时间短,检测结果清晰易分辨。本研究建立的转基因玉米双抗12-5现场快速可视化检测方法,其特异性强、灵敏度高、方便、快捷,不仅满足了转基因玉米双抗12-5的现场快速检测的需要,也为其他现场快速检测方法的开发提供了新思路。

关键词: 转基因玉米, RPA, 可视化检测, 现场检测

Abstract:

The genetically modified (GM) maize ‘Shuangkang’12-5 has good insect resistance and herbicide tolerance, which is one of the first series of GM maizes obtained a safety certificate in China, and it has broad application prospect in the future. This study established an on-site rapid detection method for GM ‘Shuangkang’12-5 based on recombinase polymerase amplification (RPA) technology, which primes and probe were designed according to the specific flank sequence. Then the best combination of primers and probe was obtained through a screeing process. The amplification results of fluorescence RPA can be directly visualized under blue light. The results showed that the visual detection system of GM ‘Shuangkang’12-5 with high specificity, and the detection sensitivity of the method could reached 10 copies. Further research found that the RPA amplification system had a wide range of temperature (34℃-46℃). According to this property, the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system. Finally, we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM ‘Shuangkang’12-5, and compared the results with the detection results of qPCR. The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR. Moreover, the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish. The rapid on-site visual detection method for GM ‘Shuangkang’ 12-5 established in this study has high specificity, high sensitivity and convenience. It not only meets the needs of on-site rapid detection of GM ‘Shuangkang’12-5, but also provides highlight for the development of other on-site rapid detection methods.

Key words: genetically modified maize, RPA, visual detection, field detection

陈欲, 陈笑芸, 彭城, 徐俊锋, 沈洁, 李玥莹, 汪小福. 转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立[J]. 遗传, 2021, 43(8): 802-812.

Yu Chen, Xiaoyun Chen, Cheng Peng, Junfeng Xu, Jie Shen, Yueying Li, Xiaofu Wang. Establishment of a field visual detection method for genetically modified maize ‘Shuangkang’12-5 by fluorescence RPA[J]. Hereditas(Beijing), 2021, 43(8): 802-812.

图/表 7

图1

表1

图2

图3

图4

图5

表2

参考文献 17

[1]	Wu WY, Liu XX, Zhou MY, Liu JX, Liu YY. Establishment of event-specific PCR detection method of transgenic maize line 2A-5. Curr Biotechnol, 2020, 10(04):363-370.
	武文艳, 刘新香, 周秒依, 刘金香, 刘亚. 转基因玉米2A-5特异性PCR方法的建立. 生物技术进展, 2020, 10(04):363-370.
[2]	Wang J, Wu FC, Liu XY, Feng SD, Song XY. Evaluation of transgenic maize ‘Shuangkang 12-5’ with complex traits of insect-resistance and glyphosate-resistance for the resistance toOstrinia furnacalis and tolerance to glyphosate. Plant Protec, 2016, 42(1):45-50.
	王江, 武奉慈, 刘新颖, 冯树丹, 宋新元. 转基因抗虫耐除草剂复合性状玉米‘双抗12-5’对亚洲玉米螟的抗性及对草甘膦的耐受性研究. 植物保护, 2016, 42(1):45-50.
[3]	Wang R. Rapid nucleic acid amplification and visual detection strategiesfor the detection of food-borne pathogen and genetically modified organisms[Dissertation]. Zhejiang University, 2019.
	王瑞. 快速核酸扩增及可视化策略用于食源性致病菌和转基因作物检测[学位论文]. 浙江大学, 2019.
[4]	Jiang ZB. The significance and way of spreading corn planting technology. Mod Agric, 2020, (23):5.
	江真兵. 玉米种植技术推广的意义及途径. 新农业, 2020, (23):5.
[5]	Jiao Y, Han Y, Yang Q, Huang YH, An JC, Yang YZ, Ye JM. Commercialization development trend of genetically modified maize and the enlightenment. Biotechnol Bull, 2021, 37(4):164-176.
	焦悦, 韩宇, 杨桥, 黄耀辉, 安吉翠, 杨亚洲, 叶纪明. 全球转基因玉米商业化发展态势概述及启示. 生物技术通报, 2021, 37(4):164-176.
[6]	Wang HQ, Xiao F, Yang L, Miu QM, Zhang XD, Zhang XJ. Analysis of verification results by PCR methods for genetically modified double-resistant 12-5 event-specific maize. Biotechnol Bull, 2020, 36(5):48-55.
	王颢潜, 肖芳, 杨蕾, 缪青梅, 张旭冬, 张秀杰. 转基因玉米双抗12-5转化体特异性PCR方法验证结果分析. 生物技术通报, 2020, 36(5):48-55.
[7]	Yu Y, Liu GQ, Su SL, Luo JX, Guo L. Detection of transgenic components in corn. Jiangsu J Agric Sci, 2020, 36(4):836-841.
	于园, 刘国强, 苏圣淋, 罗建兴, 郭梁. 玉米转基因成分的检测. 江苏农业学报, 2020, 36(4):836-841.
[8]	Meng LC, Zhou DL, Gao TT, Wang M, Lu Ming. Rapid screening of genetically modified maize based on TaqMan Real-time PCR. Mol Plant Breed, 2020, 1-7.
	孟令聪, 周德龙, 高婷婷, 王敏, 路明. 基于TaqMan实时荧光PCR技术快速筛查转基因玉米. 分子植物育种, 2020, 1-7.
[9]	Lin FF, Hou HL, Liu J, Wang XL, Deng HC, Zhou XY. Research progress in DNA component detection of transgenic plants. Chin Seed Ind, 2016, (11):19-22.
	林馥芬, 侯红利, 刘晋, 王学林, 邓汉超, 周向阳. 转基因植物DNA成分检测技术研究进展. 中国种业, 2016, (11):19-22.
[10]	Jia X. The application of recombinase polymerase amplification (RPA) technology in detection of transgenic rices[Dissertation]. Chinese Academy of Agricultural Sciences, 2017.
	贾玄. RPA等温扩增技术在转基因水稻检测中的应用 [学位论文]. 中国农业科学院, 2017.
[11]	OPiepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol, 2006, 4(7):e204. doi: 10.1371/journal.pbio.0040204
[12]	Ding X, Liu YH, Ni BX, Wang XT, Xu XZ, Ying QJ, Dai Y, Cao J. Establishment of a nucleic acid assay for detection of Echinococcus granulosus based on recombinase- aided isothermal amplification assay. Chin J Schi Contl, 2020, 32(4):340-344. doi: 10.16250/j.32.1374.2020071 pmid: 32935505
	丁昕, 刘燕红, 倪碧娴, 王晓婷, 徐祥珍, 应清界, 戴洋, 曹俊. 基于重组酶介导等温扩增技术的细粒棘球绦虫核酸检测方法的建立. 中国血吸虫病防治杂志, 2020, 32(4):340-344. doi: 10.16250/j.32.1374.2020071 pmid: 32935505
[13]	Lobato IM, O’Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. Trac Trend Anal Chem, 2018, 98:19-35. doi: 10.1016/j.trac.2017.10.015
[14]	Announcement No. 2259 of ministry of agriculture-12- 2015, Detection of genetically modified plants and derived products. Qualitative PCR method for insect-resistant and herbicide-tolerant maize Shuangkang 12-5 and its derivates. Beijing: China Agriculture Press, 2015.
	农业部2259号公告-12-2015, 转基因植物及其产品成分检测. 抗虫耐除草剂玉米12-5及其衍生品种定性PCR方法. 北京: 中国农业出版社, 2015.
[15]	Tan LQ, Zheng XC, Wang CP, Li WS, Qin ZF. Development of real time RT-RPA for rapid detection of rabies virus. Chin J Vet Med, 2019, 55(5): 44-47+122.
	谭理琦, 郑晓聪, 王翠萍, 李汶松, 秦智锋. 狂犬病病毒实时荧光RT-RPA等温检测方法的建立. 中国兽医杂志, 2019, 55(5): 44-47+122.
[16]	Zhang QL, Fu CX, Zhang W, Gao XL, Wang L, Cheng MH, Zheng XY, Liu HY, Zhou DG. Establishment and application of real-time fluorescence RT-RPA detection method for canine distemper virus. Prog Vet Med, 2020, 41(11):19-23.
	张启龙, 傅彩霞, 张玮, 高晓龙, 王林, 程敏姮, 郑雪莹, 刘海莹, 周德刚. 犬瘟热病毒实时荧光RT-RPA检测方法的建立及应用. 动物医学进展, 2020, 41(11):19-23.
[17]	Wang SH, Yuan SH, Shuai JB, Wang ZC, Cai J, Zhao ZG. Establishment and application of recombinase polymerase amplification assay for detection ofBacillus anthracis. Chin J Prev Vet Med, 2020, 42(6):590-594.
	王素华, 袁淑辉, 帅江冰, 王忠才, 蔡军, 赵治国. 炭疽杆菌RPA等温检测方法的建立和应用. 中国预防兽医学报, 2020, 42(6):590-594.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

目的	名称	序列(5ʹ→3ʹ)	扩增产物长度(bp)	啊啊啊啊
荧光RPA	12-5-F1	TCCGACGCCTCGACGCTCGTGGTGACTCGC		本研究
	12-5-F2	CGACCAAGTCATGAAGAACTCCCACTGCCG
	12-5-F3	CGACCCGAAGATGGAGGCCTACTGCGATGA
	12-5-F4	GACGTTCGGTGCCTGGAAGACAAGTTCTAC
	12-5-F5	CGTCATCGACCAAGTCATGAAGAACTCCCA
	12-5-F6	CAGCTCGTCATCGACCAAGTCATGA
	12-5-F7	CCGACGCCTCGACGCTCGTGGTGACTCGCAGC
	12-5-F8	CATCGACCAAGTCATGAAGAACTCCCACTGCCG
	12-5-R1	ATGCTAGAGCAGCTTGAGCTTGGATCAGAT
	12-5-R2	CCGATCGCCCTTCCCAACAGTTGCGCAGCC
	12-5-R3	CCAGCTGGCGTAATAGCGAAGAGGCCCGCA
	12-5-R4	TAATAGCGAAGAGGCCCGCACCGATCGCCC
	12-5-R5	TGAATGGCGAATGCTAGAGCAGCTTGAGCT
	12-5-R6	CTGAATGGCGAATGCTAGAGCAGCT
	12-5-R7	CTTGAGCTTGGATCAGATTGTCGT TTCCCGCC
	12-5-R8	CCCAACAGTTGCGCAGCCTGAATGGCGAATGC
	12-5-P	AACCACATCGCCCGACGCTACAACGAGAC[FAM-dT][THF]A [BHQ-dT]AGTTTAAACTGAA[3'-block]
普通PCR	12-5-PCR-F	CAACGTCGTGACTGGGAAAA	256	[14]
普通PCR	12-5-PCR-R	TGGAAGACAAGTTCTACGGGCT	256
qPCR	12-5qPCR-F	GTCGTTTCCCGCCTTCAGTT	94
	12-5qPCR-R	GGTGCCTGGAAGACAAGTTCTA
	12-5qPCR-P	[FAM]AGCTCAACCACATCGCCCGACGC[BHQ1]

转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立

Establishment of a field visual detection method for genetically modified maize ‘Shuangkang’12-5 by fluorescence RPA

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 17

相关文章 3

编辑推荐

Metrics

样品	检测结果		检测时间(min)
样品	荧光RPA	qPCR	荧光RPA	qPCR
1	+	+	20	60.9 (Ct=27.95)
2	+	+	20	60.1 (Ct=27.55)
3	+	+	20	64.52 (Ct=29.76)
4	+	+	20	60.48 (Ct=27.74)
5	+	+	20	60.94 (Ct=27.97)
6	+	+	20	63.18 (Ct=29.09)
7	+	+	20	62.84 (Ct=28.92)
8	+	+	20	63.64 (Ct=29.32)
9	+	+	20	60.62 (Ct=27.81)
10	+	+	20	60.58 (Ct=27.79)
11	+	+	20	62.48 (Ct=28.74)
12	+	+	20	62.66 (Ct=28.83)
13	+	+	20	62.92 (Ct=28.96)
14	+	+	20	59.8 (Ct=27.40)
15	+	+	20	59.86 (Ct=27.43)
16	+	+	20	62.9 (Ct=28.95)
17	+	+	20	63.92 (Ct=29.46)
18	+	+	20	62.36 (Ct=28.68)
19	+	+	20	58.94 (Ct=26.97)
20	+	+	20	58.64 (Ct=26.82)
21	+	+	20	60.98 (Ct=27.99)
22	+	+	20	61.02 (Ct=28.01)
23	+	+	20	61.6 (Ct=28.30)
24	+	+	20	61.84 (Ct=28.42)
其他	-	-	/	/

[1]	文雅蕾, 吕柯孬, 徐小康, 张欣, 丁良, 潘学峰. 退火解旋酶SMARCAL1在维持基因组稳定中的作用与机制[J]. 遗传, 2019, 41(12): 1084-1098.
[2]	杨爱馥，苏乔，安利佳. 利用子房滴注法获得无载体骨架序列和选择标记的转基因玉米[J]. 遗传, 2009, 31(1): 95-100.
[3]	曹际娟，覃文，朱水芳，曹远银. 用实时荧光PCR方法鉴定转基因玉米T14/T25[J]. 遗传, 2004, 26(5): 689-694.