基于环介导等温扩增的抗虫基因超灵敏比色生物传感器构建

doi:10.13560/j.cnki.biotech.bull.1985.2021-1062

摘要/Abstract

摘要：

随着转基因技术的发展,快速、稳定和简单的转基因作物检测技术的开发一直是热点。开发了以溴百里酚蓝为显色剂的基于环介导等温扩增的抗虫基因超灵敏比色生物传感器,该方法可以通用检测Cry1Ac和Cry1Ab/Ac基因,具有较好的特异性。新型pH指示剂溴百里酚蓝对于LAMP影响小,通过一步法闭管可视检测,解决了LAMP容易污染的问题,实现了自然光下裸眼可视化半定量检测。最终优化的体系为Tris终浓度3.25 mmol/L,0.1%溴百里酚蓝添加量1.5 µL,在此条件下1 h内可检测到单拷贝靶标基因。该方法兼具灵敏性、特异性和稳定性,操作简单,适合现场检测,提供了Cry1Ac和Cry1Ab/Ac基因通用检测的具体操作方法和新的LAMP比色信号输出方式,不仅完善了转基因产品的检测体系,也丰富了LAMP生物传感器信号输出方式。

关键词: 环介导等温扩增, 转基因, 抗虫基因, 溴百里酚蓝, 裸眼比色

Abstract:

With the development of genetically modified technology,the development of rapid,stable and simple detection technology for genetically modified crop has always been a hot spot. In this paper,a super-sensitive colorimetric biosensor based on loop-mediated isothermal amplification（LAMP）was developed,using bromophenol thyme blue as the chromogenic reagent. This method can detect the Cry1Ac and Cry1Ab/Ac genes in general,and has good specificity. The new pH indicator bromophenol thyme blue has little effect on LAMP,can achieve one-step closed-tube visual detection,solve the problem of easy pollution of LAMP,and realize semi-quantitative detection with naked eyes under natural light. The final optimized system is the final concentration of Tris 3.25 mmol/L,and the addition amount of 0.1% bromophenol thyme blue is 1.5 μL. Under these conditions,a single copy of the target gene can be detected within 1 h. This method combines sensitivity,specificity and stability,is of simple operation,and suitable for on-site detection. It provides specific operation methods for Cry1Ac and Cry1Ab/Ac gene universal detection and a new LAMP colorimetric signal output method,which not only improves the detection system for genetically modified products,also enriches the signal output methods of the LAMP biosensor.

Key words: loop-mediated isothermal amplification（LAMP）, transgene, insect resistance gene, bromophenol thyme l blue（BTB）, naked eye colorimetry

李佳乐, 林晟豪, 许文涛. 基于环介导等温扩增的抗虫基因超灵敏比色生物传感器构建[J]. 生物技术通报, 2022, 38(8): 69-76.

LI Jia-le, LIN Sheng-hao, XU Wen-tao. Construction of an Ultra-sensitive Colorimetric Biosensor for Insect Resistance Genes Based on Loop-mediated Isothermal Amplification[J]. Biotechnology Bulletin, 2022, 38(8): 69-76.

图/表 7

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品种 Variety	测试样品 Testing sample	Cry1A基因信息 Cry1A gene information	研发单位 R & D unit
转基因水稻	Kefeng 6	Cry1Ac	中国科学院遗传与发育研究所、福建省农业科学院
	Kefeng 8	Cry1Ac	中国科学院遗传与发育生物学研究所、福建省农业科学院生物技术研究所、山西省农业科学院棉花研究所
	TT51	Cry1Ab/Ac	华中农业大学
转基因玉米	Bt11	Cry1Ab	先正达种子有限公司
	Bt176	Cry1Ab	先正达种子有限公司
	MON 810	Cry1Ab	美国孟山都公司
转基因棉花	MON 15985	Cry1Ac	美国孟山都公司
转基因甜菜	H7-1	-	美国孟山都公司
非转基因作物	五优稻4号	-	五常市龙凤山长粒香水稻研究所
非转基因作物	粳稻糙米	-	五常市龙凤山长粒香水稻研究所

品种 Variety	测试样品 Testing sample	Cry1A基因信息 Cry1A gene information	研发单位 R & D unit
转基因水稻	Kefeng 6	Cry1Ac	中国科学院遗传与发育研究所、福建省农业科学院
	Kefeng 8	Cry1Ac	中国科学院遗传与发育生物学研究所、福建省农业科学院生物技术研究所、山西省农业科学院棉花研究所
	TT51	Cry1Ab/Ac	华中农业大学
转基因玉米	Bt11	Cry1Ab	先正达种子有限公司
	Bt176	Cry1Ab	先正达种子有限公司
	MON 810	Cry1Ab	美国孟山都公司
转基因棉花	MON 15985	Cry1Ac	美国孟山都公司
转基因甜菜	H7-1	-	美国孟山都公司
非转基因作物	五优稻4号	-	五常市龙凤山长粒香水稻研究所
非转基因作物	粳稻糙米	-	五常市龙凤山长粒香水稻研究所

引物名称 Primer name		序列 Sequence（5'-3'）
Cry1Ac&Ab/Ac -1	F3	GGTGGAAGACAAGGTTCTGT
	B3	TCTACACCGATGCTCACAGA
	FIP	GGAACTATGGGAAACGCCGCT- AGACACCCTGACCTAGTTGA
	BIP	GAGGAAAGGTAAACTCGGGCCC- TGGTCTGGACACCAGATCA
	LF	CACAACAACGTATCGTTGC
	LB	GCTGAATCCAACTGGAGAGGC
Cry1Ac&Ab/Ac -2	F3	TGATGCTCACGGAACTGTTG
	B3	TCGCCTATGGAACCTCTTCT
	FIP	CCACCCAGGCAAGGATTCTCC- TGAATCCGGAACGGAACATG
	BIP	TGTGGTGGGATTTCGTCCAAGG- AACTTGCCATCCGCTGTT
	LF	ACAGGTTGAGCCACGTGTC
	LB	AATCAACGGTACCGCTCTTTC
Cry1Ac&Ab/Ac -3	F3	GGAGCTCTGATGATGCTCAC
	B3	TCGCCTATGGAACCTCTTCT
	FIP	CAGGCAAGGATTCTCCCACAGG- GGAACTGTTGCTGAATCCGG
	BIP	TGTGGTGGGATTTCGTCCAAGG- AACTTGCCATCCGCTGTT
	LF	CCACGTGTCCATGTTCCGTT
	LB	ACGGTACCGCTCTTTCTGT

引物名称 Primer name		序列 Sequence（5'-3'）
Cry1Ac&Ab/Ac -1	F3	GGTGGAAGACAAGGTTCTGT
	B3	TCTACACCGATGCTCACAGA
	FIP	GGAACTATGGGAAACGCCGCT- AGACACCCTGACCTAGTTGA
	BIP	GAGGAAAGGTAAACTCGGGCCC- TGGTCTGGACACCAGATCA
	LF	CACAACAACGTATCGTTGC
	LB	GCTGAATCCAACTGGAGAGGC
Cry1Ac&Ab/Ac -2	F3	TGATGCTCACGGAACTGTTG
	B3	TCGCCTATGGAACCTCTTCT
	FIP	CCACCCAGGCAAGGATTCTCC- TGAATCCGGAACGGAACATG
	BIP	TGTGGTGGGATTTCGTCCAAGG- AACTTGCCATCCGCTGTT
	LF	ACAGGTTGAGCCACGTGTC
	LB	AATCAACGGTACCGCTCTTTC
Cry1Ac&Ab/Ac -3	F3	GGAGCTCTGATGATGCTCAC
	B3	TCGCCTATGGAACCTCTTCT
	FIP	CAGGCAAGGATTCTCCCACAGG- GGAACTGTTGCTGAATCCGG
	BIP	TGTGGTGGGATTTCGTCCAAGG- AACTTGCCATCCGCTGTT
	LF	CCACGTGTCCATGTTCCGTT
	LB	ACGGTACCGCTCTTTCTGT