Construction of an Ultra-sensitive Colorimetric Biosensor for Insect Resistance Genes Based on Loop-mediated Isothermal Amplification

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

Abstract

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

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.

Figures/Tables 7

References 29

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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