基于免扩增高通量测序的转基因定量检测方法研究

doi:10.19586/j.2095-2341.2024.0020

生物技术进展 ›› 2024, Vol. 14 ›› Issue (4): 610-617.DOI: 10.19586/j.2095-2341.2024.0020

• 研究论文 • 上一篇

基于免扩增高通量测序的转基因定量检测方法研究

李凯¹(), 付军², 陈锐³, 陈笑芸⁴(), 李亮¹()

^1.中国农业科学院农业质量标准与检测技术研究所，北京 100081
^2.迈克生物股份有限公司，成都 610041
^3.天津市农业科学院种质资源与生物技术研究所，蔬菜生物育种全国重点实验室，天津 300381
^4.浙江省农业科学院农产品质量安全与营养研究所，农业农村部农业转基因生物溯源重点实验室，杭州 310021

收稿日期:2024-02-05 接受日期:2024-04-18 出版日期:2024-07-25 发布日期:2024-08-07
通讯作者: 陈笑芸,李亮
作者简介:李凯 E-mail: likaij@163.com;
基金资助:
农业农村部农业转基因生物溯源重点实验室开放课题(2022KF02);科技创新2030农业生物育种重大专项(2022ZD0402013);中国农业科学院科技创新工程(CAAS-ASTIP-IQSTAP-05);中国农业科学院研究所级工作任务(1610072023101)

Transgenic Quantitative Detection Method Based on PCR-free High-throughput Sequencing Technology

Kai LI¹(), Jun FU², Rui CHEN³, Xiaoyun CHEN⁴(), Liang LI¹()

^1.Institute of Quality Standard and Testing Technology for Agro-products，Chinese Academy of Agricultural Sciences，Beijing 100081，China
^2.Maccura Biotech Co. ，Ltd，Chengdu 610041，China
^3.State Key Laboratory of Vegetable Biobreeding，Institute of Germplasm Resources and Biotechnology，Tianjin Academy of Agricultural Sciences，Tianjin 300192，China
^4.Key Laboratory of Traceability for Agricultural Genetically Modified Organisms，Ministry of Agriculture and Rural Affairs，Zhejiang Academy of Agricultural Sciences，Hangzhou 310021，China

Received:2024-02-05 Accepted:2024-04-18 Online:2024-07-25 Published:2024-08-07
Contact: Xiaoyun CHEN,Liang LI

摘要/Abstract

摘要：

转基因定量检测在食品安全监管和消费者知情权保障中扮演着重要角色。当前，数字PCR方法被广泛认可为核酸精准定量的黄金标准，但缺乏与之相互验证的新型核酸定量技术，这在一定程度上限制了其应用。然而，近年来高通量测序技术的兴起为解决这一难题提供了新的可能性。尽管高通量测序技术主要用于核酸定性序列测定，但其在核酸定量领域的应用潜力尚未充分挖掘。以含有转基因T-NOS、P-35S、CP4-EPSPS和大豆内标准基因Lectin的质粒DNA标准物质为检测对象，采用免扩增建库的方法，比较了NGS、三代测序以及数字PCR定值的差异。结果表明，NGS测序结果与数字PCR结果存在显著性差异，这一发现突显了目前核酸定量技术中的挑战和需求。然而，值得注意的是，三代测序结果与数字PCR检测结果一致性良好，显示了其作为转基因核酸精准定量方法的潜力。这一发现为未来转基因产品标识阈值的制定提供了技术上的支撑和参考，为食品安全监管提供了新的技术途径。

关键词: 质粒DNA, 标准物质, 测序技术, dPCR

Abstract:

Quantitative GMO detection is essential for ensuring food safety and protecting consumer rights to information. Although digital PCR is currently considered the gold standard for accurate nucleic acid quantification， the lack of validated new quantification technologies limits its applications. However， the emergence of high-throughput sequencing has opened up new possibilities for solving this challenge. While high-throughput sequencing is primarily used for qualitative nucleic acid sequence determination， its potential for quantitative analysis has yet to be fully explored. In this study， plasmid DNA standard materials containing transgenic T-NOS， P-35S， CP4-EPSPS， and soybean housekeeping gene Lectin were used as the detection objects. The method of library construction without amplification was adopted to compare the differences between NGS， third-generation sequencing， and digital PCR quantification. The results showed significant differences between NGS sequencing results and digital PCR results， highlighting the challenges and demands in current nucleic acid quantitative analysis technologies. However， it is worth noting that the results of third-generation sequencing were consistent with the digital PCR detection results， demonstrating its potential as a precise method for quantifying transgenic nucleic acids.

Key words: plasmid DNA, reference materials, sequencing technology, dPCR

中图分类号:

Q789

李凯, 付军, 陈锐, 陈笑芸, 李亮. 基于免扩增高通量测序的转基因定量检测方法研究[J]. 生物技术进展, 2024, 14(4): 610-617.

Kai LI, Jun FU, Rui CHEN, Xiaoyun CHEN, Liang LI. Transgenic Quantitative Detection Method Based on PCR-free High-throughput Sequencing Technology[J]. Current Biotechnology, 2024, 14(4): 610-617.

图/表 9

图1 长测序结果Matching ratio计算方法示意图

Fig. 1 Piagram of calcolation method of Matching ratio from long Reads

表1 3种基因的数字PCR定量检测结果

Table 1 The ddPCR results of three genes

靶标	单酶切				四酶切
靶标	定量结果/(copies·μL^-1)	定量均值/(copies·μL^-1)	SD	CV	定量结果/(copies·μL^-1)	定量均值/(copies·μL^-1)	SD	CV
T-NOS	4 974.65	4 910.90	55.74	1.14%	6 335.76	6 421.72	121.46	1.89%
	4 886.74				6 560.67
	4 871.33				6 368.73
P-35S	4 911.70	4 984.12	64.87	1.30%	6 242.55	6 295.91	87.13	1.38%
	5 036.90				6 396.45
	5 003.76				6 248.73
CP4-EPSPS	5 047.51	5 022.78	21.60	0.43%	6 170.94	6 213.40	116.68	1.88%
	5 013.17				6 345.36
	5 007.65				6 123.90
Lectin	4 842.36	4 806.73	39.24	0.82%	6 213.69	6 276.26	91.30	1.45%
	4 974.65				6 335.76
	4 886.74				6 560.67

图2 数字PCR检测转基因含量结果

Fig. 2 The results of digital PCR detection of transgenic content

表2 3种基因的Illumina测序结果

Table 2 The Illumina results of three genes

编号	靶标	Reads总数	匹配数	所占比例/%
Read-1	T-NOS	4 080 241	102 364	2.51
	P-35S		141 975	3.48
	CP4-EPSPS		125 462	3.07
	Lectin		107 721	2.64
Read-2	T-NOS		103 348	2.63
	P-35S		142 937	3.50
	CP4-EPSPS		108 622	3.08
	Lectin		125 866	2.66

图3 Illumina测序技术检测转基因含量结果注：粉色柱状图和蓝色柱状图分别代表的两次重复结果。

Fig.3 The results of Illumina sequencing detection of transgenic content

图4 Nanopore测序技术检测转基因含量结果

Fig. 4 The results of Nanopore sequencing detection of transgenic content

表3 Nanopore测序在不同容错情况下的Reads数量

Table 3 Reads quantity of Nanopore sequencing under different fault-tolerant

靶标	容错率
靶标	10%	20%	30%
T-NOS	1 183 010	1 238 543	1 245 965
P-35S	1 169 963	1 236 710	1 243 722
CP4-EPSPS	1 156 345	1 203 814	1 209 292
Lectin	1 158 070	1 211 675	1 216 951

图5 Nanopore技术下检测3种转基因成分含量的结果注：粉色、蓝色和红色柱状图分别代表的容错率为10%、20%和30%。

Fig. 5 The results of Nanopore sequencing detection of transgenic content

图6 数字PCR技术和测序技术检测转基因含量比较结果A：数字PCR结果与NGS二代测序技术结果比较。B：数字PCR结果与Nanopore三代测序技术结果比较。3种检测方法的靶标均为T-NOS、P-35S和CP4-EPSPS。

Fig. 6 Comparison results of transgenic content detected by digital PCR and sequencing technology

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基于免扩增高通量测序的转基因定量检测方法研究

Transgenic Quantitative Detection Method Based on PCR-free High-throughput Sequencing Technology

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