基于核酸质谱的转基因水稻多靶标检测方法

doi:10.19586/j.2095-2341.2026.0059

生物技术进展 ›› 2026, Vol. 16 ›› Issue (4): 805-821.DOI: 10.19586/j.2095-2341.2026.0059

基于核酸质谱的转基因水稻多靶标检测方法

周泽汇¹^,²^,³(), 张辰²^,³, 沈荣妍²^,³, 吴轲²^,³, 刘娜²^,³, 李瑞环²^,³, 王雅偲²^,³, 王成²^,³, 徐超²^,³, 赵新²^,³, 兰青阔²^,³, 王永²^,³, 齐欣²^,³(), 裴忠有¹()

^1.天津农学院农学与资源环境学院，天津 300392
^2.天津市农业科学院种质资源与生物技术研究所，天津 300384
^3.农业农村部农产品及加工品质量检验测试中心（天津），天津 300384

收稿日期:2026-03-09 接受日期:2026-05-19 出版日期:2026-07-25 发布日期:2026-09-11
通信作者: 齐欣,裴忠有
作者简介:周泽汇 E-mail: zhouzehui1204@163.com
基金资助:
天津市农业科学院种业创新专项(2025ZYCX007)

A Nucleic Acid Mass Spectrometry-based Multi-target Detection Method for Genetically Modified Rice

Zehui ZHOU¹^,²^,³(), Chen ZHANG²^,³, Rongyan SHEN²^,³, Ke WU²^,³, Na LIU²^,³, Ruihuan LI²^,³, Yasi WANG²^,³, Cheng WANG²^,³, Chao XU²^,³, Xin ZHAO²^,³, Qingkuo LAN²^,³, Yong WANG²^,³, Xin QI²^,³(), Zhongyou PEI¹()

^1.College of Agriculture and Resource Environment，Tianjin Agricultural University，Tianjin 300392，China
^2.Institute of Germplasm Resources and Biotechnology，Tianjin Academy of Agricultural Sciences，Tianjin 300384，China
^3.Quality Supervision，Inspection and Testing Center for Agricultural Products and Processed Products（Tianjin），Ministry of Agriculture and Rural Affairs，Tianjin 300384，China

Received:2026-03-09 Accepted:2026-05-19 Online:2026-07-25 Published:2026-09-11
Contact: Xin QI,Zhongyou PEI

摘要/Abstract

摘要：

目前转基因水稻的检测技术主要以常规PCR为主，缺乏高通量的快速筛查方法。利用基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ionization time-of-flight mass spectrometry，MALDI-TOF-MS）建立多重检测引物组，涵盖TT51水稻转化体特异性序列、Cry1A基因、bar基因、CaMV 35S启动子和NOS终止子5个靶标元件在内的转基因水稻多靶标检测方法，靶基因检测限最低为0.5%。研究建立的检测方法具有高通量、多靶标、特异性强、灵敏度高的特点，可为转基因水稻的安全监管、身份鉴定和溯源管理提供有力的技术支撑。

关键词: 转基因检测, 核酸质谱, 水稻转化体特异性序列, 外源基因

Abstract:

Current techniques for detecting genetically modified （GM）rice primarily rely on conventional PCR， with no high-throughput， rapid screening methods available. This study used matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS） to create a multiplex primer panel for detecting multiple targets in GM rice. This method covers five target elements： the TT51 rice transformant-specific sequence， the Cry1A gene， the bar gene， theCaMV 35S promoter and the NOS terminator. The minimum detection limit for target genes was 0.5%. The established detection method offers high-throughput， multi-target capability， strong specificity and high sensitivity， offerring robust technical support for safety regulation， identity authentication and traceability management of genetically modified rice.

Key words: transgenic detection, nucleic acid mass spectrometry, rice transformant-specific sequences, exogenous genes

中图分类号:

Q3-3

周泽汇, 张辰, 沈荣妍, 吴轲, 刘娜, 李瑞环, 王雅偲, 王成, 徐超, 赵新, 兰青阔, 王永, 齐欣, 裴忠有. 基于核酸质谱的转基因水稻多靶标检测方法[J]. 生物技术进展, 2026, 16(4): 805-821.

Zehui ZHOU, Chen ZHANG, Rongyan SHEN, Ke WU, Na LIU, Ruihuan LI, Yasi WANG, Cheng WANG, Chao XU, Xin ZHAO, Qingkuo LAN, Yong WANG, Xin QI, Zhongyou PEI. A Nucleic Acid Mass Spectrometry-based Multi-target Detection Method for Genetically Modified Rice[J]. Current Biotechnology, 2026, 16(4): 805-821.

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链接本文: https://swjsjz.magtechjournal.com/CN/10.19586/j.2095-2341.2026.0059

https://swjsjz.magtechjournal.com/CN/Y2026/V16/I4/805

图/表 13

表1 本研究所用引物信息

Table 1 Primers information for this study

基因名称	引物序列（5'→3'）	延伸引物（5'→3'）	延伸引物MASS值	延伸产物MASS值
TT51-1	5'-ACGTTGGATGCCATCCCCCTGTCGCCGTG-3' 3'-ACGTTGGATGGCTCAGGTGCAGGTTGGCGG-5'	5'-ACTACTGCTTATCTGCC-3'	5 096.3	5 343.5
Cry1A_1	5'-ACGTTGGATGCTATCCCATTGTTCGCAGTC-3' 3'-ACGTTGGATGGCTGAGGTGAAGATTAGCTG-5'	5'-CAAGTTCCTCTCTTGT-3'	4 798.2	5 045.4
Cry1A_2	5'-ACGTTGGATGCCATCCCACTCTTCGCCGTC-3' 3'-ACGTTGGATGGCTCAGGTGCAGGTTGGCGG-5'	5'-CCAAGTCCCGCTCCTGT-3'	5 082.4	5 329.6
Cry1A_3	5'-ACGTTGGATGCCATCCCCCTGTCGCCGTG-3' 3'-ACGTTGGATGGCTCAGGTGCAGGTTGGCGG-5'	5'-ACCAGGTGCCCCTGCTGA-3'	5 460.6	5 747.8
CRY1A_4	5'-ACGTTGGATGCCATCCCACTGTTCGCCGTG-3' 3'-ACGTTGGATGGGAAAGGTGAAGGTTGGCGG-5	5'-AGGTGCCACTGCTGT-3'	4 584	4 831.2
bar	5'-ACGTTGGATGTAGGCGTTGCGTGCCTTCCA-3' 3'-ACGTTGGATGTCCGTCTGCGGGAGCGCTAT-5'	5'-CAAGAGCGCTATCCCTGG-3'	5 484.6	5 731.8
CaMV35S	5'-ACGTTGGATGCATTGCGATAAAGGAAAGGC-3' 3'-ACGTTGGATGCTTTTTCCACGATGCTCCTC-5'	5'-CCCTATCTTTGGGACCACTGC-3'	6 043.9	6 371
NOS_Terminator	5'-ACGTTGGATGCCCATCTCATAAATAACGTC-3' 3'-ACGTTGGATGCCGGTCTTGCGATGATTATC-5'	5'-TACGTTAAGCATGTAATAATTAACA-3'	7 672	7 999.1

图1 多重引物以日本晴DNA为模板的延伸峰图A：TT51引物以日本晴为模板的延伸峰图；B：Cry1A_1引物以日本晴为模板的延伸峰图；C：Cry1A_2引物以日本晴为模板的延伸峰图；D：Cry1A_3引物以日本晴为模板的延伸峰图；E：Cry1A_4引物以日本晴为模板的延伸峰图；F: bar引物以日本晴为模板的延伸峰图；G：CaMV 35S引物以日本晴为模板的延伸峰图；H：NOS_Terminator引物以日本晴为模板的延伸峰图；第一条红线为引物延伸峰，第二、第三条红线分别为对应检测引物结果分型出峰位置

Fig. 1 The extension peak maps of multiple primers using rice Nipponbare DNA as the template

图2 多重引物以TT51 DNA为模板的延伸峰图A：TT51引物以TT51为模板的延伸峰图；B：Cry1A_1引物以TT51为模板的延伸峰图；C：Cry1A_2引物以TT51为模板的延伸峰图；D：Cry1A_3引物以TT51为模板的延伸峰图；E：Cry1A_4引物以TT51为模板的延伸峰图；F：bar引物以TT51为模板的延伸峰图；G：CaMV 35S引物以TT51为模板的延伸峰图；H：NOS_Terminator引物以TT51为模板的延伸峰图；第一条红线为引物延伸峰，蓝线为阳性结果延伸峰，第二条红线为阳性结果延伸峰，灰线为其他引物峰

Fig. 2 The extension peak maps of multiple primers using TT51 DNA as the template

图3 多重引物以5种转基因玉米混样DNA为模板的延伸峰图A：TT51引物以5种转基因玉米混样为模板的延伸峰图；B：Cry1A_1引物以5种转基因玉米混样为模板的延伸峰图；C：Cry1A_2引物以5种转基因玉米混样为模板的延伸峰图；D：Cry1A_3引物以5种转基因玉米混样为模板的延伸峰图；E：Cry1A_4引物以5种转基因玉米混样为模板的延伸峰图；F：bar引物以5种转基因玉米混样为模板的延伸峰图；G：CaMV 35S引物以5种转基因玉米混样为模板的延伸峰图；H：NOS_Terminator引物以5种转基因玉米混样为模板的延伸峰图

Fig. 3 The extension peak maps of multiple primers using five types of genetically modified corn mixed samples DNA as the template

图4 多重引物以5种转基因大豆混样为模板的延伸峰图A：TT51引物以5种转基因大豆混样为模板的延伸峰图；B：Cry1A_1引物以5种转基因大豆混样为模板的延伸峰图；C：Cry1A_2引物以5种转基因大豆混样为模板的延伸峰图；D：Cry1A_3引物以5种转基因大豆混样为模板的延伸峰图；E：Cry1A_4引物以5种转基因大豆混样为模板的延伸峰图；F：bar引物以5种转基因大豆混样为模板的延伸峰图；G：CaMV 35S引物以5种转基因大豆混样为模板的延伸峰图；H：NOS_Terminator引物以5种转基因大豆混样为模板的延伸峰图

Fig. 4 The extension peak maps of multiple primers using five types of genetically modified soybean mixed samples DNA as the template

图5 多重引物以3种非转基因玉米混样DNA为模板的延伸峰图A：TT51引物以3种非转基因玉米混样为模板的延伸峰图；B：Cry1A_1引物以3种非转基因玉米混样为模板的延伸峰图；C：Cry1A_2引物以3种非转基因玉米混样为模板的延伸峰图；D：Cry1A_3引物以3种非转基因玉米混样为模板的延伸峰图；E：Cry1A_4引物以3种非转基因玉米混样为模板的延伸峰图；F：bar引物以3种非转基因玉米混样为模板的延伸峰图；G：CaMV 35S引物以3种非转基因玉米混样为模板的延伸峰图；H：NOS_Terminator引物以3种非转基因玉米混样为模板的延伸峰图

Fig. 5 The extension peak maps of multiple primers using three types of non-GMO corn mixed samples DNA as the template

图6 多重引物以3种非转基因大豆混样为模板的延伸峰图A：TT51引物以3种非转基因大豆混样为模板的延伸峰图；B：Cry1A_1引物以3种非转基因大豆混样为模板的延伸峰图；C：Cry1A_2引物以3种非转基因大豆混样为模板的延伸峰图；D：Cry1A_3引物以3种非转基因大豆混样为模板的延伸峰图；E：Cry1A_4引物以3种非转基因大豆混样为模板的延伸峰图；F：bar引物以3种非转基因大豆混样为模板的延伸峰图；G: CaMV 35S引物以3种非转基因大豆混样为模板的延伸峰图；H：NOS_Terminator引物以TT51为模板的延伸峰图

Fig. 6 The extension peak maps of multiple primers using three types of non-GMO soybean mixed samples DNA as the template

图7 多重引物以0.1%含量的TT51 DNA为模板的延伸峰图A：TT51引物以0.1%含量的TT51为模板的延伸峰图；B：Cry1A_1引物以0.1%含量的TT51为模板的延伸峰图；C：Cry1A_2引物以0.1%含量的TT51为模板的延伸峰图；D：Cry1A_3引物以0.1%含量的TT51为模板的延伸峰图；E：Cry1A_4引物以0.1%含量的TT51为模板的延伸峰图；F：bar引物以0.1%含量的TT51为模板的延伸峰图；G：CaMV 35S引物以0.1%含量的TT51为模板的延伸峰图；H：NOS_Terminator引物以0.1%含量的TT51为模板的延伸峰图

Fig. 7 Extension peak profiles of multiple primers with 0.1% TT51 DNA as template

图8 多重引物以0.5%含量的TT51 DNA为模板的延伸峰图A：TT51引物以0.5%含量的TT51为模板的延伸峰图；B：Cry1A_1引物以0.5%含量的TT51为模板的延伸峰图；C：Cry1A_2引物以0.5%含量的TT51为模板的延伸峰图；D：Cry1A_3引物以0.5%含量的TT51为模板的延伸峰图；E：Cry1A_4引物以0.5%含量的TT51为模板的延伸峰图；F：bar引物以0.5%含量的TT51为模板的延伸峰图；G：CaMV 35S引物以0.5%含量的TT51为模板的延伸峰图；H：NOS_Terminator引物以0.5%含量的TT51为模板的延伸峰图

Fig. 8 Extension peak profiles of multiple primers with 0.5% TT51 DNA as template

图9 多重引物在以1% TT51 DNA为模板下的延伸峰散点图A：TT51引物以1% TT51为模板的延伸峰散点图；B：Cry1A_1引物以1% TT51为模板的延伸峰散点图；C：Cry1A_2引物以1% TT51为模板的延伸峰散点图；D：Cry1A_3引物以1% TT51为模板的延伸峰散点图；E：Cry1A_4引物以1% TT51为模板的延伸峰散点图；F：bar引物以1% TT51为模板的延伸峰散点图；G：CaMV 35S引物以1% TT51为模板的延伸峰散点图；H：NOS_Terminator引物以1% TT51为模板的延伸峰散点图

Fig. 9 Scatter plot of extension peak profiles of multiple primers with 1% TT51 DNA as template

图10 多重引物以0.5% TT51 DNA为模板的延伸峰散点图A：TT51引物以0.5% TT51为模板的延伸峰散点图；B：Cry1A_1引物以0.5% TT51为模板的延伸峰散点图；C：Cry1A_2引物以0.5% TT51为模板的延伸峰散点图；D：Cry1A_3引物以0.5% TT51为模板的延伸峰散点图；E：Cry1A_4引物以0.5% TT51为模板的延伸峰散点图；F：bar引物以0.5% TT51为模板的延伸峰散点图；G：CaMV 35S引物以0.5% TT51为模板的延伸峰散点图；H：NOS_Terminator引物以0.5% TT51为模板的延伸峰散点图

Fig. 10 Scatter plot of extension peak profiles of multiple primers with 0.5% TT51 DNA as template

图11 多重引物以T1C-19 DNA为模板的延伸峰图A：Cry1A_1引物以T1C-19为模板的延伸峰图；B：Cry1A_2引物以T1C-19为模板的延伸峰图；C：Cry1A_3引物以T1C-19为模板的延伸峰图；D：Cry1A_4引物以T1C-19为模板的延伸峰图；E：bar引物以T1C-19为模板的延伸峰图；F：CaMV 35S引物以T1C-19为模板的延伸峰图；G：NOS_Terminator引物以T1C-19为模板的延伸峰图

Fig. 11 Extension peak profiles of multiple primers with T1C-19 DNA as template

图12 多重引物以散装大米DNA为模板的延伸峰图A：TT51引物以散装大米DNA为模板的延伸峰图；B：Cry1A_1引物以散装大米DNA为模板的延伸峰图；C：Cry1A_2引物以散装大米DNA为模板的延伸峰图；D：Cry1A_3引物以散装大米DNA为模板的延伸峰图；E：Cry1A_4引物以散装大米DNA为模板的延伸峰图；F：bar引物以散装大米DNA为模板的延伸峰图；G：CaMV 35S引物以散装大米DNA为模板的延伸峰图；H：NOS_Terminator引物以散装大米DNA为模板的延伸峰图

Fig. 12 The extension peak maps of multiple primers using bulk rice DNA as the template

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基于核酸质谱的转基因水稻多靶标检测方法

A Nucleic Acid Mass Spectrometry-based Multi-target Detection Method for Genetically Modified Rice

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