应用LNA-TaqMan探针实时荧光PCR检测大米制品中转基因成分

doi:10.7506/spkx1002-6630-201510042

食品科学

应用LNA-TaqMan探针实时荧光PCR检测大米制品中转基因成分

潘广1，章桂明1，陈枝楠2，程颖慧1，向才玉1，包先雨2，凌杏园1,*

1.深圳出入境检验检疫局动植物检验检疫技术中心，广东深圳 518045；
2.深圳市检验检疫科学研究院，广东深圳 518010

出版日期:2015-05-25 发布日期:2015-05-08
通讯作者: 凌杏园
基金资助:
深圳市科技研发资金基础研究计划重点项目（JC201105190969A）；国家转基因重大专项（2014ZX08012-001）

Ultra-Sensitive Detection of Genetically Modified Ingredients in Rice-Derived Products Using Real-Time PCR with Locked Nucleic Acid TaqMan Probe

PAN Guang1, ZHANG Guiming1, CHEN Zhinan2, CHENG Yinghui1, XIANG Caiyu1, BAO Xianyu2, LING Xingyuan1,*

1. Animal and Plant Inspection and Quarantine Technology Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau,
Shenzhen 518045, China; 2. Shenzhen Academy of Inspection and Quarantine, Shenzhen 518010, China

Online:2015-05-25 Published:2015-05-08
Contact: LING Xingyuan

摘要/Abstract

摘要：

大米制品中痕量转基因成分的检测需要特异和超灵敏的检测方法。本研究以行业标准中转基因大米筛查位点CaMV35S启动子、NOS终止子和Cry1A基因为目标，利用在常规TaqMan探针中掺入锁核苷酸提高探针退火温度和杂交特异性等特点，经比较以上位点不同LNA-TaqMan探针实时荧光聚合酶链式反应（polymerase chain reaction，PCR）检测效果，建立了针对上述筛查位点的基于LNA-TaqMan探针的新型实时荧光PCR检测方法。该方法特异性强，检测灵敏度超高；与普通TaqMan实时荧光PCR方法相比，其反应Ct值可提前1～3 个循环（Cry1A位点除外），检测低限可达3 pg。该检测方法可以用以检测大米制品中常规实时荧光PCR难以检测到的痕量转基因大米成分。

关键词: 大米制品, 锁核酸, 锁核酸探针, 实时荧光PCR

Abstract:

This study aimed to establish a specific and ultra-sensitive detection method for trace genetically modified (GMO)
ingredients in rice-derived products. In this study, the CaMV35S promoter, NOS terminator and Cry1A gene included in the
industrial standard for screening the GM components of rice were selected as the targets. By substituting a few nucleotides
of the TaqMan probe with locked nucleic acid (LNA) nucleotide and comparing the performances of these LNA-TaqMan
probes in real-time PCR (RT-qPCR), a novel real-time PCR method based on LNA-TaqMan probe for the above genes and
gene elements was established with high specificity. Compared to the conventional RT-PCR with TaqMan probe, this RTPCR
with LAN-Taqman probe was much more sensitive, had lower limit of detection (LOD) (0.001% by mass) and 1-3 less
Ct value cycles except for Cry1A. This reported new PCR method can be applied for the detection of trace GM components
in rice-derived products that could not be detected with the conventional RT-PCR probe.

Key words: rice-derived products, locked nucleic acid (LNA), LNA-TaqMan probe, real-time PCR

中图分类号:

S511

潘广1，章桂明1，陈枝楠2，程颖慧1，向才玉1，包先雨2，凌杏园1,*. 应用LNA-TaqMan探针实时荧光PCR检测大米制品中转基因成分[J]. 食品科学, doi: 10.7506/spkx1002-6630-201510042.

PAN Guang1, ZHANG Guiming1, CHEN Zhinan2, CHENG Yinghui1, XIANG Caiyu1, BAO Xianyu2, LING Xingyuan1,*. Ultra-Sensitive Detection of Genetically Modified Ingredients in Rice-Derived Products Using Real-Time PCR with Locked Nucleic Acid TaqMan Probe[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201510042.

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应用LNA-TaqMan探针实时荧光PCR检测大米制品中转基因成分

Ultra-Sensitive Detection of Genetically Modified Ingredients in Rice-Derived Products Using Real-Time PCR with Locked Nucleic Acid TaqMan Probe

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