应用LNA-TaqMan探针实时荧光PCR检测大米制品中转基因成分

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Ca MV35S启动子、NOS终止子和Cry1A基因为目标,利用在常规Taq Man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-Taq Man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-Taq Man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Taq Man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。

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

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.