中文摘要：随着转基因技术的发展和转基因生物的日益普及，转基因生物面临越来越多的法规和压力来遵守严格的标签准则。单粒子检测方法无法满足最优转基因生物检测所需的高通量分析。结合多重检测和液滴数字聚合酶链反应（DDPCR）的优点，本文提出了一个同于精确筛选和定量转基因生物的单一通用引物多重DDPCR（SUV-M DDPCR）策略。SUP提高了PCR引物的效率，在建立高通量、多重检测方法中起着重要的作用。新兴的DDPCR技术已被用于准确地定量核酸分子。作者选择玉米的四个异源序列（35S、NOS、NPTII、PAT）来评价该策略的可行性和适用性。研究发现，这四个基因作为初步筛选序列覆盖了超过93%的转基因玉米品系。这种五聚体筛选方法是转基因生物筛选的新进展。作者利用最佳扩增分析，验证了该方法的特异性、检测限（LOD）和定量限（LOQ）。研究结果表明，该方法可作为在不同市售产品中检测转基因玉米的重要工具。此外，该筛选方法可应用于需要可靠和灵敏检测DNA靶标的其他领域。
外文摘要：As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences (35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.
作者：Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao
作者单位：China Agr Univ
期刊名称：ANALYTICAL CHEMISTRY
期刊影响因子：6.042
出版年份：2018
出版刊次：5
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