SYBRA®Green qPCR screening methods for the presence of "35S promoter" and "NOS terminator" elements in food and feed products
SYBRA®Green qPCR screening methods for the presence of "35S promoter" and "NOS terminator" elements in food and feed products
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DOI：
10.1007/s00217-009-1170-5
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发表时间：
2010-01-01
影响因子：
3.3
通讯作者：
Van den Bulcke, Marc
中科院分区：
文献类型：
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作者：
Barbau-Piednoir, Elodie;Lievens, Antoon;Van den Bulcke, Marc
花椰菜花叶病毒的35S启动子(P35S)和农杆菌的诺帕林合成酶终止子(TNOS)是迄今为止在商业转基因作物中最具代表性的通用重组元件。针对“P35s”和“Tnos”核心元件，开发了一套四种新的SYBR(A(R))Green定量聚合酶链式反应方法。这些定量聚合酶链式反应方法对“P35S”元件产生147和75bp的短扩增片段，对“Tnos”元件产生172和69bp的短扩增产物。构建了包含这些扩增子的单靶向质粒，并允许测定每个扩增子的标称熔融温度(T(M)值)。这四种方法对各自的靶标都是特异的，而且其中三种方法高度敏感(可检测到1-2个拷贝)，聚合酶链式反应效率在95%到100%之间。后一种方法可以在0.1%(w/w)的gDNA水平上检测到各自的靶标，适用于检测含有“P35s”和/或“Tnos”元素的低水平转基因材料。
The Cauliflower Mosaic Virus "35S promotor" (p35S) and the Agrobacterium "Nopaline Synthase" terminator (tNOS) are the most represented generic recombinant elements in commercial genetically modified crops to date. A set of four new SYBR(A (R))Green qPCR methods targeting the "p35S" and "tNOS" core elements have been developed. These qPCR methods generate short amplicons of 147 and 75 bp for the "p35S" element and 172 and 69 bp for the "tNOS" element. Single target plasmids containing these amplicons were constructed and allow determining the nominal melting temperature (T (m) value) of each amplicon. The four methods are specific for their respective targets, and moreover, three of them are highly sensitive (up to 1-2 copies detectable) at a PCR efficiency ranging between 95 and 100%. The latter methods can detect their respective targets at 0.1% (w/w) gDNA levels and are suitable for detecting low levels of genetically modified materials containing the "p35S" and/or "tNOS" elements.