GMO Detection Laboratory, SJTU-Bor Luh Food Safety Center, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
J Agric Food Chem. 2009 Aug 26;57(16):7205-12. doi: 10.1021/jf901198x.
Genetically modified (GM) papaya (Carica papaya L.), Huanong No. 1, was approved for commercialization in Guangdong province, China in 2006, and the development of the Huanong No. 1 papaya detection method is necessary for implementing genetically modified organism (GMO) labeling regulations. In this study, we reported the characterization of the exogenous integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. The results suggested that one intact copy of the initial construction was integrated in the papaya genome and which probably resulted in one deletion (38 bp in size) of the host genomic DNA. Also, one unintended insertion of a 92 bp truncated NptII fragment was observed at the 5' end of the exogenous insert. Furthermore, we revealed its 5' and 3' flanking sequences between the insert DNA and the papaya genomic DNA, and developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5' integration flanking sequence. The relative limit of detection (LOD) of the qualitative PCR assay was about 0.01% in 100 ng of total papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the limits of detection and quantification (LOD and LOQ) were as low as 12.5 and 25 copies of papaya haploid genome, respectively. In practical sample quantification, the quantified biases between the test and true values of three samples ranged from 0.44% to 4.41%. Collectively, we proposed that all of these results are useful for the identification and quantification of Huanong No. 1 papaya and its derivates.
转基因为 Huanong No.1 的木瓜（Carica papaya L.），于 2006 年在中国广东省获得商业化批准，为实施转基因生物（GMO）标签法规，开发 Huanong No.1 木瓜检测方法是必要的。在这项研究中，我们通过常规聚合酶链反应（PCR）和热不对称交错（TAIL）-PCR 策略，报道了 GM Huanong No.1 木瓜的外源整合特征。结果表明，初始构建物的一个完整拷贝被整合到木瓜基因组中，这可能导致宿主基因组 DNA 的一个缺失（大小为 38bp）。此外，还观察到外源插入物的 5'端存在一个未预期的 92bp 截断 NptII 片段的插入。此外，我们揭示了外源插入物和木瓜基因组 DNA 之间的 5'和 3'侧翼序列，并基于 5'整合侧翼序列，开发了 GM Huanong No.1 木瓜的事件特异性定性和定量 PCR 检测方法。定性 PCR 检测的相对检测限（LOD）约为 100ng 总木瓜基因组 DNA 中的 0.01%，相当于约 25 个木瓜单倍体基因组拷贝。在定量 PCR 中，检测限和定量限（LOD 和 LOQ）低至 12.5 和 25 个木瓜单倍体基因组拷贝。在实际样品定量中，三个样品的测试值与真实值之间的定量偏差范围为 0.44%至 4.41%。总的来说，这些结果可用于 Huanong No.1 木瓜及其衍生物的鉴定和定量。
