Wei Jiaojun, Li Feiwu, Guo Jinchao, Li Xiang, Xu Junfeng, Wu Gang, Zhang Dabing, Yang Litao
National Center for Molecular Characterization of Genetically Modified Organisms, SJTU-Bor Luh Food Safety Center, School of Life Science and Biotechnology, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, People's Republic of China.
J Agric Food Chem. 2013 Nov 27;61(47):11363-70. doi: 10.1021/jf403338a. Epub 2013 Nov 15.
The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.
在先前的研究中，木瓜（番木瓜科番木瓜属）的木瓜凝乳蛋白酶（CHY）基因已被报道为转基因木瓜检测的合适内参基因。在此，我们通过实验室间协作环试进一步验证了CHY基因及其定性和定量聚合酶链反应（PCR）检测方法的应用。共有12个从事转基因生物检测的实验室参与了环试并返回了检测结果。对返回结果的统计分析证实了CHY基因在不同木瓜品种间的物种特异性、低异质性和单拷贝数。CHY定性PCR检测的检测限为0.1%，而定量PCR检测的定量限为单倍体木瓜基因组约25个拷贝，PCR效率和线性度可接受。10个盲样中木瓜含量的检测值与真实值之间的差异在0.84%至6.58%之间。这些结果表明，CHY基因适合作为转基因木瓜鉴定和定量的内参基因。
