Noguchi Akio, Nakamura Kosuke, Sakata Kozue, Kobayashi Tomoko, Akiyama Hiroshi, Kondo Kazunari, Teshima Reiko, Ohmori Kiyomi, Kasahara Masaki, Takabatake Reona, Kitta Kazumi
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
J AOAC Int. 2013 Sep-Oct;96(5):1054-8. doi: 10.5740/jaoacint.12-442.
Genetically modified (GM) papaya line 55-1 (55-1) is resistant to papaya ringspot virus infection, and is commercially available in several countries. A specific detection method for 55-1 is required for mandatory labeling regulations. An event-specific real-time PCR method was developed by our laboratory. To validate the method, interlaboratory validation of event-specific qualitative real-time PCR analysis for 55-1 was performed in collaboration with 12 laboratories. DNA extraction and real-time PCR reaction methods were evaluated using 12 blind samples: six non-GM papayas and six GM papayas in each laboratory. Genomic DNA was highly purified from all papayas using an ion-exchange column, and the resulting DNA sample was analyzed using real-time PCR. Papaya endogenous reference gene chymopapain (CHY) and the event-specific 55-1 targeted sequence were detected in GM papayas whereas CHYalone was detected in non-GM papayas in all laboratories. The cycle threshold values of CHYand the 55-1 targeted sequence showed high repeatability (RSD, 0.6-0.8%) and reproducibility (RSDR 2.2-3.6%). This study demonstrates that the 55-1 real-time PCR detection method is a useful and reliable method to monitor 55-1 papaya in foods.
转基因（GM）番木瓜品系55 - 1对番木瓜环斑病毒感染具有抗性，并且已在多个国家上市销售。由于强制标签法规的要求，需要一种针对55 - 1的特异性检测方法。我们实验室开发了一种事件特异性实时荧光定量PCR方法。为验证该方法，与12个实验室合作对55 - 1事件特异性定性实时荧光定量PCR分析进行了实验室间验证。使用12个盲样评估DNA提取和实时荧光定量PCR反应方法：每个实验室有6个非转基因番木瓜和6个转基因番木瓜。使用离子交换柱从所有番木瓜中高度纯化基因组DNA，并使用实时荧光定量PCR分析所得DNA样本。在所有实验室中，转基因番木瓜中检测到番木瓜内参基因木瓜凝乳蛋白酶（CHY）和事件特异性55 - 1靶向序列，而非转基因番木瓜中仅检测到CHY。CHY和55 - 1靶向序列的循环阈值显示出高重复性（相对标准偏差，0.6 - 0.8%）和重现性（相对标准偏差，2.2 - 3.6%）。本研究表明，55 - 1实时荧光定量PCR检测方法是监测食品中55 - 1番木瓜的一种有用且可靠的方法。
