转基因油菜RT73品系特异性检测标准分子协同实验验证

Inter-laboratory Validation of a Plasmid Reference Molecule Suitable for Genetically Modified Canola RT73 Event-specific Detection

对构建的适于转基因油菜RT73品系特异性检测标准分子pEASY-RT73在普通PCR和实时荧光PCR检测中的适用性进行了实验室间协同实验验证。对8家实验室结果的统计分析表明,标准分子pEASY-RT73对转基因油菜RT73品系特异性检测及油菜内源基因HMG和PEP检测具有高特异性。在普通PCR扩增中,标准分子对PEP和RT73品系特异性序列的检测下限均为20拷贝,对HMG基因的检测下限为50拷贝;在实时荧光PCR扩增中,HMG和RT73品系特异性序列的检测下限均为10拷贝,PEP检测下限为50拷贝。虽然各实验室所用仪器和试剂有较大差异,但对检测结果影响不大。因此,标准分子pEASY-RT73可稳定的作为新型标准物质用于转基因油菜RT73品系特异性普通PCR和实时荧光PCR检测。

In this study, the applicability of a reference molecule pEASY-RT73 suitable for event-specific detection of genetically modified （GM） canola RT73 in conventional and real-time PCR assays was validated by inter-laboratory trail. The analysis of 8 labs reports showed that the plasmid pEASY-RT＇/3 was highly specific to GM canola RT73 rapeseed endogenous gene HMG and PEP detections. In qualitative PCR, the limits of detection （LODs） that could be detected by all 8 participants were 20 copies of plasmid DNA for PEP and RT＇/3 event-specific sequence detection and 50 copies for HMG detection. In real-time PCR assay, LODs were determined to be about 10 copies for HMG and RT73 event-specific sequence detection and 50 copies for PEP. The PCR instruments and reagents have little impact on the results even though there are many differences among 8 labs. These results indicate that reference molecule pEASY-RT73 can be used as a new type of reference material for conventional and real-time event-specific PCR detections of GM canola RT73.