转基因油菜RT73品系特异性检测标准分子协同实验验证

doi:10.3969/j.issn.1008-0864.2011.06.06

›› 2011, Vol. 13 ›› Issue (6): 33-40.DOI: 10.3969/j.issn.1008-0864.2011.06.06

转基因油菜RT73品系特异性检测标准分子协同实验验证

李想1,褚庆华1,高琴1,吕蓉1,潘良文1,李俊毅2,张其刚2

(1.上海出入境检验检疫局, 上海 200135|2.华东理工大学, 上海 200237)

收稿日期:2011-05-20 修回日期:2011-06-14 出版日期:2011-12-15 发布日期:2011-10-20
通讯作者: 通讯作者:潘良文,研究员,博士,从事转基因作物安全性相关领域研究。Tel:021-38620631; E-mail:panlw888@126.com
作者简介:李想,高级工程师,博士,从事转基因作物检测相关技术研究。Tel:021-38620634|E-mail:idealne@163.com。
基金资助:
国家科技支撑计划项目(2008BAK41B01-2);国家转基因重大专项(2008ZX08012-001;2009ZX08012-002B);上海市科委研发平台专项(10DZ2294102)资助。

Inter-laboratory Validation of A Plasmid Reference Molecule Suitable For Genetically Modified Canola RT73 Event-Specific Detection

LI Xiang1, ZHU Qing-hua1, GAO Qin1, LV Rong1, PAN Liang-wen1, LI Jun-yi2, ZHANG Qi-gang2

(1.Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135|2.East China University of Science and Technology, Shanghai, 200237, China)

Received:2011-05-20 Revised:2011-06-14 Online:2011-12-15 Published:2011-10-20

摘要/Abstract

摘要：

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。

关键词: RT73;标准分子;品系特异性;普通PCR;实时荧光PCR

Abstract:

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-RT73 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 RT73 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.

Key words: RT73, reference molecule, event-specific, conventional PCR, real-time PCR

中图分类号:

S565.4
Q789

李想1,褚庆华1,高琴1,吕蓉1,潘良文1,李俊毅2,张其刚2. 转基因油菜RT73品系特异性检测标准分子协同实验验证[J]. , 2011, 13(6): 33-40.

LI Xiang1, ZHU Qing-hua1, GAO Qin1, LV Rong1, PAN Liang-wen1, LI Jun-yi2, ZHANG . Inter-laboratory Validation of A Plasmid Reference Molecule Suitable For Genetically Modified Canola RT73 Event-Specific Detection[J]. , 2011, 13(6): 33-40.

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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

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