A Real-Time Quantitative PCR Detection Method Specific to Widestrike Transgenic Cotton (Event 281-24-236/3006-210-23)
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- 作者：Stefan Baeumler ; Dö ; rte Wulff ; Laura Tagliani ; Ping Song
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2006
- 出版时间：September 6, 2006
- 年：2006
- 卷：54
- 期：18
- 页码：6527 - 6534
- 全文大小：86K
- 年卷期：v.54,no.18(September 6, 2006)
- ISSN：1520-5118
文摘
In compliance with global regulations on transgenic crops, a real-time quantitative PCR method specificto Widestrike transgenic cotton (event 281-24-236/3006-210-23, OECD Unique Identifier DAS-24236-5/DAS-21023-5) was established on the basis of the DNA sequences in the junction between thetransgene insert and cotton genome. The optimized method consists of a DNA extraction method forcotton seeds and three PCR systems corresponding to a cotton-specific endogenous reference DNAsequence SAH7 (Sinapis Arabidopsis Homolog 7) and specific detection of event 281-24-236 andevent 3006-210-23. The method performance including specificity, sensitivity, accuracy, and precisionwas determined at a dynamic range of Widestrike DNA levels from 0.04% to 5.0%. The limits ofdetection (LOD) and quantification (LOQ) were 
0.04% and 0.09%, respectively, at 100 ng of DNAsample per reaction. The quantification results using either the event 281-24-236 or 3006-210-23system were consistent, and the relative deviation from the expected (true) value was in the rangeof ±25%. The robustness of the method was demonstrated by a series of tests with deviations fromthe optimized assay parameters such as annealing temperature, extension time, PCR instrument,interlaboratory transferability, etc. All the measurements from these tests met the criteria set by EUJRC-CRL (European Commission Joint Research Centre-Community Reference Lab). This real-timequantitative PCR method is accurate and robust, and is recommended as a global benchmark methodfor the detection and quantification of Widestrike cotton. The method including description, protocol,and performance results is available on the JRC-CRL website (http://gmo-crl.jrc.it/statusofdoss.htm).
0.04% and 0.09%, respectively, at 100 ng of DNAsample per reaction. The quantification results using either the event 281-24-236 or 3006-210-23system were consistent, and the relative deviation from the expected (true) value was in the rangeof ±25%. The robustness of the method was demonstrated by a series of tests with deviations fromthe optimized assay parameters such as annealing temperature, extension time, PCR instrument,interlaboratory transferability, etc. All the measurements from these tests met the criteria set by EUJRC-CRL (European Commission Joint Research Centre-Community Reference Lab). This real-timequantitative PCR method is accurate and robust, and is recommended as a global benchmark methodfor the detection and quantification of Widestrike cotton. The method including description, protocol,and performance results is available on the JRC-CRL website (http://gmo-crl.jrc.it/statusofdoss.htm).