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首页> 外文期刊>Quality Assurance and Safety of Crops & Foods >Bt11 event detection by real-time PCR: single-laboratory validation, comparison of DNA extraction and quantification techniques and application
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Bt11 event detection by real-time PCR: single-laboratory validation, comparison of DNA extraction and quantification techniques and application

机译：Bt11事件实时PCR的检测:single-laboratory验证、比较的DNA提取和量化技术和应用程序

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摘要

The detection of genetically modified organisms (GMO) by real-time polymerase chain reaction (PCR) is recommended due to its effectiveness in GMO analysis. A complete in-house validation method was applied to the detection of Bt11 events by real-time PCR. A full factorial design was used to compare DNA extraction (cetyltrimethyl ammonium bromide; CTAB, and NucleoSpin (R) Plant II Kit) and DNA quantification techniques (conventional GENESYS (TM) 10S UV-Vis spectrophotometer and confined drop-based NANOVUE (TM) Plus spectrophotometer). In the validation, various levels (0.0007 to 0.0315%) of Bt11 maize were formulated with blank maize and certified Bt11 reference material. A false-positive rate of 0% was obtained for blank samples, which corresponded to selectivity and reliability rates of 100%. The false-negative rate varied from 0 to 83.3%, consistent with sensitivity and reliability rates ranging from 16.7 to 100%. The Bt11 level that presented 100% positive results was 0.0315%, which indicated the sensitivity of the method. Non-linear models were used to estimate the region of unreliability and to calculate the detection limit of 0.014%. Accordance and concordance values of 1.0 were obtained for the 0.0315% level, which indicated method standardisation. Selectivity in the presence of interference was confirmed by the detection of Bt11 maize in the presence of other events. The method was considered robust for different DNA extraction and DNA quantification techniques. Higher DNA concentration values were obtained using CTAB. The absorbance ratio of A260/A230 was negatively influenced by quantification using a conventional spectrophotometer. Both DNA extraction techniques gave values of A260/A280 higher than 1.7, which indicated DNA of great purity. This validated method was applied to routine samples.
机译：转基因生物的检测(GMO)实时聚合酶链反应建议(PCR)由于其有效性转基因生物分析。方法应用于Bt11的检测事件的实时PCR。是用来比较DNA提取(cetyltrimethyl溴化铵;NucleoSpin (R)植物II工具包)和DNA量化技术(常规GENESYS(TM) 10年代紫外可见分光光度计和局限drop-based NANOVUE (TM) +分光光度计)。在验证,不同层次(0.00070.0315%)的Bt11玉米制定了空白玉米和认证Bt11参考资料。假阳性率为0%,获得了空白样本,选择性和对应可靠性水平的100%。率不同,从0到83.3%,符合从利率敏感性和可靠性16.7到100%。积极的结果是0.0315%,这说明该方法的灵敏度。用来估计该地区的不可靠性计算检出限为0.014%。根据和一致性值1.0获得了0.0315%的水平,这说明方法标准化。证实了干扰的存在检测Bt11玉米的事件。不同的DNA DNA提取和量化技术。用CTAB获得。A260 / A230是负面的影响使用传统的量化分光光度计。给A260 / A280高于1.7的值表示DNA的纯度。方法应用常规样品。

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