转基因抗虫大豆转化体CAL16定性PCR检测方法
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科技创新2030重大项目(2022ZD040190902、2022ZD0401815)


Qualitative PCR detection method for transgenic insect-resistant soybean transformant CAL16
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    摘要:

    【目的】 转基因大豆CAL16的抗虫融合蛋白Cry1Ab/Vip3Da对大豆鳞翅目害虫有很好的抗性效果,但目前针对抗虫大豆CAL16及其衍生品种的定性检测的方法尚未建立,本研究旨在根据外源片段插入位置,设计CAL16转化体的特异的定性PCR检测引物,弥补标准上的空白。【方法】 根据技术资料公布的插入位置和载体信息,在左边界(LB端)和右边界(RB端)设计多对引物,分别对引物进行特异性测试、扩增反应优化、测序验证、稳健性测试、灵敏度测试、检出限测试等。【结果】 数据显示,引物组合CAL16-F/R具有很好的特异性和稳健性,检出限为0.10%,符合相关转基因分子检测标准的要求,国内的8家转基因生物安全检测机构能够重复试验结果,因此,该方法可以在检测行业中推广使用。【结论】 转基因抗虫大豆CAL16定性PCR检测方法的建立,为我国CAL16抗虫大豆及其衍生品种的安全监管提供技术支撑。

    Abstract:

    【Aim】 The insect fusion protein Cry1Ab/Vip3Da of transgenic soybean CAL16 has shown good resistance against soybean lepidopteran pests; however, a qualitative detection method for insect-resistant soybean CAL16 and its derivatives has not yet been established. The purpose of this study was to design a specific qualitative polymerase chain reaction (PCR) detection primer for CAL16 transformants based on the insertion position of exogenous fragments to fill the gap in the standard. 【Method】 Based on the insertion position and vector information published in the technical data, multiple pairs of primers were designed at the left boundary (LB terminus) and right boundary (RB terminus). The primers were subjected to specificity testing, amplification reaction optimization, sequencing verification, robustness testing, sensitivity testing, and detection limit testing. 【Result】 The data showed that the primer combination CAL16-F/R had good specificity and robustness and that the detection limit was 0.10%, which met the requirements of relevant transgenic molecular testing standards. Eight genetically modified organism (GMO) safety testing institutions in China were able to replicate the test results. Therefore, this method can be used in the testing industry. 【Conclusion】 The establishment of a qualitative PCR detection method for CAL16 in transgenic insect-resistant soybean provides technical support for the safety supervision of this transformant and its derivatives in China.

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陆佳雨,王沛然,许学,吴爽,胡笑林,李晨,潘伟芹,江雅婷,汪秀峰.转基因抗虫大豆转化体CAL16定性PCR检测方法[J].生物安全学报中文版,2025,34(2):137-144

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  • 收稿日期:2024-02-18
  • 最后修改日期:2024-09-11
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  • 在线发布日期: 2025-05-16
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