中文摘要：本发明涉及分子生物学及生物检测技术领域，具体公开了基于环介导等温扩增消光技术的转基因玉米MIR604检测方法。本发明针对转基因玉米MIR604的cry3A基因序列的6个区域设计4条特异性引物，其上下游外引物序列如SEQ?ID?NO.1?2所示，其上下游内引物序列如SEQ?ID?NO.3?4所示。利用环介导等温扩增消光技术使用所述引物对样品的DNA进行扩增，通过反应颜色或吸光值的变化判断是否有扩增产物，从而检测转基因玉米MIR604。本发明方法具有灵敏度高、特异性强、操作简单、可肉眼判定结果等优点，为转基因玉米MIR604检测提供了有效、快速的检测方法。
 
外文摘要：NOVELTY - Primer comprising an upstream outer primer, downstream outer primers, upstream inner primers and downstream inner primers, includes fully defined 34 nucleotides (SEQ ID NO: 1) as given in the specification, fully defined 35 nucleotides (SEQ ID NO: 2) as given in the specification, fully defined 57 nucleotides (SEQ ID NO: 3) as given in the specification and fully defined 54 nucleotides (SEQ ID NO: 4) as given in the specification, respectively, is new.
USE - The primer and kit are useful for detecting transgenic maize MIR604 based on loop-mediated isothermal amplification extinction method (claimed).
ADVANTAGE - The method has high sensitivity, strong specificity, simple operation and determines the results by naked eyes, and has rapid detection performance.
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for:
(1) use of primer in detecting transgenic maize MIR604;
(2) detecting transgenic maize MIR604, comprising amplifying DNA of the sample by loop-mediated isothermal amplification extinction process using the primer, determining whether or not there is an amplification product by the change of the reaction color or the absorbance value, so as to detect transgenic maize MIR604; and
(3) kit comprising primer.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Method: The method comprises (a) extracting the genome template for the sample to be tested; (b) mixing hemin working solution with the prime, adding ABTS color solution with hydrogen peroxide to obtain a color reaction mixture, immediately recording the color or absorbance values measured at 419 nm; (c) carrying out loop-mediated isothermal amplification extinction reaction by adding the sample genome template to the color reaction mixture, and carrying out excitation reaction at 60-65 degrees C for 40-60 minutes; and (d) visually determining the reaction color, or the absorbance at 419 nm, comparing with the results of step (b) to determine whether there is amplification products, when the color changes from blue to light or colorless or the OD 419nm value becomes smaller, indicating the purpose of the product, and samples to be tested were genetically modified maize MIR604 positive. In step (v), 40-60 mu M hemin working fluid added with 1.25 mu M upstream outer primers and downstream outer primer and 10 mu M upstream inner primer and downstream inner primer, mixing, incubated at 35-45 degrees C for 20-30 minutes, adding 30-40 mM (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) coloring solution with 50-60 mM hydrogen peroxide, to obtain a color reaction mixture. In the step (c), using 1.4-2 mM dNTP to the color reaction mixture, 3-8 mM magnesium sulfate, 0.6-0.8 M betaine, 3.6-10 U Bst DNA polymerase large fragment, 1X Thermopol buffer, 2 mu l sample genome template, and then loop-mediated isothermal amplification of extinction reaction. Preferred Components: The hemin working solution is prepared by dissolving hemin in the working fluid, contains 10 mM sodium dihydrogen phosphate, 100 mM potassium chloride, 2 mM magnesium chloride and 0.003% Triton X-100 (RTM: Nonionic surfactant). The kit further comprises working fluid, hemin, ABTS powder, dimethyl sulfoxide (DMSO) buffer, 30% hydrogen peroxide, and 1XThermopol buffer, dNTP, magnesium sulfate, betaine consisting of a reaction solution and Bst DNA polymerase, the working solution composed of 10 mM sodium dihydrogen phosphate, 100 mM potassium chloride, 2 mM magnesium chloride and 0.003% Triton X-100 (RTM: Nonionic surfactant).
 
主权项：基于环介导等温扩增消光技术的检测转基因玉米MIR604的引物，其特征在于，所述引物包括上游外引物、下游外引物、上游内引物和下游内引物，其序列分别如SEQ?ID?NO.1?4所示。
 
申请号：CN201610950696.3
专利号：CN106399544-A
公开/公告号：CN106399544-A
申请/专利权人：中国农业大学
发明/设计人：罗云波;黄昆仑;许文涛;徐瑗聪;陈冰
分类号：C12Q1/68;C12N15/11
主分类号：C12Q1/68
法律状态：实质审查
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