中文摘要：本发明涉及分子生物学及生物检测领域，具体公开了基于环介导等温扩增消光技术的转基因大豆GTS40?3?2检测引物及检测方法与试剂盒。本发明针对转基因大豆GTS40?3?2转化事件3’端基因的部分区域设计4条特异性引物，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，通过判定反应颜色或吸光值的变化判断是否有扩增产物，从而判断是否存在转基因大豆GTS40?3?2转。所述方法具有灵敏度高、特异性强、操作简单、肉眼判定结果等优点，为转基因大豆GTS40?3?2检测提供了有效、快速的检测方法。
外文摘要：NOVELTY - Transgenic soybean GTS40-3-2 detection primer based on loop-mediated isothermal amplification, comprises an upstream outer primer and downstream outer primer; and upstream inner primer and downstream inner primer. The upstream outer primer contains nucleobase sequence of 37 nucleobases (SEQ ID NO: 1), given in the specification. The downstream outer primer contains nucleobase sequence of 35 nucleobases (SEQ ID NO: 2), given in the specification. The downstream inner primer contains nucleobase sequence of 60 nucleobases (SEQ ID NO: 3), given in the specification.
USE - Transgenic soybean GTS40-3-2 detection primer based on loop-mediated isothermal amplification for detecting transgenic soybean GTS40-3-2 (claimed).
ADVANTAGE - The transgenic soybean GTS40-3-2 detection primer based on loop-mediated isothermal amplification is effective and fast; and has high sensitivity, strong specificity, and easy to operate.
DETAILED DESCRIPTION - Transgenic soybean GTS40-3-2 detection primer based on loop-mediated isothermal amplification, comprises an upstream outer primer and downstream outer primer; and upstream inner primer and downstream inner primer. The upstream outer primer contains nucleobase sequence of 37 nucleobases (SEQ ID NO: 1), given in the specification. The downstream outer primer contains nucleobase sequence of 35 nucleobases (SEQ ID NO: 2), given in the specification. The downstream inner primer contains nucleobase sequence of 60 nucleobases (SEQ ID NO: 3), given in the specification. The downstream inner primer contains nucleobase sequence of 58 nucleobases (SEQ ID NO: 4), given in the specification. INDEPENDENT CLAIMS are included for:
(1) method for detecting transgenic soybean GTS40-3-2, which involves extracting genomic DNA from sample, mixing hemin working solution with primer, incubating for 20-35 minutes at 30-45 degrees C, adding 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) color developing solution and hydrogen peroxide to obtain the mixed color reaction solution, immediately determining color visually or measuring the light absorption value at 419nm by ultraviolet spectrophotometer, then adding genomic DNA in mixed color reaction solution; performing loop-mediated isothermal amplification at 60-65 degrees C for 40-60minutes, visually determining the reaction color, or measuring the light absorption value at 419nm by ultraviolet spectrophotometer, comparing obtained result with the previous result where presence or absence of transgenic soybean GTS40-3-2 is detected by determining whether there is an amplified product or not; and
(2) kit for detecting transgenic soybean GTS40-3-2, comprises transgenic soybean GTS40-3-2 detection primer based on loop-mediated isothermal amplification.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Conditions: The hemin working solution is prepared by dissolving hemin in a working solution where working solution contains 10mM monosodium phosphate, 100mM potassium chloride, 2mM magnesium chloride and 0.003% Triton (RTM: nonionic surfactant) X-100. The kit further comprises a working solution, hemin, ABTS powder, dimethyl sulfoxide buffer solution, 30% hydrogen peroxide, 1x Thermopol (RTM: Not defined) buffer, deoxynucleotide, magnesium sulfate, betaine, and Bst DNA polymerase.
主权项：基于环介导等温扩增消光技术的转基因大豆GTS40?3?2检测引物，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，序列分别为：上游外引物GTS40?3?2?F3：CTGGGAGGGAGGGAGGGGGAGTAGTACACTCACCAGT；下游外引物GTS40?3?2?B3：CTGGGAGGGAGGGAGGGGCATTCGAGCTTCTTCAC；上游内引物GTS40?3?2?FIP：ACAACGAGAAGCTATATGTAGACTGGGAGGGAGGGAGGGGCGACCCTAATAGGCAACAGC；下游内引物GTS40?3?2?BIP：CAAAACTATTTGGGATCGGAGCTGGGAGGGAGGGAGGGGAGAACTTCTCGACGATGGC。
申请号：CN201610909381.4
专利号：CN106319076-A
公开/公告号：CN106319076-A
申请/专利权人：中国农业大学
发明/设计人：黄昆仑;许文涛;徐瑗聪;王晨光;罗云波;田文莹
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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