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TC1507检测方法。本发明针对转基因玉米TC1507转化事件的5’端基因的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TC1507。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TC1507检测提供了有效、快速的检测方法。
 
外文摘要：NOVELTY - Primer based on extinction technology of loop-mediated isothermal amplification comprises an upstream outer primer and a downstream outer primer, upstream inner primer and downstream inner primer and the primer containing nucleotide sequence of 35, 36, 56 and 57 nucleobases (SEQ ID NOs: 1-4), given in the specification.
USE - Primer for detecting transgenic corn TC1507(all claimed).
ADVANTAGE - The primer provides effective and fast detection method for detecting transgenic corn TC1507.
DETAILED DESCRIPTION - An INDEPENDENT CLAIM is included for a method for detecting transgenic corn TC1507, involves:
(A) extracting the genome template of the sample to be tested;
(B) mixing the hemin working solution with the primer, hydrogen peroxide and 2,2'-azino-bis color developing solution to obtain the colored solution 1;
(C) recording the absorbance value of the obtained colored solution 1 at 419 nm;
(D) subjecting the colored solution 1 to ring-mediated isothermal expansion of extinction reaction treatment and mixing the sample genome template at 60-65 degrees C for 40-60 minutes to obtain coloring solution 2;
(E) recording the absorbance value of the color solution 2 at 419nm; and
(F) comparing the absorbance value of colored solution 1 and color solution 2 for determining amplification products, where the solution color changes from blue to light or colorless or absorbance value decreases then the genetically modified maize TC1507 is detected in the test sample.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Conditions: The amplification extinction technique of using loop-mediated isothermal amplification of DNA involves changing of reaction color or the absorbance value and the amplified products helps in detecting transgenic maize TC1507. The upstream of the hemin working liquid of 40-60 mu M and 1.25 mu M outer primer and a downstream outer primer and 10 mu M of upstream inner primer and downstream inner primer are incubated for 20-35 particularly 30 minutes at 45 degrees C and after adding 30-40mM of 2,2'-azino-bis color developing solution and 50 to 60mM of hydrogen peroxide to obtain the color development reaction mixed solution. The color development reaction solution is mixed with 1.4-2.0mM deoxynucleotides, 3-8mM magnesium sulfate, 0.6-0.8M betaine, 3.6-10.0 atomic mass unit Bacillus stearothermophilus DNA polymerase, 1 x thermopol buffer, 2 mu /L sample genome template and then performing the extinction loop-mediated isothermal amplification reaction to obtain the colored solution for detecting transgenic corn TC1507. Preferred Components: The hemin working solution comprises hemin, 10mM monosodium phosphate, 100mM potassium chloride, 2mM magnesium chloride and 0.003 wt.% Triton (RTM: nonionic surfactant) X-100. The hemin working solution further comprises 2,2'-azino-bis powder dimethyl sulfoxide buffer liquid, 30 wt.% hydrogen peroxide and 1 x thermopol buffer, deoxynucleotides, magnesium sulfate, betaine and components of reaction solution.
主权项：基于环介导等温扩增消光技术的检测转基因玉米TC1507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：CN201610951619.X
专利号：CN106381336-A
公开/公告号：CN106381336-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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