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水稻KMD1检测引物及检测方法与试剂盒。本发明针对转基因水稻KMD1转化事件3’端基因的6个区域设计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水稻KMD1。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，为转基因水稻KMD1检测提供了有效、快速的检测方法。
外文摘要：NOVELTY - Loop-mediated isothermal amplification kit comprises outer upstream and downstream primers, and inner upstream and downstream primers.
USE - Loop-mediated isothermal amplification kit used for detecting transgenic rice KMD1 (claimed).
ADVANTAGE - The product is easy to use, and has high sensitivity and specificity, simple results, and short detection time.
DETAILED DESCRIPTION - Loop-mediated isothermal amplification kit comprises upstream primer KMD1-F3: ctgggagggagggagggcggcgatggcgatgc; downstream primer KMD1-B3: ctgggagggagggagggctgttgcccgtctcactggt; upstream primer KMD1-FIP: tatcccgagatgggcaggcactgggagggagggagggccaactgcggcgggt; and downstream primer KMD1-BIP: ttgtggtgtaaacaaattgacgcctgggagggagggaggggaaaagaaaaaccaccccagtac. An INDEPENDENT CLAIM is included for application of loop-mediated isothermal amplification kit for detecting transgenic rice KMD1 comprising extracting sample genome template; mixing hemin solution and detection primers, incubating at 35-45 degrees C for 20-30 minutes, adding 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) coloring solution and hydrogen peroxide to obtain color reaction mixture, and determining color using UV spectrophotometer at 419 nm absorbance value; performing loop-mediated isothermal amplification by adding sample genome template into color reaction mixture, and reacting at 60-65 degrees C for 40-60 minutes; and measuring absorbance at 419 nm using UV spectrophotometer, and comparing results from second step.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Components: The loop-mediated isothermal amplification kit further comprises hemin, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) powder, dimethyl sulfoxide buffer, 30% hydrogen peroxide, 1xThermopol buffer, deoxynucleotide, magnesium sulfate, betaine, Bst DNA polymerase, 10 mM monosodium phosphate, 100 mM potassium chloride, 2 mM magnesium chloride, and 0.003 wt.% Triton X-100 (RTM: polyethylene glycol tert octylphenyl ether).
主权项：基于环介导等温扩增消光技术的转基因水稻KMD1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KMD1?F3：CTGGGAGGGAGGGAGGGCGGCGATGGCGATGC；下游外引物KMD1?B3：CTGGGAGGGAGGGAGGGCTGTTGCCCGTCTCACTGGT；上游内引物KMD1?FIP：TATCCCGAGATGGGCAGGCACTGGGAGGGAGGGAGGGCCAACTGCGGCGGGT；下游内引物KMD1?BIP：TTGTGGTGTAAACAAATTGACGCCTGGGAGGGAGGGAGGGGAAAAGAAAAACCACCCCAGTAC。
申请号：CN201610912986.9
专利号：CN106367522-A
公开/公告号：CN106367522-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;张莉
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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