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MON89788检测引物及检测方法与试剂盒。本发明针对转基因大豆MON89788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MON89788。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MON89788检测提供了有效、快速的检测方法。
外文摘要：NOVELTY - A primer comprising forward outer primer, reverse outer primer, forward inner primer and reverse inner primer, is new. The forward outer primer comprises a 38 base pair sequence (SEQ ID NO: 1) fully defined in the specification. The reverse outer primer comprises a 39 base pair sequence (SEQ ID NO: 2) fully defined in the specification. The forward inner primer comprises a 59 base pair sequence (SEQ ID NO: 3) fully defined in the specification. The reverse inner primer comprises a 57 base pair sequence (SEQ ID NO: 4) fully defined in the specification.
USE - The primer is useful in kit for detecting transgenic soybean MON89788 by loop-mediated isothermal amplification technique (all claimed).
ADVANTAGE - The primer enables to detect transgenic soybean MON89788 simply and rapidly with high sensitivity and strong specificity.
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the following:
(1) method for detecting transgenic soybean MON89788, involves amplifying DNA of the sample by loop-mediated isothermal amplification technique using the above-mentioned primer, and detecting existence of amplified product to determine presence or absence of transgenic soybean MON89788 conversion event; and
(2) kit comprising the primer.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Components: The hemin working solution comprises 10 mM monosodium phosphate, 100 mM potassium chloride, 2 mM magnesium chloride and 0.003% Triton X-100 (RTM: Nonionic surfactant that has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group). Preferred Method: The method involves extracting genomic DNA as template from the sample, performing chromogenic reaction by mixing hemin working solution with the primer, incubating at 35-45 degrees C for 20-30 minutes, adding 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) coloring liquid and hydrogen peroxide, reacting and determining color using UV spectrophotometer by measuring absorbance at 419 nm, performing loop-mediated isothermal amplification, and inspecting amplified product. The method involves performing chromogenic reaction by adding 40-60 mu M hemin working solution, 1.25 mu M forward outer primer, 1.25 mu M reverse outer primer, 10 mu M forward inner primer, 10 mu M reverse inner primer, incubating at 35-45 degrees C for 20-30 minutes, adding 30-40 mM ABTS coloring liquid and 50-60 mM hydrogen peroxide to obtain color developing mixed reaction solution, adding 1.4-2 mM deoxynucleoside triphosphate, 3-8 mM magnesium sulfate, 0.6-0.8 M betaine, 3.6-10 U DNA polymerase, buffer, and 2 mu l template, reacting at 60-65 degrees C for 40-60 minutes, measuring and determining color.
主权项：基于环介导等温扩增消光技术的转基因大豆MON89788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MON89788?F3：CTGGGAGGGAGGGAGGGTTGTTTATTGCTTTCGCCTAT；下游外引物MON89788?B3：CTGGGAGGGAGGGAGGGCTATCACATATGGTTGAAGTCG；上游内引物MON89788?FIP：AGTCACTAGAGTCTGAGCGTTCTGGGAGGGAGGGAGGGGAGACGGATCGTAATTTGTCG；下游内引物MON89788?BIP：ACCTTCACTCTCCTCAAGCACTGGGAGGGAGGGAGGGAACGTAAGGGATAATTTGGT。
申请号：CN201610913115.9
专利号：CN106319077-A
公开/公告号：CN106319077-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;田文莹
分类号：C12Q1/68;C12N15/11
主分类号：C12Q1/68
法律状态：实质审查
点击下载：基于环介导等温扩增消光技术的转基因大豆MON89788检测引物及检测方法与试剂盒