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KF6检测引物及检测方法与试剂盒。本发明针对转基因水稻KF6转化事件5’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KF6。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KF6检测提供了有效、快速的检测方法。
 
外文摘要：NOVELTY - A primer capable of detecting transgenic rice KF6, is new. The detection primer comprises outer and inner upstream primer, and downstream primer. The outer upstream primer KF6-F3 comprises base pair sequence of SEQ ID NO: 1, and outer downstream primer KF6-B3 comprises base pair sequence of SEQ ID NO: 3, and inner upstream primer KF6-FIP comprises base pair sequence of SEQ ID NO: 4, and inner downstream primer KF6-BIP comprises base pair sequence SEQ ID NO: 5.
USE - The primer in kit is useful for detecting transgenic rice KF6 (all claimed).
ADVANTAGE - The primer enables to detect transgenic rice KF6 in a rapid manner with high sensitivity, and specificity, and is easy to use.
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the following:
(1) method for detecting transgenic rice KF6 involves subjecting DNA of the sample to loop-mediated isothermal amplification using primer, and detecting presence or absence of transgenic rice KF6 in the amplified product; and
(2) kit for detecting transgenic rice KF6, comprising primer.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Method: The method involves (a) extracting the sample genomic template, (b) uniformly mixing 40-60 mu M hemin working solution with 1.25 mu M KF6-F3 and KF6-B3 and 10 mu M KF6-FIP and KF6-BIP primer, incubating at 35-45 degrees C for 20-30 minutes, adding 30-40 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) coloring liquid and 50-60 mM hydrogen peroxide, and reading absorbance under UV spectrophotometer at 419 nm, (c) subjecting sample genomic template to loop-mediated isothermal amplification at 60-65 degrees C for 40-60 minutes, and (d) visually determining the reaction color. The method involves adding color reaction mixture comprising 1.4-2 mM deoxynucleotide (dNTP), 3-8 mM magnesium sulfate, 0.6-0.8M betaine, 3.6-10 U Bst DNA polymerase large fragment, 1X Thermopol (RTM: Reaction buffer containing nonionic detergent), and 2 mu l sample genome template, and reacting at 60-65 degrees C for 40-60 minutes. The hemin working solution is prepared by dissolving hemin in a working fluid containing 10 mM sodium phosphate monobasic, 100 mM potassium chloride, 2 mM magnesium chloride and 0.003% Triton X-100 (RTM: t-Octyl phenoxy polyethoxy ethanol). Preferred Kit: The kit further comprises working fluid, hemin, ABTS powder, dimethyl sulfoxide (DMSO) buffer, 30% hydrogen peroxide, 1X Thermopol (RTM: Reaction buffer containing nonionic detergent), dNTP, magnesium sulfate, betaine and Bst DNA polymerase, where the working solution comprises sodium phosphate monobasic, potassium chloride, magnesium chloride and Triton X-100 (RTM: t-Octyl phenoxy polyethoxy ethanol) in a volume ratio of 5:8:1:10.
 
主权项：基于环介导等温扩增消光技术的转基因水稻KF6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KF6?F3：CTGGGAGGGAGGGAGGGACCATGCTGCGATTCATG；下游外引物KF6?B3：CTGGGAGGGAGGGAGGGATCTTCATCCCTGGACTTG；上游内引物KF6?FIP：GAGTGACACGAATTCAACCTGACTGGGAGGGAGGGAGGGGCACTAAATCAATACCTCCT；下游内引物KF6?BIP：GGCTTGCAAATCCTGCATGCTGGGAGGGAGGGAGGGGTAACAGAAATCAGCAACGT。
申请号：CN201610913824.7
专利号：CN106367523-A
公开/公告号：CN106367523-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;张莉
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水稻KF6检测引物及检测方法与试剂盒