中文摘要：本发明提供一种基于环介导等温扩增消光技术的转基因油菜GT73检测方法，首先针对转基因油菜GT73外源基因Epsps的部分区域设计一套LAMP引物组合，包括GT73?F3/B3和GT73?FIP/BIP(Seq?ID?No:1?4)，在所述引物组合基础上结合环介导等温扩增消光技术检测转基因油菜GT73转化事件。本方法具有灵敏度高、特异性强、操作简单，结果易于观察等优点，为转基因油菜GT73检测提供了有效、快速的检测方法，非常适于基层检测使用。
外文摘要：NOVELTY - A primer combinations capable of detecting transgenic rapeseed GT73 by performing loop-mediated isothermal amplification (LAMP), is new. The primers are designed based on exogenous gene Epsps of GT73. The primer combination contains outer forward primer GT73-F3 comprising a 37 base pair sequence (SEQ ID NO: 1) fully defined in the specification, and outer reverse primer GT73-B3 comprising a 35 base pair sequence (SEQ ID NO: 2) fully defined in the specification, and inner forward primer GT73-FIP comprising a 58 base pair sequence (SEQ ID NO: 3) fully defined in the specification.
USE - The primer combinations in kit is useful for detecting transgenic rapeseed GT73 (all claimed).
ADVANTAGE - The primer combinations enables to detect transgenic rapeseed GT73 in a simple and easy manner with high sensitivity, and specificity.
DETAILED DESCRIPTION - A primer combinations capable of detecting transgenic rapeseed GT73 by performing loop-mediated isothermal amplification (LAMP), is new. The primers are designed based on exogenous gene Epsps of GT73. The primer combinations comprise outer forward primer GT73-F3 comprising a 37 base pair sequence (SEQ ID NO: 1) fully defined in the specification, and outer reverse primer GT73-B3 comprising a 35 base pair sequence (SEQ ID NO: 2) fully defined in the specification, and inner forward primer GT73-FIP comprising a 58 base pair sequence (SEQ ID NO: 3) fully defined in the specification, and inner reverse primer GT73-BIP comprising a 59 base pair sequence (SEQ ID NO: 4) fully defined in the specification. INDEPENDENT CLAIMS are included for the following:
(1) kit, comprising LAMP primer combinations; and
(2) method for detecting transgenic rapeseed GT73 involves (a) collecting transgenic rapeseed GT73, grinding the sample into powder in liquid nitrogen, weighing 100 mg plus minus 10 mg sample, and extracting genomic DNA as DNA template using cetyltrimethylammonium bromide (CTAB) method, (b) observing the color of the reaction mixture, or determining the optical density (OD) of the reaction mixture using UV spectrophotometer at 419 nm, (c) performing LAMP amplification of DNA extract using primer combinations, and (d) analyzing the PCR amplified product.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Kit: The kit further comprises deoxynucleotides (dNTPs), Bacillus stearothermophilus (Bst) DNA polymerase, betaine, ABTS (RTM: 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) coloring solution, hemin working solution, hydrogen peroxide solution, Thermopol (RTM: Reaction buffer containing nonionic detergent), and standard positive template. The ABTS (RTM: 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) coloring solution (36 mM) comprises 1X dimethyl sulfoxide (DMSO) solvent. The hemin working solution comprises 50 mu M hemin, 10 mM monosodium phosphate, 100 mM potassium chloride, 2 mM magnesium chloride, and 0.003% Triton X-100 (RTM: t-Octyl phenoxy polyethoxy ethanol). Preferred Method: The method involves using reaction system of color reaction comprising 90 mu l 40-60 mu M hemin working solution, 10 mu M primers GT73-F3 and GT73-B3, 10 mu M primers GT73-FIP and GT73-BIP, 30 mu l 30-40 mM ABTS color developing solution, 1 mu l 30% hydrogen peroxide, and 130 mu l double-distilled water, and amplification reagent comprising 1.8-3.0 mM dNTP, 6-9 mM magnesium sulfate, 1-2 M betaine, 3.6-10 U Bst DNA polymerase, 1X Thermopol (RTM: Reaction buffer containing nonionic detergent), and 2 mu l DNA template.
主权项：用于检测转基因油菜GT73的LAMP引物组合，其特征在于，所述引物组合根据GT73的外源基因Epsps设计，包括：外侧正向引物GT73?F3：5’?CTGGGAGGGAGGGAGGGCGACGTCACCATCCTTAACG?3’；外侧反向引物GT73?B3：5’?CTGGGAGGGAGGGAGGGCAGCTGCAACAGCGAGAA?3’；以及内侧正向引物GT73?FIP：5’?AGCAAGACGTGGGTTGATCACTCTGGGAGGGAGGGAGGGCCAACCCGTACTGGTCTCA?3’；内侧反向引物GT73?BIP：5’?GGAGAAGACGTGGCTGACTTGCCTGGGAGGGAGGGAGGGGAAGGAGCACGGTCTTCTGG?3’。
申请号：CN201610907561.9
专利号：CN106282388-A
公开/公告号：CN106282388-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
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