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水稻TT51?1检测方法，首先针对转基因水稻TT51?1转化事件3’端基因的部分区域设计一套LAMP引物组合，包括TT51?1?F3/B3和TT51?1?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水稻TT51?1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，为转基因水稻TT51?1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 - Loop-mediated isothermal amplification (LAMP) primer combination comprises outer forward primer TT51-1-F3 comprising sequence of 36 base pairs (SEQ ID NO: 1) as given in the specification, outer reverse primer TT51-1- B3 comprising sequence of 35 base pairs (SEQ ID NO: 2) as given in the specification , inner forward primer TT51-1- FIP comprising sequence of 58 base pairs (SEQ ID NO: 3) as given in the specification and inner reverse primer TT51-1-BIP comprising sequence of 58 base pairs (SEQ ID NO: 4) as given in the specification, is new.
USE - The LAMP primer combination is useful for detecting transgenic rice TT51-1 (claimed).
ADVANTAGE - The LAMP primer combination has high sensitivity, strong specificity and simple operation; and is suitable for substrate detection.
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for:
(1) kit comprising LAMP primer for detecting transgenic rice TT51-1;
(2) use of LAMP primer combination or kit in detection of transgenic rice TT51-1; and
(3) detecting transgenic rice TT51-1 by extinction loop-mediated isothermal amplification technology comprising (a) crushing sample into powder, to obtain 100 plus minus 10mg sample and extracting genomic DNA by cetrimonium bromide method, (b) carrying out color reaction, visually observing color of the reaction mixture or determining OD value of the reaction mixture at 419 nm using an ultraviolet spectrophotometer, (c) carrying out loop-mediated isothermal amplification using step (a) extracted DNA as a template and LAMP primer combination, and (d) analyzing amplification products.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Components: The kit further comprises deoxynucleotide, Bst DNA polymerase, betaine, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hemin working solution, hydrogen peroxide solution, thermopol buffer solution and standard positive template. The concentration of the ABTS color developing solution was 36 mu M. The solvent is 1X dimethyl sulfoxide. The composition of the hemin working fluid comprises 50 mu M hemin, 10 mu M monosodium phosphate, 100 mu M potassium chloride, 2 mu M magnesium chloride and 0.003% Triton X-100 (RTM: Octyl phenol ethoxylate). Preferred Method: The step (b) specifically comprises hemin working solution is combined with the LAMP primer, incubating at 35-45 degrees C for 20-30 minutes, then adding ABTS color liquid and hydrogen peroxide solution, immediately observe the color with the naked eye or determining using ultraviolet spectrophotometer at OD 419 nm value, where the reaction system of color reaction comprises 90 mu l 40-60 mu M hemin working solution, 10 mu M primer TT51-1-F3, 0.5 mu l TT51-1-B3, 10 mu M primer TT51-1-FIP 3-4 mu l TT51-1-BIP, 30 mu l 30-40 mu M ABTS color developing solution, 1 mu l 30% hydrogen peroxide, and making up the total system to 130 mu l using double distilled water. The step (c) specifically comprises adding 16 mu l amplification reagent to step (b) product, where the amplification reagent comprises 1.8-3.0 mu M deoxynucleotide, 6-9 mu M magnesium sulfate, 1-2 M betaine, 3.6-10 U Bst DNA polymerase, 1X Thermopol buffer and 2 mu l DNA template, and the reaction system is incubated at 60-65 degrees C for 40-60 minutes, then incubating at 80-85 degrees C for 2-3 minutes, and terminating the reaction using ice. The step (d) specifically comprises the color of the reaction mixture was visually judged before and after the reaction or measuring the OD value of the resulting reaction mixture at 419 nm using an ultraviolet spectrophotometer, if the color changes from blue before the reaction to light blue or colorless or after the reaction OD 419 nm value becomes smaller, it indicates that the sample to be tested contains transgenic rice TT51-1. The transgenic rice TT51-1 is detected by (i) crushing sample into powder, to obtain 100 plus minus 10mg sample and extracting genomic DNA by cetrimonium bromide method with as a DNA template; (ii) carrying out color reaction by uniformly mixing hemin working fluid with LAMP primer combination, incubating at 40 degrees C for 30 minutes, then adding ABTS color liquid and hydrogen peroxide, immediately observing color with the naked eye or using ultraviolet spectrophotometer to determine the OD at 419 nm; (iii) carrying out loop-mediated isothermal amplification using step (i) extracted DNA as a template and LAMP primer combination and incubating at 65 degrees C for 40 minutes, then subjecting to 85 degrees C for 3 minutes, and terminating the reaction with ice; and (iv) visually judging color of the reaction mixture before and after the reaction or measuring step (iii) product at 419 nm using an ultraviolet spectrophotometer, if the color changes from blue before the reaction to light blue or colorless or after the reaction OD419 nm if value becomes smaller, it indicates that sample to be tested contains transgenic rice TT51-1.
主权项：用于检测转基因水稻TT51?1的LAMP引物组合，其特征在于，所述引物组合根据TT51?1的外源基因3’端设计，包括：外侧正向引物TT51?1?F3：5’?CTGGGAGGGAGGGAGGGCCGGCGTCAATACGGGATA?3’；外侧反向引物TT51?1?B3：5’?CTGGGAGGGAGGGAGGGTCGTAGCCCCACCACTAC?3；’以及内侧正向引物TT51?1?FIP：5’?CGGTCATTGACTGGAGCGAGGCTGGGAGGGAGGGAGGGATACCGCGCCACATAGCA?3’；内侧反向引物TT51?1?BIP：5’?AGAGACTGGTGATTTCAGCGGGCTGGGAGGGAGGGAGGGCTTATCTGCCCCAGCACTC?3’。
申请号：CN201610913848.2
专利号：CN106319078-A
公开/公告号：CN106319078-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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