CN103451292B - Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology - Google Patents
Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology Download PDFInfo
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Abstract
本发明公开了一种适用于重组酶聚合酶等温扩增技术（Recombinase Ploymerase Amplification,RPA）鉴定含有转基因抗虫水稻华恢1号（TT51-1）的引物及探针组合，其正向引物序列如SEQIDNo.1所示，反向引物序列如SEQIDNo.2所示，探针序列如SEQIDNo.3所示。同时，本发明还公开了一种鉴定含有转基因抗虫水稻TT51-1的方法：提取待测样品的DNA作为模板，利用所述的引物进行RPA快速扩增及实时荧光检测，如果得到明显的扩增曲线，则证明所检样品中含有转基因抗虫水稻TT51-1的成分。本发明首次提供转基因抗虫水稻TT51-1品系特异性的RPA检测方法。The invention discloses a combination of primers and probes suitable for identification of transgenic insect-resistant rice Huahui No. 1 (TT51-1) by Recombinase Polymerase Amplification (RPA). The sequence of the forward primer is As shown in SEQ ID No.1, the sequence of the reverse primer is shown in SEQ ID No.2, and the sequence of the probe is shown in SEQ ID No.3. At the same time, the invention also discloses a method for identifying the transgenic insect-resistant rice TT51-1: extract the DNA of the sample to be tested as a template, use the primers to perform RPA rapid amplification and real-time fluorescence detection, if obvious amplification If the increase curve shows that the tested sample contains components of the transgenic insect-resistant rice TT51-1. The invention provides for the first time a specific RPA detection method for the transgenic insect-resistant rice TT51-1 strain.
Description
Technical field
The invention belongs to biological technical field, relate to the strain specificity method of application recombinase polysaccharase isothermal amplification technique (Recombinase Ploymerase Amplification, RPA) technical evaluation transgenic pest-resistant rice China extensive No. 1 (TT51-1).
Background technology
DNA cloning is the main method of detection of nucleic acids at present, and conventional PCR detects needs accurate instrument and loaded down with trivial details testing sequence, is difficult to meet the requirement of Site Detection under non-lab environment.In recent years, nucleic acid constant-temperature amplification technology has obtained significant progress, and comparing nucleic acid constant-temperature amplification technology with normal PCR does not need expensive PCR instrument, and rapid amplifying goes out object fragment at short notice, has the advantages such as easy, quick, sensitive.RPA technology is DNA replication dna in simulation organism, based on the polymerase-mediated amplification principle of recombinase, develops, while utilizing recombinase and primer to form microfilament to search the sequence of complete complementary with it on template DNA, under the help of single-stranded DNA binding protein, template DNA is unwind, primer and template DNA start pairing formation and copy required 3 ' C-terminal freely, under the effect of archaeal dna polymerase, copy extension, forming new DNA complementary strand reaction product is also to increase with exponential.Different from conventional PCR reaction, RPA reacts required primer length and is generally 30-35nt.During design of primers for fear of form primer inner and between secondary structure, the increase of its length also makes design of primers and selects difficulty to increase, the design of primer sequence with select most important to the result of RPA.In RPA amplification system, add a fluorescently-labeled probe just can realize the Real-Time Monitoring of template amplification, each mark Yi Ge fluorescence group (FAM and BHQ1) in these two T bases in probe middle part, between Liang Ge group, there is an abasic site (dSpacer), this site can be identified from colibacillary exonuclease by a kind of, this enzyme has 3 '-5 ' 5 prime excision enzyme activity, can Shi Liangge fluorescence group separated, thus make the accumulation synchronised of fluorescent signal and amplified production.In conjunction with a portable amplified fluorescence detector, just can in 10-20 minute, fluorescence curve be detected.RPA technology has greatly shortened detection time, has simplified response procedures, combines make field detection become possibility with DNA rapid extraction technology, is with a wide range of applications.
The method that round pcr detects transgenic product has screening to detect, and gene specific detects, and builds specific detection and specificity of transformant and detects.Due to the uniqueness characteristic of foreign gene in acceptor gene group insertion point, therefore according to the connecting zone design RPA primer of external source insertion DNA sequence dna and rice genome, detect and there is very high specificity, can detect specifically this transformant and derivative strain thereof.
In August, 2009, the Ministry of Agriculture provided the safety certificate of transgenic pest-resistant rice TT51-1 in Hubei Province, caused the extensive concern of the public to its security.At present China not yet ratifies the commercialization plantation of TT51-1, but its spread condition happens occasionally, in the urgent need to setting up fast and convenient detection method, for market surpervision and routine monitor.
At present, in the transgenic plant detection method of having reported, be mainly to utilize PCR instrument in laboratory, to carry out conventional detection, the method can't further meet the rapid detection of transgenic product.Also not utilizing at present RPA technology to do strain specificity to transgenic paddy rice China extensive No. 1 (TT51-1) both at home and abroad identifies.
Summary of the invention
For the blank in above-mentioned field, the invention provides the RPA detection method of extensive No. 1 (TT51-1) strain specificity of accurate, quick, easy detection transgenic paddy rice China.
Technical scheme provided by the invention is: a kind of for identify the primer that contains transgenic pest-resistant rice China extensive No. 1 (TT51-1) by recombinase polysaccharase isothermal amplification technique, its forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence is as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
The present invention also provides a kind of test kit contain extensive No. 1 of transgenic pest-resistant rice China of identifying by recombinase polysaccharase isothermal amplification technique, and this test kit comprises above-mentioned primer and probe.
The present invention also provides a kind of method that contains transgenic pest-resistant rice TT51-1 of identifying by recombinase polysaccharase isothermal amplification technique: extract the DNA of testing sample as template, utilize the primer described in claim 1 to carry out fluorescence rapid detection, if obtain obvious amplification curve, prove that institute's sample product are extensive No. 1 of transgenic pest-resistant rice China.Implementation step is: in the 50mL amplification system of RPA amplification kit recommendation response, add each 2mL(10mmol/L of primer), probe 0.5mL(10mmol/L), template DNA 50ng.39 degrees Celsius of reactions of RPA augmentation detection instrument (or quantitative real time PCR Instrument) 15 minutes.
The inventive method is to design a large amount of RPA Auele Specific Primers according to the connecting zone of external source insertion DNA sequence dna and rice genome, therefrom filters out a set of primer and probe combinations that can effectively detect fast extensive No. 1 (TT51-1) composition of transgenic paddy rice China.Utilize this to primer, to carry out fluorescence rapid detection, extensive No. 1 (TT51-1) genomic dna of transgenic paddy rice China of take can obtain obvious amplification curve as template, but not the genomic dna of transgenic paddy rice bright extensive 63 and 3 kinds of rice materials of No. 6 grades of other strain Kefengs is that template amplification does not all have amplification curve.Template is diluted with water to 2000,400,200,100,20 copies, result has amplification curve, and the method has higher sensitivity, and the present invention provides the RPA detection method of extensive No. 1 (TT51-1) strain specificity of transgenic pest-resistant rice China first.The method is improved the ability of biological technology products aspect accurate, rapid detection.
Accompanying drawing explanation
Fig. 1 is TT51-1 specific detection figure, wherein, and 1: transgenic paddy rice TT51-1; 2: rich No. 6 of transgenic paddy rice section; 3: transgenic paddy rice Kemingdao 1; 4: non-transgenic paddy rice bright extensive 63; 5: water
Fig. 2 is sensitivity test figure, and from 1 to 6 template copy number is followed successively by 2000; 400; 200; 100; 20; 0
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does example explanation.
The experimental technique of unreceipted actual conditions in embodiment below, conventionally according to normal condition, < < molecular cloning such as Sambrook etc.: laboratory manual > > (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or the condition of advising according to instrument or reagent manufacturer.
First, design primer: according to TT51-1 transformant distinguished sequence (GenBank No. EU880444) design primer, and probe.RPA requires as 30-35nt primer length, just thereby easily causing producing a large amount of primer dimers under constant temperature affects experiment effect for this, RPA experiment need to design multipair primer and be optimized from target sequence two ends, screening, the replacement of indivedual bases or increase and decrease all can produce material impact to experimental result.In this experiment, designed a large amount of primers, and therefrom filtered out a pair of highly sensitive and primer that specificity is good for RPA fluoroscopic examination, primer and probe sequence are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3(table 1).
Note: FAM: luminophore; DSpacer: abasic site; BHQ1: quenching group; Phosphate: phosphate group
1. experiment material
(1) vegetable material
Transgenic paddy rice TT51-1, rich No. 6 of transgenic paddy rice section, transgenic paddy rice Kemingdao 1, non-transgenic paddy rice bright extensive 63.
(2) enzyme and reagent
Molecular biology reagent, TwistAmp DNA Amplification Exo Kits is purchased from TwistDX company, and other biochemical reagents are import packing or domestic analytical pure.Primer is synthetic by Beijing Sheng Gong Bioisystech Co., Ltd.
(3) laboratory apparatus
DNA process instrumentation: low-temperature mixed ball milling instrument MM400(Retsch)
Fluorescence detector: RPA augmentation detection instrument (Twista) or quantitative real time PCR Instrument.
Other Instruments comprises: thermostat water bath, electronic balance, whizzer, vortex instrument, pure water instrument, constant incubator etc.
2. experimental technique and process
(1) extraction of oryza sativa genomic dna
Single-seed rice plantation, gets young leaflet tablet as DNA extraction material, according to TianGen Plant Genomic DNA Kit(Cat#DP-305) operational manual of test kit, carries out the extraction of rice total dna.
(2) DNA concentration and purity testing
Use NanoDrop 1000 spectrophotometers (Thermo Scientific) to measure purity and the concentration of DNA, and regulate DNA concentration to 50ng/mL with deionization distilled water.
(3) primer amplification
The transformant distinguished sequence inserting between DNA and plant genome DNA for PRA method amplification external source in the present embodiment is identified transgenic paddy rice TT51-1, and template concentrations is 50ng/mL.
RPA amplification system is: total system 50mL, in the 0.2mL TwistAmp Exo reaction tubes that contains lyophozyme powder, add rehydration damping fluid 29.5mL, magnesium acetate solution 2.5mL(280mmol/L), each 2mL(10mmol/L of primer), probe 0.5mL(10mmol/L), template DNA 50ng, residue water is supplied;
Primer amplification program: 39 degrees Celsius of reactions of RPA augmentation detection instrument 15 minutes.
3. experimental result
According to forward primer and the reverse primer of the design of transformant distinguished sequence, to bright extensive 63, rich No. 6 of section, Kemingdao 1 and TT51-1 oryza sativa genomic dna carry out RPA fluoroscopic examination, can identify rapidly and accurately transgenic paddy rice TT51-1, wherein at transgenic paddy rice TT51-1, have obvious fluorescence curve, but not rich the grade in material for No. 6 of transgenic paddy rice Ming Hui63He transgenic paddy rice section all do not have amplification curve (Fig. 1).Template is diluted with water to 2000,400,200,100,20 copies, result has amplification curve (Fig. 2).Explanation establishes primer with the present invention and method identifies that transgenic paddy rice TT51-1 has higher sensitivity and accuracy, and easy and simple to handle.
<110> Biological Technology institute, Chinese Academy of Agricultural Sciences
<120> application RPA technology is identified extensive No. 1 strain specificity of transgenic paddy rice China
<160>?3
<210>?1
<211>?35
<212>?DNA
<400>?1
CCCGGCGTCA?ATACGGGATA?ATACCGCGCC?ACATA
<210>?2
<211>?35
<212>?DNA
<400>?2
GGAACATATG?AGTGGTAGCG?TCCAGAAGGA?AAAGG
<210>?3
<211>?46
<212>?DNA
<400>?3
CTTTAACCCCCGAACATCGCCTCGCTCCAGCAGACC?GCTGTTATGC
Claims (4)
1. primer pair and the probe combinations for identifying to contain transgenic pest-resistant rice by recombinase polysaccharase isothermal amplification technique, it is characterized in that: its forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence is as shown in SEQ ID No.2, probe sequence is as shown in SEQ ID No.3, and wherein said paddy rice is extensive No. 1 of China.
2. the test kit that evaluation contains extensive No. 1 of transgenic pest-resistant rice China, is characterized in that: this test kit comprises primer pair claimed in claim 1 and probe combinations.
3. a method of identifying to contain extensive No. 1 of transgenic pest-resistant rice China by recombinase polysaccharase isothermal amplification technique, it is characterized in that: extract the DNA of testing sample as template, utilize the primer pair described in claim 1 and probe combinations is carried out rapid amplifying and real-time fluorescence detects, if obtain obvious amplification curve, prove that institute's sample product contain extensive No. 1 composition of transgenic pest-resistant rice China.
4. method as claimed in claim 3, is characterized in that:
RPA amplification system is: total system 50mL adds rehydration damping fluid 29.5mL, 280mmol/L magnesium acetate solution 2.5mL in the 0.2mL TwistAmp Exo reaction tubes that contains lyophozyme powder, each 2mL of 10mmol/L primer, 10mmol/L probe 0.5mL, template DNA 50ng, residue water is supplied;
Amplification program is: RPA augmentation detection instrument or quantitative real time PCR Instrument were in 39 degrees Celsius of reactions 15 minutes.
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| CN107016258B (en) * | 2016-01-27 | 2020-06-05 | 应清界 | Method for fluorescence quantitative calculation based on recombinase-mediated isothermal nucleic acid amplification (RAA) method |
| CN106755488A (en) * | 2017-01-22 | 2017-05-31 | 中国农业科学院生物技术研究所 | Transgenic corn BT 11 strain specificity is identified using RPA technologies |
| CN106868137B (en) * | 2017-03-01 | 2021-01-26 | 浙江省农业科学院 | Multiple digital PCR quantitative detection method for transgenic rice |
| CN109136339A (en) * | 2018-10-19 | 2019-01-04 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic paddy rice TT51-1 |
| CN109234434A (en) * | 2018-10-19 | 2019-01-18 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic paddy rice G6H1 |
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| CN101240277A (en) * | 2007-02-09 | 2008-08-13 | 中国农业科学院植物保护研究所 | The flanking sequence of the exogenous insert fragment of the transgenic rice line Bt Shanyou 63 |
| CN101302520A (en) * | 2008-07-01 | 2008-11-12 | 中国农业科学院油料作物研究所 | The complete sequence of the exogenous vector integration site of transgenic rice TT51-1 transformation event and its application |
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| CN101240277A (en) * | 2007-02-09 | 2008-08-13 | 中国农业科学院植物保护研究所 | The flanking sequence of the exogenous insert fragment of the transgenic rice line Bt Shanyou 63 |
| CN101302520A (en) * | 2008-07-01 | 2008-11-12 | 中国农业科学院油料作物研究所 | The complete sequence of the exogenous vector integration site of transgenic rice TT51-1 transformation event and its application |
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