CN103451292A - 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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- CN103451292A CN103451292A CN2013103915258A CN201310391525A CN103451292A CN 103451292 A CN103451292 A CN 103451292A CN 2013103915258 A CN2013103915258 A CN 2013103915258A CN 201310391525 A CN201310391525 A CN 201310391525A CN 103451292 A CN103451292 A CN 103451292A
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Abstract
The invention discloses a primer and probe combination which is suitable for identifying transgenic insect-resistant rice Huahui 1 (TT51-1) by using a recombinase polymerase amplification (RPA) technology. A forward primer sequence of the primer and probe combination is shown by SEQ ID No.1, a reverse primer sequence is shown by SEQ ID No.2, and a probe sequence is shown by SEQ ID No.3. Meanwhile, the invention also discloses a method for identifying the transgenic insect-resistant rice TT51-1. The method comprises the following steps: extracting DNA (Deoxyribonucleic Acid) of a sample to be tested as a template; performing RPA rapid amplification and real-time fluorescence detection by using the primer; if an obvious amplification curve is obtained, proving that the tested sample contains the ingredients of the transgenic insect-resistant rice TT51-1. The invention provides an RPA detection method for the specificity of the transgenic insect-resistant rice TT51-1 for the first time.
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 PCR commonly used 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, the nucleic acid constant-temperature amplification technology has obtained significant progress, and comparing the nucleic acid constant-temperature amplification technology with normal PCR does not need expensive PCR instrument, and rapid amplifying goes out the purpose fragment at short notice, has the advantages such as easy, quick, sensitive.The RPA technology is that in the simulation organism, DNA replication dna, the amplification principle polymerase-mediated based on recombinase develop, 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, copied extension under the effect of archaeal dna polymerase, 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.Secondary structure between reaching for fear of formation primer inside during design of primers, the increase of its length also makes design of primers and selects difficulty to increase, and the design of primer sequence and selection are most important to the result of RPA.In the RPA amplification system, add a fluorescently-labeled probe just can realize the Real-Time Monitoring of template amplification, fluorescence group of each mark (FAM and BHQ1) on this two, middle part of probe T base, an abasic site (dSpacer) is arranged between two groups, this site can be identified from colibacillary exonuclease by a kind of, this enzyme has 3 '-5 ' 5 prime excision enzyme activity, can make two fluorescence groups separate, thereby make the accumulation synchronised of fluorescent signal and amplified production.Just can in 10-20 minute, fluorescence curve be detected in conjunction with a portable amplified fluorescence detector.The RPA technology has greatly shortened detection time, has simplified response procedures, combines with DNA rapid extraction technology and makes the field detection become possibility, 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 the connecting zone that inserts DNA sequence dna and rice genome according to external source designs the RPA primer and detects and have 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 the PCR instrument to carry out conventional detection in laboratory, the method can't further meet the rapid detection of transgenic product.Also not utilizing at present the 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, the 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 primer claimed in claim 1 to carry out the 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 centigrade 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 the 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.
The 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 the example explanation.
The experimental technique of unreceipted actual conditions in following embodiment, usually according to normal condition, " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press such as Sambrook etc., 2001) condition described in, or the condition of advising according to instrument or reagent manufacturer.
At first, the design primer: according to TT51-1 transformant distinguished sequence (GenBank No. EU880444) design primer, and probe.RPA requires as 30-35nt primer length, thereby just easily causing producing a large amount of primer dimers under constant temperature, this affects experiment effect, the RPA experiment need to design multipair primer and be optimized from the target sequence two ends, screening, the replacement of indivedual bases or increase and decrease all can produce material impact to experimental result.Designed a large amount of primers in this experiment, and therefrom filtered out a pair of highly sensitive and primer that specificity is good for the 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).
Annotate: 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, get young leaflet tablet as the DNA extraction material, according to TianGen Plant Genomic DNA Kit(Cat#DP-305) operational manual of test kit, carry 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 the deionization distilled water.
(3) primer amplification
The transformant distinguished sequence inserted 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.
The RPA amplification system is: total system 50mL, add rehydration damping fluid 29.5mL in the 0.2mL TwistAmp Exo reaction tubes that contains the lyophozyme powder, magnesium acetate solution 2.5mL(280mmol/L), each 2mL(10mmol/L of primer), probe 0.5mL(10mmol/L), template DNA 50ng, the residue water is supplied;
Primer amplification program: 39 degrees centigrade of reactions of RPA augmentation detection instrument 15 minutes.
3. experimental result
Forward primer and reverse primer according to 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 the RPA fluoroscopic examination, can identify rapidly and accurately transgenic paddy rice TT51-1, wherein at transgenic paddy rice TT51-1, obvious fluorescence curve is arranged, but not all there is no amplification curve (Fig. 1) in the material such as rich No. 6 of transgenic paddy rice bright extensive 63 and transgenic paddy rice section.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. one kind for identifying primer and the probe combinations contain extensive No. 1 of transgenic pest-resistant rice China by recombinase polysaccharase isothermal amplification technique, it is characterized in that: its forward primer sequence is as shown in SEQ ID No.1, the reverse primer sequence is as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
2. the test kit that an evaluation contains extensive No. 1 of transgenic pest-resistant rice China, it is characterized in that: this test kit comprises primer 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 primer claimed in claim 1 and probe combinations to carry out rapid amplifying and real-time fluorescence detection, 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:
To each 2mL of primer that adds 10mmol/L in the 50mL amplification system of RPA amplification kit recommendation response, the probe 0.5mL of 10mmol/L, template DNA 50ng;
Amplification program is: RPA augmentation detection instrument or quantitative real time PCR Instrument were in 39 degrees centigrade of reactions 15 minutes.
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Cited By (5)
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| 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 |
| CN106868137A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Transgenic rice multiple digital pcr quantitative detecting method |
| CN107016258A (en) * | 2016-01-27 | 2017-08-04 | 应清界 | One kind is based on recombinase-mediated isothermal nucleic acid amplification（RAA）The method that method carries out fluorescent quantitation calculating |
| 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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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107016258A (en) * | 2016-01-27 | 2017-08-04 | 应清界 | One kind is based on recombinase-mediated isothermal nucleic acid amplification（RAA）The method that method carries out fluorescent quantitation calculating |
| 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 |
| CN106868137A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Transgenic rice multiple digital pcr quantitative detecting method |
| 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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