CN106995841A - A kind of genetically engineered soybean detection multiple PCR reagent kit and detection method - Google Patents
A kind of genetically engineered soybean detection multiple PCR reagent kit and detection method Download PDFInfo
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- CN106995841A CN106995841A CN201710167334.1A CN201710167334A CN106995841A CN 106995841 A CN106995841 A CN 106995841A CN 201710167334 A CN201710167334 A CN 201710167334A CN 106995841 A CN106995841 A CN 106995841A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses a kind of genetically engineered soybean detection multiple PCR reagent kit and detection method, can the approval of import of the Ministry of Agriculture of quick detection China 12 kinds of genetically engineered soybeans, including MON87701, GTS40 32, MON89788, CV127, A2704, A5547, DP356043, DP305423, MON87769, MON87708,305423 × 40 32, MON87701 × MON89788；Multiple PCR reagent kit disclosed by the invention is based on the multiple PCR method that " consensus primer " is mediated, including the detection of consensus primer, multi-primerses, multiplex PCR system；Double annealing temperature method improves specificity, the sensitiveness of multiplex PCR system, and the multiple PCR method has pardon, can continue increase detection target, be conducive to improving the detection flux of multiplex PCR.The present invention has sensitivity high, economic, quick, simple operation and other advantages, can be applied to food inspection.
Description
Technical field
The invention belongs to food/field of gene detection, and in particular to genetically engineered soybean detection multiple PCR reagent kit and inspection
Survey method.
Background technology
At present, the genetically modified crops detection technique reported both at home and abroad is broadly divided into three classes, and a class is the detection based on nucleic acid
Technology；Two be zymetology and immunology detection technology based on protein；3rd class is detection GMOs new technology, including to automatic
The biology sensor and biochip, near infrared spectrum and analytical technique of mass spectrum based on Modern Analytical Instrument of change technology development
Deng.Detection technique of the Equations of The Second Kind based on protein is that the albumen of exogenous gene expression in genetically modified plants is detected, these
Albumen is vulnerable to destruction and inactivates, decomposes or disappear in process, and the repetition of false negative and method easily occurs in inspection result
Property is poor.The detection GMOs new technology developed in recent years such as biology sensor, chromatography and near infrared spectroscopy etc., such technology
At present still in conceptual phase, have the shortcomings that some temporarily can not effectively be solved or objective reality, such as chromatography is only applicable to
Qualitative detection analysis during great change occurs for the composition of genetically modified plants and products thereof, and such detection technique still can not in a short time
Effective popularization and application.Therefore, the detection of GMOs method standard of the Ministry of Agriculture of China issue, should based on nucleic acid detection technique
Class detection technique includes round pcr, isothermal amplification technology, southern hybridization, biochip technology, quantitative fluorescent PCR etc..
Multiplex PCR is that on the basis of regular-PCR, multistage is expanded simultaneously using multipair primer in same reaction system
The round pcr of target sequence.Since multiplex PCR report, due to its high efficiency, sensitivity and economical and convenient, have been widely used
In the multiple fields of diagnostic nucleic acid, such as diagnosis of genetic disorders, mutation and polymorphism analysis, pathogen detection, transgenosis identification；
Multiplex PCR system, generally comprises following reagent：PCR buffer solutions, Taq enzyme, dNTP, MgCl2, multi-primerses, template and deionization
Water.
China allows the genetically engineered soybean of import to be up to 12 kinds at present（Specifically include GTS-40-3-2, MON87701,
MON89788、MON87701×MON89788、CV127-9、A2704-12、DP356043、DP305423、A5547、305423×
40-3-2, MON87769, MON87708, wherein MON87701 × MON89788 and 305423 × 40-3-2 are that complex character turns base
Because of soybean）.
External source target gene is transferred in plant by genetically modified crops using technique for gene engineering to be cultivated and goes out, and they have
There are the good characteristics such as pest-resistant, antiweed, degeneration-resistant and nutrition improvement.The cultivated area of global genetically modified crops was from 1996
0.17 hundred million hectares increase to 1.797 hundred million hectares in 2015.As transgenic technology is continued to develop, genetically modified crops industrialization with
Commercialized degree is constantly deepened, and China has formulated relevant laws and regulations for genetically modified crops successively, however, the public is to turning base
Because of the worry of food security and to institute's edible food, whether the right to know containing transgene component is still growing day by day, therefore, quick,
Accurately, sensitive GM food detection technique is the base that the laws and regulations such as all transgenosis safe evaluations are really fulfilled
One of plinth and key.
The content of the invention
It is an object of the invention to provide a kind of multiple PCR reagent kit for detecting genetically engineered soybean, ratify at present for China
Import（Safety certificate is issued）12 kinds of genetically engineered soybeans single or simultaneously multiplex PCR detect, it is public by designing
Primer and genetically engineered soybean specific primer, limit multiplex PCR detection architecture and condition, can detect food（Such as soybean, soybean
Product, food containing soybean composition etc.）In whether contain genetically engineered soybean composition.
To achieve the above object of the invention, the technical solution adopted by the present invention is：
A kind of genetically engineered soybean detection multiple PCR reagent kit, including consensus primer, the chimeric primers for genetically engineered soybean；
The sequence of the consensus primer is SEQ ID NO.1；The chimeric primers for genetically engineered soybean be SEQ ID NO.2 extremely
The one or more in nucleotide sequence shown in SEQ ID NO.18.
In above-mentioned technical proposal, the genetically engineered soybean detection with multiple PCR reagent kit also include Taq polymerases,
dNTP、MgCl2, PCR buffer solutions, deionized water, positive control primers pair；The sequence of wherein described positive control primers pair is preferred
For SEQ ID NO.19, SEQ ID NO.20；It can be used to detect DNA profiling quality and reaction system amplification efficiency；
SEQ ID NO.19：GCCCTCTACTCCACCCCCATCC
SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG .
In above-mentioned technical proposal, when entering performing PCR reaction, using double annealing temperature method, preceding 10 cycle annealing temperature are
61℃；30 cycle annealing temperature are 54 DEG C afterwards.First step annealing temperature is higher than 5~10 DEG C of the annealing temperature of second step；Such as
The annealing temperature of preceding 10 circulations is higher than rear 7 DEG C of 30 cycle annealing temperature, can effectively improve the specificity of detection；Carry out multiple
PCR react when, for genetically engineered soybean every chimeric primers final concentration between 8~80nM, the final concentration of consensus primer
For 1600nM.
The invention also discloses a kind of method for detecting product GMOs, product gene group to be detected is extracted
DNA is used as template（The insertion gene of such as genetically engineered soybean is target sequence with the bonding pad of soybean gene group）, draw chimeric
Thing using double annealing temperature method in the presence of consensus primer with entering performing PCR reaction, and it is to complete production that electrophoretic analysis is carried out to PCR primer
The detection of product GMOs；Electrophoretic analysis finally is carried out to PCR primer amplification, that is, completes genetically engineered soybean in product
The detection of composition；The sequence of the consensus primer is SEQ ID NO.1；The chimeric primers are SEQ ID NO.2 to SEQ ID
The one or more in nucleotide sequence shown in NO.18.
In above-mentioned technical proposal, the first step（I.e. preceding 10 circulations）The annealing temperature of multiplexed PCR amplification is higher than second step（I.e.
30 circulations afterwards）5~10 DEG C of the annealing temperature of multiplexed PCR amplification；The final concentration of every chimeric primers is public between 8~80nM
The final concentration of 1600nM of common primer；The product is the food comprising soybean component.Specifically it may include following steps：95 DEG C pre-
It is denatured 5min；95 DEG C of denaturation 30s, 61 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 10 circulations；72 DEG C of extension 2min；95
DEG C denaturation 30s, 54 DEG C annealing 30s, 72 DEG C extension 30s, totally 30 circulation；Last 72 DEG C of extensions 7min.Preceding 10 circulations,
Under high annealing temperature, special primer expands target sequence in the chimeric primers of low concentration, produces with consensus primer end
PCR primer；30 circulations afterwards, under low temperature thermal oxidation, the consensus primer of high concentration is last with consensus primer to produce in early days
The PCR primer at end is that template is largely expanded.The method according to the invention can be in detection product effectively, easy, especially
It is whether to contain genetically engineered soybean composition in food, is that national right to know and food security make tremendous contribution.
Genetically engineered soybean component nucleic acid detection method disclosed by the invention is accurate, target gene in can detecting 12 simultaneously, and
And testing result is accurate, it is possible to resolve the public to the worry of Safety of GM Food and to institute's edible food whether containing transgenosis into
The right to know divided.
The present invention further discloses a kind of genetically engineered soybean detection primer, including it is consensus primer, big for transgenosis
The chimeric primers of beans；The sequence of the consensus primer is SEQ ID NO.1；The chimeric primers for genetically engineered soybean are
The one or more in nucleotide sequence shown in SEQ ID NO.2 to SEQ ID NO.18.
The multiplex PCR examination of detection genetically engineered soybean is being prepared the invention also discloses above-mentioned genetically engineered soybean detection primer
Application of the genetically engineered soybean detection primer described in application or claim 8 in agent box in detection genetically engineered soybean.
In the present invention, the sequence SEQ ID NO.1 of the consensus primer and the nucleotide sequence of the chimeric primers are as follows
It is shown：
The chimeric primers are the chimeric primers for 12 kinds of genetically engineered soybeans；A2704 and A5547 shares sense primer SEQ
ID NO.11, MON89788, MON87769 and MON87708 share anti-sense primer SEQ ID NO.18.305423×40-3-2、
MON87701 × MON89788 is the genetically engineered soybean of two kinds of gene stackings, if detecting genetically engineered soybean simultaneously in sample
DP305423 and GTS40-3-2, then be possible to containing 305423 × 40-3-2 of genetically engineered soybean in sample；If examined in sample simultaneously
Measure genetically engineered soybean MON87701 and MON89788, then be possible in sample containing genetically engineered soybean MON87701 ×
MON89788。
In above-mentioned technical proposal, consensus primer and genetically engineered soybean related gene group are without homology, when with genetically engineered soybean
, all should be without specific product no matter condition when genome is that template is expanded；When with the amplified production of chimeric primers
During for template, consensus primer can amplify specific product；And the Tm of consensus primer draws than genetically engineered soybean target-specific
The Tm values of thing are low 5~10 DEG C.
In above-mentioned technical proposal, when entering performing PCR reaction, the final concentration of 1600nM of consensus primer, for genetically engineered soybean
Chimeric primers final concentration of 8~80nM, for expand 12 kinds of genetically engineered soybean MON87701, GTS40-3-2,
MON89788、CV127、A2704、A5547、DP356043、DP305423、MON87769、MON87708、305423×40-3-
2nd, MON87701 × MON89788, such as SEQ ID NO.2/3:80nM, SEQ ID NO.4/5:60nM, SEQ ID NO.6/
7:64nM, SEQ ID NO.8/9:8nM, SEQ ID NO.10:32nM, SEQ ID NO.11:48nM, SEQ ID
NO.12:16nM, SEQ ID NO.13/14:24nM, SEQ ID NO.15/16:8nM, SEQ ID NO.17: 16nM,
SEQ ID NO.18: 32nM；When entering performing PCR reaction, using double annealing temperature method, first step annealing temperature is moved back than second step
Fiery temperature is high 5~10 DEG C.
Kit disclosed by the invention can be used for the detection of product especially food GMOs, and the present invention is public
The multiplex PCR opened is the multiplex PCR combination double annealing temperature method of consensus primer mediation.Specifically such as use double annealing temperature
Method, is connected region sequence with soybean gene as target sequence using genetically engineered soybean insertion gene order, 10 is first expanded with chimeric primers
Individual circulation, annealing temperature is 61 DEG C, carries out first step multiplexed PCR amplification, can produce the PCR primer of consensus primer end；It is public
The PCR primer with consensus primer end that primer is expanded using the first step is template, and the second steps of 30 circulations are multiple after progress
PCR is expanded, and annealing temperature is 54 DEG C；Complete the multiplexed PCR amplification of genetically engineered soybean；The multiplexed PCR amplification of preceding 10 circulations
Annealing temperature is higher 5~10 DEG C than the multiplexed PCR amplification annealing temperature of 30 below circulations.Circulation early stage, in high annealing temperature
Under, the chimeric primers amplification target sequence of low concentration produces the PCR primer with consensus primer end；The later stage is circulated, is moved back low
At fiery temperature, the consensus primer of high concentration is largely expanded using the PCR primer produced in early days as template.What multiplex PCR was used
25 μ L reaction systems include：10 μM of the μ L of consensus primer 4, chimeric primers mixture 2 μ L, 2 × Dream Taq of various concentrations
The μ L of Green Mix 12.5, the μ L of DNA profiling 1, sterilize ddH2O 4.5μL；PCR reaction conditions are：95 DEG C of pre-degeneration 5min；95
DEG C denaturation 30s, 61 DEG C annealing 30s, 72 DEG C extension 30s, totally 10 circulation；72 DEG C of extension 2min；95 DEG C are denatured 30s, 54
DEG C annealing 30s, 72 DEG C extension 30s, totally 30 circulation；Last 72 DEG C of extensions 7min.Finally enter row agarose gel electrophoresis point
Analysis, as a result shows that the multiple PCR reagent kit of the detection genetically engineered soybean of the present invention can simultaneously or independently 10 kinds of specific detection
The genome of genetically engineered soybean；Using the plasmid template of structure, the multiple PCR reagent kit of detection genetically engineered soybean of the invention
Sensitiveness reaches as high as 0.1%（w/w）Genetically engineered soybean component content, i.e. 0.1ng/100ngDNA samples.
Because above-mentioned technical proposal is used, the present invention has advantages below compared with prior art：
（1）It is disclosed by the invention detection genetically engineered soybean multiple PCR reagent kit, can simultaneously also individually detect China ratify into
Mouthful 12 kinds of genetically engineered soybeans, i.e. MON87701, GTS40-3-2, MON89788, CV127, A2704, A5547,
DP356043, DP305423, MON87769, MON87708,305423 × 40-3-2, MON87701 × MON89788, not only may be used
To save amount of reagent and reduction testing cost, while detection efficiency is improved, economic, quick, operation high with sensitivity
Simple the advantages of；
（2）When kit disclosed by the invention is detected, chimeric primers concentration, most as little as 8nM/ every, consensus primer are reduced
Concentration is 1600nM；The concentration of reduction chimeric primers is conducive to addition more to chimeric primers, improves multiplex PCR detection flux；
Using wall scroll consensus primer, the formation of dimer is advantageously reduced, high concentration is conducive to the amount of specific amplification product, Jin Erti
High detection sensitiveness；
（3）Kit disclosed by the invention is used using the double annealing temperature method in a PCR reaction, preceding 10 cyclic amplifications
High annealing temperature（61℃）Be conducive to the specific binding of chimeric primers, reduce nonspecific products；30 cyclic amplifications are low afterwards
Annealing temperature（54℃）Consensus primer is set to play amplification；Middle 72 DEG C of 2 min of extension of extra increase, are conducive to drawing to be public
Thing obtains enough templates；
（4）In kit disclosed by the invention, consensus primer is connected with the 5' ends of every multiplex PCR special primer, together structure
Into chimeric primers；The multiplex PCR of consensus primer mediation can reduce the concentration of primer pair, reduce the formation of dimer, so that
Improve the detection flux of multiplex PCR；The non-specific expansion that existing multiplex PCR system is present is overcome by creative design
Increasing, the defect that easily there is dimer；
（5）The multiplex PCR detection architecture specificity of the present invention is high, and sensitiveness is up to 0.1%（w/w）(genetically engineered soybean composition contains
Amount), i.e. 0.1ng/100ngDNA samples；Single tube is carried out, simple to operate, and has the advantages that sensitivity is high, economical, quick, is had
There is actual application value；
（6）Multiplex PCR (mutiplex PCR) disclosed by the invention detects multiple target molecules simultaneously in a PCR pipe, than
Southern hybridization, method for gene chip are more simple and efficient, more more economical than quantitative fluorescent PCR, with isothermal amplification technology（Including
Ring mediated isothermal amplification method）Compared to be difficult pollution and high flux.High efficiency, sensitiveness and the economy letter having by the technology
Just the advantages of property, it can be received significant attention in nucleic acid detection technique.
Brief description of the drawings
Fig. 1 is the specific detection electrophoretogram of consensus primer of the present invention；
Fig. 2 is consensus primer susceptibility results figure of the present invention；
Fig. 3 is the electrophoresis result figure of the specific detection of multiple system of the invention；
Fig. 4 is present invention detection genetically engineered soybean sequencing result figure；
Fig. 5 is the susceptibility results figure of Multiple detection system of the present invention.
Embodiment
With reference to embodiment, the invention will be further described with accompanying drawing
Embodiment
1. the sensitiveness of screening and its detection of consensus primer
Consensus primer directly affects the success or failure of technology as the core technology in whole technical system, its quality.This hair
Bright used consensus primer SEQ ID NO.1 enter performing PCR amplification with genetically engineered soybean and Non-transgenic soybean genomic DNA,
In 45 DEG C of annealing temperature gradient -- amplifying specific product, such as Fig. 1 are unable in the range of 65 DEG C, a-k is consensus primer and transgenosis
Soybean and Non-transgenic soybean genomic DNA amplification result, as a result show, consensus primer is unable to amplifying specific product, wherein,
Swimming lane 0 is blank control, and swimming lane M is 100bp DNA Marker, and swimming lane 1-8 is 45 DEG C of annealing temperature gradient -- 65 DEG C；A templates
For Non-transgenic soybean genomic DNA, b templates are GTS 40-3-2 genomic DNAs, and c templates are A2704-12 genomic DNAs, d
Template is MON89788 genomic DNAs, and e templates are DP356043 genomic DNAs, and f templates are DP305423 genomic DNAs, g moulds
Plate is CV127-9 genomic DNAs, and h templates are MON87701 genomic DNAs, and i templates are A5547-127 genomic DNAs, j templates
For MON87708 genomic DNAs, k templates are MON87769 genomic DNAs.
By the sensitiveness of the recombinant plasmid template of structure, further detection consensus primer SEQ ID NO.1 detections, it is examined
Survey sensitiveness and reach 100 copies, such as Fig. 2, swimming lane M is 100bp DNA marker, and swimming lane 0 is blank control, swimming lane 1-8
The corresponding plasmid copy number of template is followed successively by 101、102、103、104、105、106、107、108。
In the present invention, when being expanded using genetically engineered soybean and Non-transgenic soybean genomic DNA as template, no matter expanding
How increasing condition optimizes, all should be without amplified production；When the multiple chimeric primers with consensus primer have amplified production, pass through
Consensus primer can necessarily amplify specific product.
2. the multiple PCR reagent kit of genetically engineered soybean is detected, including conventional multiplex PCR component, in addition to for this 10 kinds
The chimeric primers and consensus primer of genetically engineered soybean；Chimeric primers and consensus primer sequence are shown in Table 1, conventional multiplex PCR component bag
Include Taq polymerases, dNTP, MgCl2, PCR buffer solutions, deionized water, positive control primers pair；Positive control primers are to available
To detect DNA profiling quality and reaction system amplification efficiency, particular sequence is：SEQ ID NO.19：
GCCCTCTACTCCACCCCCATCC、
SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG.
Table 1 is directed to the 12 kinds of genetically engineered soybean chimeric primers and consensus primer of China's import
3. the specific detection of multiple system
Using the PCR method of many primer+consensus primer+single mode plates, the genome or target of various detection genetically engineered soybeans are expanded
Gene, DNA profiling is using the corresponding 10 kind transgenic soybean gene group DNA extracted, and concentration is about 25ng/ μ L.It is multiple
The 25 μ L reaction systems that PCR is used include：10 μM of the μ L of consensus primer 4, different final concentrations（SEQ ID NO.2/3:80nM, SEQ
ID NO.4/5:60nM, SEQ ID NO.6/7:64nM, SEQ ID NO.8/9:8nM, SEQ ID NO.10:32nM, SEQ
ID NO.11:48nM, SEQ ID NO.12:16nM, SEQ ID NO.13/14:24nM, SEQ ID NO.15/16:8nM,
SEQ ID NO.17: 16nM, SEQ ID NO.18: 32nM）μ L, the 2 × Dream Taq Green of chimeric primers mixture 2
The μ L of Mix 12.5, the μ L of DNA profiling 1, sterilize ddH2O 5.5μL；PCR reaction conditions are：95 DEG C of pre-degeneration 5min；95 DEG C of changes
Property 30s, 61 DEG C annealing 30s, 72 DEG C extension 30s, totally 10 circulation；72 DEG C of extension 2min；95 DEG C of denaturation 30s, 54 DEG C are moved back
Fiery 30s, 72 DEG C of extension 30s, totally 30 circulations；Last 72 DEG C of extensions 7min.Fig. 3 is the electricity of the specific detection of multiple system
Swimming result figure, wherein 0 is blank control, N is negative control, and 1-10 swimming lanes are the agarose gel electrophoresis results of PCR primer, according to
It is secondary for MON87701, DP356043, DP305423, CV127, MON87769, A2704, GTS40-3-2, MON87708,
MON89788, A5547, N are negative control, and M is DNA standard molecular weights（From top to bottom purpose band size be followed successively by 100,200,
300th, 400,500,600,700,800,900 and 1000bp）, PM is product marker, and stripe size, which is shown on Fig. 3, to be indicated；As a result
It has been shown that, detects target DNA with the multiple system of the present invention, the specificity of expected size can be amplified from corresponding template
PCR primer, and band is clear.Amplified production is through further sequencing, it was demonstrated that be corresponding genetically engineered soybean detection sequence（Figure
4）.This part test result indicates that, multiple system of the invention has high specific.
4. the sensitivity Detection of Multiple detection system
System sensitivity Detection is using 10 kinds of genetically engineered soybeans A2704-12, MON89788, CV127, MON87701, A5547-
127th, MON87708, MON87769, DP356043, DP305423, GTS 40-3-2 Genomic DNA solutions are mixed in equal volume（Its
Middle DP356043, DP305423 and GTS40-3-2 initial concentration is 2.5ng/ μ L, and remaining 7 kinds of initial concentration is 25ng/ μ L）,
Then by this mixed genomic DNA solution using TE buffer solutions carry out 10 times of gradient dilutions, successively obtain 10,100,1000 and
10000 times of genomic DNAs dilute mixed liquor, and every kind of dilution takes 4 μ L as template（Every kind of transgenic soybean gene group DNA moulds
Plate concentration is respectively 10ng, 1ng, 0.1ng, 0.01ng, and this 3 kinds of DP356043, DP305423, GTS40-3-2 is corresponding dense
Degree is respectively 1ng, 0.1ng, 0.01ng, 0.001ng）, template grads PCR reaction, ddH are carried out to 10 target genes respectively2O
Negative control is used as template.Multiplex PCR is included using 25 μ L reaction systems：10 μM of the μ L of consensus primer 4,1 μM for corresponding
The chimeric primers of kind genetically engineered soybean are to 2 μ L, 2 × Dream Taq Green Mix of mixture 12.5 μ L, the μ of DNA profiling 4
L, sterilize ddH2O 2.5μL；PCR reaction conditions are：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 61 DEG C of anneal 30s, 72
DEG C extension 30s, totally 10 circulation；72 DEG C of extension 2min；95 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 30s, altogether
30 circulations；Last 72 DEG C of extensions 7min.PCR reaction products carry out electrophoresis, gel imaging system point by 2% Ago-Gel
It is 100bpDNA Ladder to analyse result such as Fig. 5, swimming lane M, and swimming lane 0 is the blank negative control that deionized water is template, 1~4 point
Not Wei genomic DNA template be respectively that dilution 10000,1000,100 and 10 times of 10 kinds of transgenic soybean gene group DNA dilution are mixed
Close liquid.As a result show, 1000 times 10 kinds of the detectable dilution of sensitiveness of the multiplex PCR for the consensus primer mediation that the present invention is set up turns
Transgenic soybean genomic DNA dilutes mixed liquor, and system sensitiveness is more than 0.1%, better than country for GM food examination criteria
It is required that.
SEQUENCE LISTING
<110>University Of Suzhou
<120>A kind of genetically engineered soybean detection multiple PCR reagent kit and detection method
<160> 20
<210> 1
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 1
CTCGTCAACTCCGCAAG 17
<210> 2
<211> 42
<212> DNA
<213>It is artificial synthesized
<400> 2
CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG 42
<210> 3
<211> 40
<212> DNA
<213>It is artificial synthesized
<400> 3
CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT 40
<210> 4
<211> 37
<212> DNA
<213>It is artificial synthesized
<400> 4
CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG 37
<210> 5
<211> 39
<212> DNA
<213>It is artificial synthesized
<400> 5
CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG 39
<210> 6
<211>42
<212> DNA
<213>It is artificial synthesized
<400> 6
CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA 42
<210> 7
<211> 42
<212> DNA
<213>It is artificial synthesized
<400> 7
CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA 42
<210> 8
<211> 41
<212> DNA
<213>It is artificial synthesized
<400> 8
CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG 41
<210> 9
<211> 39
<212> DNA
<213>It is artificial synthesized
<400> 9
CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT 39
<210> 10
<211> 43
<212> DNA
<213>It is artificial synthesized
<400> 10
CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG 43
<210> 11
<211> 40
<212> DNA
<213>It is artificial synthesized
<400> 11
CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC 40
<210> 12
<211> 42
<212> DNA
<213>It is artificial synthesized
<400> 12
CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG 42
<210> 13
<211> 39
<212> DNA
<213>It is artificial synthesized
<400> 13
CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC 39
<210> 14
<211> 39
<212> DNA
<213>It is artificial synthesized
<400> 14
CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT 39
<210> 15
<211> 42
<212> DNA
<213>It is artificial synthesized
<400> 15
CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC 42
<210> 16
<211>42
<212> DNA
<213>It is artificial synthesized
<400> 16
CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT 42
<210> 17
<211> 43
<212> DNA
<213>It is artificial synthesized
<400> 17
CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG 43
<210> 18
<211> 36
<212> DNA
<213>It is artificial synthesized
<400> 18
CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG 36
<210> 19
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 19
GCCCTCTACTCCACCCCCATCC 22
<210> 20
<211> 23
<212> DNA
<213>It is artificial synthesized
<400> 20
GCCCATCTGCAAGCCTTTTTGTG 23
Claims (10)
1. a kind of genetically engineered soybean detection multiple PCR reagent kit, it is characterised in that：The genetically engineered soybean is detected with multiple
PCR kit includes consensus primer, the chimeric primers for genetically engineered soybean；The sequence of the consensus primer is SEQ ID
NO.1；The chimeric primers for genetically engineered soybean are the nucleotide sequence shown in SEQ ID NO.2 to SEQ ID NO.18
In one or more：
SEQ ID NO.1：CTCGTCAACTCCGCAAG
SEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG
SEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT
SEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG
SEQ ID NO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG
SEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA
SEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA
SEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG
SEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT
SEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG
SEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC
SEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG
SEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC
SEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT
SEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC
SEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT
SEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG
SEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG.
2. genetically engineered soybean detection multiple PCR reagent kit according to claim 1, it is characterised in that：The transgenosis is big
Beans detection also includes Taq polymerases, dNTP, MgCl with multiple PCR reagent kit2, PCR buffer solutions, deionized water, positive control
Primer pair.
3. genetically engineered soybean detection multiple PCR reagent kit according to claim 2, it is characterised in that：The positive control
The sequence of primer pair is SEQ ID NO.19, SEQ ID NO.20；
SEQ ID NO.19：GCCCTCTACTCCACCCCCATCC
SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG .
4. genetically engineered soybean detection multiple PCR reagent kit according to claim 1, it is characterised in that：Enter performing PCR reaction
When, using double annealing temperature method, first step annealing temperature is higher than 5~10 DEG C of second step annealing temperature；When entering performing PCR reaction,
For genetically engineered soybean every chimeric primers final concentration between 8~80nM, the final concentration of 1600nM of consensus primer.
5. a kind of method for detecting product GMOs, it is characterised in that extract product gene group DNA to be detected and make
For template, performing PCR reaction is entered using double annealing temperature method in the presence of chimeric primers and consensus primer, PCR primer is carried out
Electrophoretic analysis is the detection for completing product GMOs；
The sequence of the consensus primer is SEQ ID NO.1；The chimeric primers are SEQ ID NO.2 to SEQ ID NO.18 institutes
One or more in the nucleotide sequence shown：
SEQ ID NO.1：CTCGTCAACTCCGCAAG
SEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG
SEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT
SEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG
SEQ ID NO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG
SEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA
SEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA
SEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG
SEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT
SEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG
SEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC
SEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG
SEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC
SEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT
SEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC
SEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT
SEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG
SEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG.
6. the method for product GMOs according to claim 5, it is characterised in that：First step annealing temperature is high
In 5~10 DEG C of the annealing temperature of second step；The final concentration of every chimeric primers is between 8~80nM, the final concentration of consensus primer
For 1600nM；The product is soy food product.
7. the multiplexed PCR amplification method of genetically engineered soybean according to claim 5, it is characterised in that：The multiplex PCR expands
Increase and use 25 μ L reaction systems；Specifically PCR reaction conditions are：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 61 DEG C of annealing
30s, 72 DEG C of extension 30s, totally 10 circulations；72 DEG C of extension 2min；95 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extensions
30s, totally 30 circulations；Last 72 DEG C of extensions 7min.
8. genetically engineered soybean detection primer, it is characterised in that：The genetically engineered soybean detection with primer include consensus primer,
For the chimeric primers of genetically engineered soybean；The sequence of the consensus primer is SEQ ID NO.1；It is described for genetically engineered soybean
Chimeric primers are the one or more in the nucleotide sequence shown in SEQ ID NO.2 to SEQ ID NO.18：
SEQ ID NO.1：CTCGTCAACTCCGCAAG
SEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG
SEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT
SEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG
SEQ ID NO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG
SEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA
SEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA
SEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG
SEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT
SEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG
SEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC
SEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG
SEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC
SEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT
SEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC
SEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT
SEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG
SEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG.
9. genetically engineered soybean detection primer described in claim 8 is in the multiple PCR reagent kit for preparing detection genetically engineered soybean
Application or claim 8 described in genetically engineered soybean detection primer detection genetically engineered soybean in application.
10. genetically engineered soybean detection multiple PCR reagent kit described in claim 1 is in detection bean product GMOs
In application.
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