CN106755557A - A kind of transgenic wheat and the detection method of cotton - Google Patents
A kind of transgenic wheat and the detection method of cotton Download PDFInfo
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Abstract
The present invention provides the detection method of a kind of transgenic wheat and cotton, wherein the sequence of the primer combination of probe thing for using respectively SEQ ID NO:1‑18.The present invention uses real-time fluorescence PCR technology, and DNA molecular can be marked, and is capable of achieving to wheat, and cotton detects the quick and precisely detection of gene.In the design aspect of primed probe, the present invention between the primer and its primer of designed six pairs to having carried out analysis below：Interference between primer, hairpin structure and dimer.And the BLAST functions by NCBI carry out Preliminary Identification to its specificity.Through screening, the single-stranded primer that can form secondary structure that there is complementary series, amplified production between primer itself and primer is eliminated, finally filter out the six pairs of primer combination of probe that can be met and be reacted in same reaction system.
Description
Technical field
The invention belongs to detection GMOs technical field, and in particular to the detection method of a kind of transgenic wheat and cotton.
Background technology
Since nineteen eighty-three world's the first genetically modified plants appearance, genetically modified plants are gradually increased for commercialization, are thus derived
GM food out is also more and more.GM food is to be produced and processed by genetically modified organism to be formed.From human body peace
From the point of view of in all directions, perhaps certain transfer of gene can produce new toxin and anaphylactogen, cause unexpected poisoning or allergy
Reaction, some nutritional ingredients of this food or nutritional quality make human body certain illness occur there may be change, and disease can
There can be the incubation period of some time, and harm of the toxicant to human body is also required to a process for accumulating and could realize.Therefore
Its safety issue causes global common concern.China also formulates for this《Agriculture GMO bio-safety manages bar
Example》, and consideration carries out tag identifier to GM food.Indicating label are pasted to GM food, transgenosis and non-transgenic is distinguished
Food, or the number of food transfer gene content is any limitation as, this is required for accurate and effective detection method.With turn
The continuous marketization of gene prod, sets up effective detection method, be further strengthen China in terms of GM food supervision
Important technology guarantee.Conventional transgenic detection method mainly has two major classes：One class is the detection of protein level, such as enzyme-linked
Immunization (ELISA method) and colloid gold test paper method.Another kind of is the detection of nucleic acid level, such as PCR qualitative methods and real-time fluorescence
Quantifying PCR method.With the research and constantly appearance of transgenosis new varieties, it is still necessary to deeply, expand and carry out GM food inspection
The research of survey technology, sets up new, practical, high-throughout detection method.Particularly in the feelings known nothing transgene background
, it is necessary to improve to the various candidates target to be checked operating efficiency that examination is detected one by one under condition.
The content of the invention
It is an object of the invention to provide a kind of transgenic wheat and the detection method of cotton, that is, use primer combination of probe thing
Come in quick detection wheat, cotton whether contain foreign gene, so as to make up the deficiencies in the prior art.
Present invention firstly provides a kind of primer combination of probe thing detected for wheat and cotton transgenic, wherein including
Have:
The primer and probe of endogenous Wx012 genes are detected, its sequence is respectively SEQ ID NO:1-3；
The primer and probe of external source bar genes are detected, sequence is respectively SEQ ID NO:4-6；
The primer and probe of external source no genes are detected, sequence is SEQ ID NO:7-9；
The primer and probe of endogenous Acp1 genes are detected, its sequence is respectively SEQ ID NO:10-12；
The primer and probe of external source Mon531 genes are detected, its sequence is respectively SEQ ID NO:13-15；
The primer and probe of external source Mon15985 genes are detected, its sequence is respectively SEQ ID NO:16-18；
5 ' ends of each gene specific probe described above are modified with reporter group, and 3 ' ends are modified with quenching group.
Reporter group includes FAM, FITC, JOE, CY3, CY5, ROX, and quenching group includes TAMRA, BHQ,
Eclipse etc..
Above-mentioned primer combination of probe thing can also be used to prepare the product of wheat and cotton transgenic detection.
Another aspect of the present invention provides a kind of discriminating wheat, the detection method of cotton transgenic, including following step
Suddenly：
1) genomic DNA of sample to be checked is extracted,
2) by step 1) extract genomic DNA be added to differentiate wheat, cotton transgenic detection reaction system in, profit
Detected with sixfold quantitative real-time PCR, whether interpretation detects transgene component；Above-mentioned primer is with the addition of in reaction system
And probe groups；
3) fluorescence signal in PCR amplification procedures is collected, whether foreign gene is contained in differentiating sample by fluorescence signal；
Above-mentioned steps 2) reaction condition it is as follows：95 DEG C of 3min, 1 circulation.95 DEG C of 15s, 60 DEG C of 60s, 40 circulations.
In the above method, whether interpretation in the following manner detects transgene component：
1) endogenous gene detection Ct values are less than or equal to 36 in test sample, and foreign gene detection Ct values are more than or equal to 40,
Judge that the sample is free of and examine foreign gene.
2) endogenous gene detection Ct values are less than or equal to 36 in test sample, and foreign gene detection Ct values are less than or equal to 40,
Judge that the sample contains and examine foreign gene.
3) endogenous gene detects that Ct values between 36~40, should adjust template concentrations, real-time fluorescence of reforming in test sample
PCR, the foreign gene Ct values after expanding again are still less than 40, and negative control, and positive control and blank result are normal then
Can determine that as the sample is examined foreign gene.If the foreign gene Ct values after expanding again are still greater than or equal to 40, and cloudy
Property control, positive control and blank result normally then can determine that as the sample does not detect foreign gene.
The present invention uses real-time fluorescence PCR technology, and DNA molecular can be marked, and is capable of achieving to wheat, and cotton is detected
The quick and precisely detection of gene.In the design aspect of primed probe, this experiment is between the primer and its primer of designed six pairs
Analysis below is carried out：Interference between primer, hairpin structure and dimer.And it is specific to it by the BLAST functions of NCBI
Carry out Preliminary Identification.Through screening, the single-stranded energy formation that there is complementary series, amplified production between primer itself and primer is eliminated
The primer of secondary structure, finally filters out the six pairs of primer combination of probe that can be met and be reacted in same reaction system.The present invention
Method by the detection to its sensitivity, when fluorescent quantitation DNA profiling consumption is 0.01ng, the specific probe of each gene is still
There is amplification curve, it is 0.01ng to be capable of achieving sensitivity.By the checking of replica test, the present invention to 3 times of each sample solely
The standard variance of the vertical Ct values for repeating experiment is respectively less than 0.5, illustrates the reproducible of testing result, and the stability of detection is high.
Specific embodiment
Applicant is detected the sequence difference of cls gene in analyzing wheat, cotton, and analyzes the interference between primer, hair fastener knot
Structure and dimer；Screening and optimizing corresponding primer and probe, so as to facilitating the present invention.
In following embodiments, experiment material used, reagent is as follows with instrument：
(1) experiment material
Non- transgene cotton, turn Mon531 genes cotton, turn Mon15985 genes cotton, non-transgenic wheat, turn bar bases
Because wheat, turn no DNA triticums, non-genetically engineered soybean, non-transgenic corns, Chinese cabbage, spinach, pork, mutton, duck, the flesh of fish,
Rabbit meat, donkey meat, chicken, goose.
(2) agents useful for same
Animal tissue's genome DNA extracting reagent kit (Tiangeng board), plant genome DNA extracts kit (Tiangeng board),
The Premix Ex Taq of TAKARA, thermal starting enzyme, dNTP, Mg2+Deng PCR reaction reagents.Primer must synthesize and DNA with probe
Sequencing is completed by raw work bioengineering (Dalian) Co., Ltd.
(3) laboratory apparatus
ABI ViiA7 quantitative real time PCR Instruments, supercentrifuge, semi-automatic instrument for extracting nucleic acid, metal bath, ultramicron light splitting
Photometer.
Embodiment 1：Design primed probe
Wheat endogenous Wx012 genes, external source bar genes, no genes, the endogenous Acp1 genes of cotton, external source are directed to respectively
Mon531 genes, external source Mon15985 genes, using PrimerExpress3.0 Software for Design, RTi-PCR primers and Taq man
Probe sequence, uses fluorophor FAM, FITC, JOE, CY3, CY5, ROX respectively.As the luminophore of probe, with corresponding
The different wave length section of quantitative real time PCR Instrument is for detecting.Analysis below is carried out to designed primer：Interference between primer, hair
Card structure and dimer.And the BLAST functions by NCBI carry out Preliminary Identification to its specificity.Through screening, primer is eliminated
There is the single-stranded primer that can form secondary structure of complementary series, amplified production between itself and primer, select and remain as shown in table 1
Primer and probe sequence.
Primer and probe sequence such as following table：
Substance real-time fluorescence quantitative PCR uses 25 μ L reaction systems：0.25 μ L (5U/ μ L) Taq enzyme, 2.5 10 × PCR of μ L
Buffer, 2 μ L dNTPs mixtures, 2.5 μ LMgcl2(25mM), forward and reverse each 1 μ L of primer, the μ L of probe 2, the μ L of template DNA 2
(DNA concentration is 50ng/ μ L), plus distilled water complements to 25 μ L.Added primer final concentration is 0.2pmol/ μ L.
Embodiment 2：Detection method
Base is turned using primed probe of the invention and multiple real time fluorescence quantifying PCR detection method detection wheat and cotton
Because the method and step of situation is as follows：
1st, DNA is extracted：
Take sample to be tested 50g liquid nitrogen to be fully ground, therefrom taking 50mg carries out DNA extractions, can use extracting genome DNA reagent
Box is extracted, and the concentration and purity for carrying DNA is detected with ultramicrospectrophotometer, it is desirable to which OD values are between 1.7-1.8.
2nd, the real-time fluorescence quantitative PCR amplification of testing sample DNA
(1) foundation of substance real-time fluorescence quantitative PCR system and condition optimizing
With the Premix Ex Taq reagents of TAKARA as reaction system, different annealing temperatures are selected：
PCR reaction conditions one are：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 15s, 1min is extended after 55 DEG C of annealing.
PCR reaction conditions two are：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 15s, 1min is extended after 60 DEG C of annealing.
PCR reaction conditions three are：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 15s, 1min is extended after 62 DEG C of annealing.
Each reaction system collects fluorescence, 40 circulations in annealing stage.Then to six primed probes of gene
Concentration is optimized, and primer concentration optimization range is 0.1 μm of ol/L-1.0 μm of ol/L, and gradient is 0.1 μm of ol/L.Concentration and probe concentration is excellent
Change scope is 0.1 μm of ol/L-1.0 μm of ol/L, and gradient is 0.1 μm of ol/L.Ct values and △ Rn values according to amplification curve are selected
Suitable primer and concentration and probe concentration are combined.
When annealing temperature is 55 DEG C, six genes do not occur smooth amplification curve, when annealing temperature is 62 DEG C,
It is relatively low that △ Rn values occur in no genes, Mon531 genes, the phenomenon such as Ct values increase.Therefore the PCR reaction conditions selected by the present invention
For：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 15s, extend 1min after 60 DEG C of annealing, annealing stage collects fluorescence, 40 circulations.
Under this reaction condition, six genes can amplify that Ct values are relatively low, the result that △ Rn values are maximum and amplification curve is smooth.
Through comparing Ct values, △ Rn values and amplification curve.Finally the primed probe concentration range of six genes of determination is：
The primer concentration of Wx012 genes is 0.2 μm of ol/L-0.6 μm of ol/L, and concentration and probe concentration is 0.2 μm of ol/L-0.3 μm of ol/L；Bar bases
The primer concentration of cause is 0.2 μm of ol/L-0.5 μm of ol/L, and concentration and probe concentration is 0.2 μm of ol/L-0.4 μm of ol/L；The primer of no genes
Concentration is 0.2 μm of ol/L-0.6 μm of ol/L, and concentration and probe concentration is 0.2 μm of ol/L-0.5 μm of ol/L；The primer concentration of Acp1 genes is
0.3 μm of ol/L-0.6 μm of ol/L, concentration and probe concentration is 0.2 μm of ol/L-0.5 μm of ol/L.The primer concentration of Mon531 genes is 0.2 μ
Mol/L-0.5 μm of ol/L, concentration and probe concentration is 0.3 μm of ol/L-0.6 μm of ol/L.The primer concentration of Mon15985 genes is 0.3 μ
Mol/L-0.7 μm of ol/L, concentration and probe concentration is 0.2 μm of ol/L-0.4 μm of ol/L.PCR reaction systems are the Premix Ex of TAKARA
The μ L of taq reagents 12.5, each primer combination of probe of respective amount, the μ L of template 2 (DNA concentration is 50ng/ μ L), plus without RNase water
To 25 μ L.
The foundation of multiple real time fluorescence quantifying PCR and condition optimizing
The optimization of 3.1 primers, concentration and probe concentration
PCR reaction systems are the μ L of Premix Ex Taq reagents 12.5 of TAKARA, with the wheat containing institute's transgenosis and cotton
Carried DNA carries out multiple real time fluorescence quantifying PCR experiment for template, and amount of each genomic DNA in PCR reaction systems is
100ng, each primer combination of probe of respective amount, plus without RNase water to 25 μ L.PCR reaction conditions are：95 DEG C of predegenerations
3min, 95 DEG C of denaturation 15s, extend 1min after 60 DEG C of annealing, annealing stage collects fluorescence, 40 circulations.In substance real-time fluorescence
On the basis of quantitative PCR, take rational primed probe concentration combination and optimize.First determine wx012 genes and bar genes most
Good primed probe concentration, then the primed probe optium concentration of no genes is determined based on this, set up triple real time fluorescent quantitatives
PCR reaction systems.Then determine that the optimal primed probe of acp1 genes is dense on the basis of triple real-time fluorescence quantitative PCRs herein
Degree, sets up quadruple real-time fluorescence quantitative PCR reaction system.Determine Mon531 on the basis of this quadruple real-time fluorescence quantitative PCR
The optimal primed probe concentration of gene, sets up five heavy real-time fluorescence quantitative PCR reaction systems.Last five weight real-time fluorescences herein are determined
The optimal primed probe concentration of Mon15985 genes is determined on the basis of amount PCR.Set up the sixfold reality of detectable cotton and wheat
When quantitative fluorescent PCR reaction system.In the concentration of optimizational primer probe, upstream and downstream primer concentration all same is selected, with Ct values
Minimum primer and concentration and probe concentration are mark when minimum, △ Rn values maximum and amplification curve are smoothed, have obvious logarithmic phase and plateau
Standard carries out choosing optimal primed probe concentration.
Find that wx012 genes and bar genes can be expanded simultaneously in same reaction system through experiment.When wx012 genes
Primer concentration is 0.5 μm of ol/L, and concentration and probe concentration is 0.3 μm of ol/L, and bar gene primers concentration is 0.3 μm of ol/L, and concentration and probe concentration is
During 0.3 μm of ol/L, both Ct values are relatively low and △ Rn values are higher, have smooth amplification curve and obvious logarithmic phase and platform
Phase；Ct values and △ Rn the value differences opposite sex of two genes are minimum simultaneously, and amplification efficiency is closest, it is thus determined that for optimal dual is glimmering in real time
The primed probe concentration of Fluorescent Quantitative PCR.When add no genes primed probe when, in primer concentration be 0.3 μm of ol/L, probe
Concentration be 0.4 μm of ol/L locate, three genes in same reaction △ Rn value differences the opposite sex minimum, and wx012 genes and bar genes Ct
Value and △ Rn values and amplification curve with it is dual when it is most like.When add acp1 genes primed probe when, in primer concentration be 0.3
μm ol/L, concentration and probe concentration is that 0.4 μm of ol/L locates, and Ct values are relatively low and △ Rn values are higher, have smooth amplification curve and significantly right
Number phase and plateau；And the Ct values and △ Rn values and amplification curve of wx012 genes and bar genes and no genes with it is triple when most
It is similar.It is 0.2 μm of ol/L in primer concentration when the primed probe of Mon531 genes is added, concentration and probe concentration is 0.5 μm of ol/L
Place, Ct values are relatively low and △ Rn values are higher, have smooth amplification curve and obvious logarithmic phase and plateau；And wx012 genes,
It is most like when the Ct values and △ Rn values and amplification curve and quadruple of bar genes, no genes and acp1 genes.Work as addition
It is 0.3 μm of ol/L in primer concentration during the primed probe of Mon15985 genes, concentration and probe concentration is that Ct values are relatively low at 0.4 μm of ol/L
And △ Rn values are higher, have smooth amplification curve and obvious logarithmic phase and plateau；And wx012 genes, bar genes, no bases
It is most like when the Ct values and △ Rn values and amplification curve of cause, acp1 genes and Mon531 genes are with five weights.Therefore the present invention is final
Primer combination of probe be：Wx012 gene primers concentration is 0.5 μm of ol/L, and concentration and probe concentration is 0.3 μm of ol/L；Bar gene primers
Concentration is 0.3 μm of ol/L, and concentration and probe concentration is 0.3 μm of ol/L；The primer concentration of no genes is 0.3 μm of ol/L, and concentration and probe concentration is
0.4μmol/L；The primer concentration of acp1 genes is 0.3 μm of ol/L, and concentration and probe concentration is 0.4 μm of ol/L；The primer of mon531 genes
Concentration is 0.2 μm of ol/L, and concentration and probe concentration is 0.5 μm of ol/L；The primer concentration of mon15985 genes is 0.3 μm of ol/L, and probe is dense
It is 0.4 μm of ol/L to spend.
3.2Mg2+With Taq DNA polymerase concentration optimization
Mg2+Concentration optimization scope is 1.0mmol/L-9.0mmol/L, and gradient is 1.0mmol/L, archaeal dna polymerase optimization model
Enclose and expand as 1.0U-5.0U, gradient is 1.0U；Experimental result shows works as Mg2+Concentration is 5.0mmol/L, and Taq DNA polymerase is dense
Spend during for 4U, reaction acquired results are optimal, therefore the five heavy real-time fluorescence quantitative PCR reaction systems such as following table institute for finally giving
Show：
Table 3：Five heavy real-time fluorescence quantitative PCR reaction systems
Reaction condition is 95 DEG C of predegenerations 1min, 95 DEG C of denaturation 3s, and 1min is extended after 60 DEG C of annealing, and annealing stage is collected glimmering
Light, 40 circulations.Examining six kinds of genes of interest has smooth amplification curve and obvious logarithmic phase and plateau, and Ct values are
Less than 36, illustrate that this reaction system can meet the reaction requirement of sixfold real-time fluorescence quantitative PCR.
3rd, using ABI ViiA7 quantitative real time PCR Instrument analysis software amplifications, to ensure the accurate of testing result
Property, while setting negative control experiment and positive control experiment.
3.1 negative control experiments
While DNA sample to be measured addition PCR reaction systems are carried out into multiple real time fluorescence PCR reactions, distilled water is used
Control experiment is carried out instead of sample DNA to be checked, as negative control.When negative control experiment result shows normal, experiment is carried out
Normally.When negative control experiment result shows abnormal, after experiment condition need to being remodified, then tested.
Negative control experiments testing result criterion is as shown in the table：
3.2 positive control experiments
While DNA sample to be measured addition PCR reaction systems are carried out into multiple real time fluorescence PCR reacting, with pure
Cotton DNA, Wheat DNA carries out control experiment instead of sample DNA to be checked, used as positive control.Positive control experiment result shows
When normal, experiment carries out normal.When positive control experiment result shows abnormal, after experiment condition need to being remodified, then carry out
Experiment.
Positive control experiment testing result criterion is as shown in the table：
3.3rd, sample to be tested testing result judges：
In the case of the feminine gender that carries out at the same time, positive control experiment result are normal, experimental result has availability.Test
Endogenous gene detection Ct values are less than or equal to 36 in sample, and foreign gene detection Ct values are more than or equal to 40, judge that the sample is free of
Examined foreign gene.Endogenous gene detection Ct values are less than or equal to 36 in test sample, and foreign gene detection Ct values are less than or equal to
40, judge that the sample contains and examine foreign gene.Endogenous gene detects that Ct values between 36~40, should adjust template in test sample
Concentration, real-time fluorescence PCR of reforming, the foreign gene Ct values after expanding again are still less than 40, and negative control, positive control and sky
White results of comparison normally then can determine that as the sample is examined foreign gene.If the foreign gene Ct values after expanding again are still big
In or equal to 40, and negative control, positive control and blank result normally then to can determine that and examined outer by the sample is not detected
Source gene.
Embodiment 3：In order to verify the performance of probe primer, specific test is carried out：
Extract non-transgene cotton respectively with plant DNA extraction kit respectively, turn Mon531 genes cotton, turn
Mon15985 genes cotton, non-transgenic wheat, turn bar DNA triticums, turn no DNA triticums, non-genetically engineered soybean, do not turn base
Because corn, Chinese cabbage, spinach, pork, mutton, duck, the flesh of fish, rabbit meat, donkey meat, chicken, goose genomic DNA, with extract
Genomic DNA is template, and fluorescence quantitative PCR detection is carried out respectively according to the reaction system and reaction condition of above-mentioned optimization, checking
The specificity of primer and probe.Testing result is as shown in the table.
By result above as can be seen that 6 pairs of primed probes have a Ct values only for corresponding species, and other samples
Without amplification.The primed probe of this experimental design has species specificity very high as can be seen here.
Embodiment 4：The sensitivity test of primer
Six kinds of corresponding plant genome DNAs are extracted using plant genome DNA extracts kit, it is fixed with Nanodrop
Measure 50ng, do 10 × gradient dilution (10-1,10-2,10-3,10-4,10-5), each gradient takes 2 μ l for template amount, according to upper
State method to six kinds of genomic DNAs detect and sensitivity for analysis successively.
Learnt by experiment：When fluorescent quantitation DNA profiling consumption is 0.01ng, the specific probe of each gene still has expansion
Increase curve；When fluorescent quantitation DNA profiling consumption is 0.001ng, substantially without amplification curve, Ct values ＞ 35, so this method
The sensitivity of primed probe group is 0.01ng.
Replica test
Extract respectively non-transgene cotton, turn Mon531 genes cotton, to turn Mon15985 genes cotton, non-transgenosis small
Wheat, the genomic DNA for turning bar DNA triticums, turning no DNA triticums, each species respectively extract 3 sample DNAs, according to above-mentioned excellent
The system changed, PCR conditions carry out real-time fluorescence quantitative PCR, and each sample carries out 3 the independent of multiple holes and repeats, and investigates method
Stability, as a result such as following table：
Ctmean represents the average value of Ct in upper table, and Ct SD represent the standard variance of Ct, as can be seen from the table, each
The standard variance of the independent Ct values for repeating experiment of 3 times of sample is respectively less than 0.5, illustrates the reproducible of testing result, detection
Stability is high.
Embodiment 5：Detection embodiment
Selection is actually subjected to the sample of detection, and sample is the wheat products from the major supermarkets in Qingdao, the market of farm produce etc. buying
And cotton products, detection GMOs are carried out to wheat and cotton products using method of the present invention, to verify this method
Use value.
Step is as follows：
1st, DNA is extracted：Testing sample is extracted into DNA, detects that DNA sample concentration is with Nanodrop detection of nucleic acids instrument
50ng/ μ L, OD values are between 1.7-1.8.
2nd, the real-time fluorescent PCR amplification of testing sample DNA adds PCR reaction systems as follows in PCR reaction tubes,
PCR reaction tubes are put into quantitative real time PCR Instrument, complete to expand by following condition：95 DEG C of predegeneration 3min, 95 DEG C are denatured 15s, 60
Extend 1min after DEG C annealing, annealing stage collects fluorescence, 40 circulations, while carrying out positive control experiment and negative control reality
Test.
PCR reaction systems：
3rd, ABI ViiA7 quantitative real time PCR Instrument analysis software amplifications, positive control and negative control reality are used
Test display result normally, the testing result of various measuring samples is as shown in the table.As can be seen here, commercially available wheat products and cotton
In product, there is transgenosis phenomenon.
SEQUENCE LISTING
<110>Qingdao JLA Testing Technology Services Co., Ltd.
<120>A kind of transgenic wheat and the detection method of cotton
<130>
<160> 18
<170> PatentIn version 3.5
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Claims (10)
1. a kind of primer combination of probe thing detected for wheat and cotton transgenic, it is characterised in that described primed probe
Composition includes primer and probe, the primer of detection external source no genes and probe, the detection external source of detection external source bar genes
Any one or several in the primer and probe of the primer and probe of Mon531 genes or detection external source Mon15985 genes,
Wherein：The primer and probe of external source bar genes are detected, its nucleotide sequence is respectively SEQ ID NO:4-6；
The primer and probe of external source no genes are detected, its nucleotide sequence is SEQ ID NO:7-9；
The primer and probe of external source Mon531 genes are detected, its nucleotide sequence is respectively SEQ ID NO:13-15；
The primer and probe of external source Mon15985 genes are detected, its nucleotide sequence is respectively SEQ ID NO:16-18.
2. primer combination of probe thing as claimed in claim 1, it is characterised in that described primer combination of probe thing also includes
The primer and probe that detect endogenous Wx012 genes and/or the primer and probe that detect endogenous Acp1 genes.
3. primer combination of probe thing as claimed in claim 2, it is characterised in that the described endogenous Wx012 genes of detection draw
Thing and probe, its nucleotide sequence are respectively SEQ ID NO:1-3；
The primer and probe of endogenous Acp1 genes are detected, its nucleotide sequence is respectively SEQ ID NO:10-12.
4. the primer combination of probe thing as described in claim any one of 1-3, it is characterised in that repair at 5 ' ends of described probe
Reporter group is decorated with, 3 ' ends are modified with quenching group.
5. primer combination of probe thing as claimed in claim 4, it is characterised in that described reporter group include FAM,
FITC、JOE、CY3、CY5、ROX。
6. primer combination of probe thing as claimed in claim 4, it is characterised in that described quenching group includes TAMRA,
BHQ、Eclipse。
7. application of the primer combination of probe thing described in claim 1 in detection wheat and cotton transgenic product is prepared.
8. a kind of product for detecting wheat and cotton transgenic, it is characterised in that comprising have the right will in described product
Seek the primer combination of probe thing described in 1.
9. it is a kind of to differentiate wheat, the detection method of cotton transgenic, it is characterised in that described method includes the steps：
1) genomic DNA of sample to be checked is extracted,
2) by step 1) extract genomic DNA be added to differentiate wheat, cotton transgenic detection reaction system in, using six
Whether weight quantitative real-time PCR detection, interpretation detects transgene component；Be with the addition of described in claim 1 in reaction system
Primer combination of probe thing；
3) fluorescence signal in PCR amplification procedures is collected, whether foreign gene is contained in differentiating sample by fluorescence signal.
10. method as claimed in claim 9, it is characterised in that described step 3) in the following manner interpretation whether detect
Transgene component：
1) endogenous gene detection Ct values are less than or equal to 36 in test sample, and foreign gene detection Ct values are more than or equal to 40, judge
The sample is free of and examines foreign gene；
2) endogenous gene detection Ct values are less than or equal to 36 in test sample, and foreign gene detection Ct values are less than or equal to 40, judge
The sample contains examines foreign gene；
3) endogenous gene detects that Ct values between 36~40, should adjust template concentrations in test sample, real-time fluorescence PCR of reforming,
Foreign gene Ct values after expanding again are still less than 40, and negative control, and positive control and blank result normally can then be sentenced
It is set to the sample and examines foreign gene；If the foreign gene Ct values after expanding again are still greater than or equal to 40, and negative right
According to positive control and blank result normally then can determine that as the sample does not detect foreign gene.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060258A (en) * | 2018-01-18 | 2018-05-22 | 中国农业科学院植物保护研究所 | For primer sets, kit and the method for the pure heterozygosis identification of cotton MON15985 transformant |
| CN112725423A (en) * | 2020-12-31 | 2021-04-30 | 镇江华大检测有限公司 | Detection method of transgenes in rice |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706969A (en) * | 2005-05-09 | 2005-12-14 | 曹际娟 | Primer series and reagent kit for real-time PCR detection of transgenic cotton (seed) |
| EP2107126A1 (en) * | 2008-03-31 | 2009-10-07 | Eppendorf Array Technologies SA | Detection of unspecified genetically modified organism (GMO) on micro-arrays |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706969A (en) * | 2005-05-09 | 2005-12-14 | 曹际娟 | Primer series and reagent kit for real-time PCR detection of transgenic cotton (seed) |
| EP2107126A1 (en) * | 2008-03-31 | 2009-10-07 | Eppendorf Array Technologies SA | Detection of unspecified genetically modified organism (GMO) on micro-arrays |
Non-Patent Citations (3)
| Title |
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| 李俊等: "商品化转基因植物的外源基因及其检测技术", 《中国农业科技导报》 * |
| 白月等: "小麦中转基因成分PCR与实时荧光PCR的定性检测低限", 《麦类作物学报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060258A (en) * | 2018-01-18 | 2018-05-22 | 中国农业科学院植物保护研究所 | For primer sets, kit and the method for the pure heterozygosis identification of cotton MON15985 transformant |
| CN112725423A (en) * | 2020-12-31 | 2021-04-30 | 镇江华大检测有限公司 | Detection method of transgenes in rice |
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