CN107142322A - Transgenic corns MON87403 strain specificities real-time fluorescent PCR testing primer, probe, method and kit - Google Patents
Transgenic corns MON87403 strain specificities real-time fluorescent PCR testing primer, probe, method and kit Download PDFInfo
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Abstract
The invention discloses transgenic corns MON87403 strain specificities real-time fluorescent PCR testing primer, probe, method and kit.The end of box gene 5 ' and Maize genome adjacent area primers and probe that the present invention is transferred to for transgenic corns MON87403 strains, the transgenic corns MON87403 strain specificity real-time fluorescence PCR detection methods of foundation, with specific good, sensitivity is high, repeatable good the advantages of, amplification efficiency is 94%, and minimum quantitative Monitoring lower-cut is 32copies.Effective the problem of solve quantitative measurement technology accurate without transgenic corns MON87403 strain specificities in the prior art, this method can be applied to pass in and out the detection of transgenic corns MON87403 and products thereof in Check and Examination of Port quarantine, domestic agricultural products foods supervision.
Description
Technical field：
The invention belongs to GMO detection field, and in particular to transgenic corns MON87403 strain specificities are real-time
Fluorescent PCR detecting primer, probe, method and kit.
Background technology：
MON87403 is by agriculture bacillus mediated insertion arabidopsis ATHB17 gene coding regions full length sequence.ATHB17 is HD-Zip
Family plant transcription factor member, its protein binding is in specific DNA, controlling gene expression.HD-Zip protein families are planted in regulation
Thing grows and played an important role in evolving.In MON87403 plant, the specific cutting ATHB17 of corn translates into it
Truncated albumin A THB17 Δs 113, it lost 113 N-terminal amino acid of starting.ATHB17 Δs 113 can adjust heading group
The activity of middle HD-Zip II albumen is knitted, causes increase (increase of fringe and the photosynthesis generation in early stage raw reproductive phase fringe
Dry qualitative correlation).
MON87403 is ground by agriculture bacillus mediated plasmid PV-ZMAP5714 insertion corn strain LH224 inbred strais immature embryos
System is formed, and PV-ZMAP5714 plasmids include three box genes：ATHB17 expression cassettes, cp4 epsps screening mark boxs and aadA tables
Up to box.PV-ZMAP5714 produces unmarked plant using series connection T-DNA methods, and (cp4 epsps box genes are being initially used to carry out
Separated after the screening of resistance glyphosate herbicidal properties by traditional breeding method, without cp4 epsps genes in transfer-gen plant), turn
ATHB17 expression cassettes in gene plant only containing 1 copy.
MON87403 was listed in 2015, and there are Australia's big sharp (food), New Zealand's (food) in the country of currently acquired approval, added
Put on airs (food and feed and plantation), South Korea's (food and feed) and the U.S. (food, feed and plantation) 5 countries.
At present to transgenic product majority state using corresponding mark management system, strain specificity PCR (conversion things
Part specificity) (Event-specific PCR) detection target sequence be between external source insetion sequence and Plant Genome connect
Area, compared to screening PCR (Screening PCR), gene specific PCR (Gene-specific PCR), builds specificity
PCR (Construct-specific PCR) has that more increases to have high specific, can be used for the identical plasmid of Testing and appraisal and turn
The specific strain of transgenosis of change, is the important side in current transgenic strain identification detection technique research and actually detected work
Method.
To break the transgenic product tradeing mutual compensation that other countries and area are set, improve and China's transgenic product
Quantitative measurement technology system, protection consumer is to the right to know and right to choose of transgenic product, for the port supervision department that passes in and out
The method for providing the identification of transgenosis different lines label detection, sets up transgenic corns MON87403 strain specificity quantitative PCRs
Accurate detection method is very necessary.
The content of the invention：
It is an object of the invention to provide a species specificity is good, sensitivity is high, stability is strong, quick and precisely differentiate that transgenosis is beautiful
Transgenic corns MON87403 strain specificities real-time fluorescent PCR testing primer, probe, method and the examination of rice MON87403 strains
Agent box.
First purpose of the present invention is to provide transgenic corns MON87403 strain specificity real-time PCR detections and drawn
Thing, it is characterised in that described detection primer is as follows：
MON87403-F：5 '-ATAATAACGCTGCGGACATCTAC-3 ' (as shown in SEQ ID NO.1)；
MON87403-R：5 '-GAATGAGTGCTCTGTATCCTCCAC-3 ' (as shown in SEQ ID NO.2).
Second object of the present invention is to provide a kind of transgenic corns MON87403 strain specificities real-time fluorescence PCR inspection
Probing pin, it is characterised in that described detection probe is as follows：
MON87403-P：5 '-CCATCATACTCATTGCGATCCACATTTC-3 ' (as shown in SEQ ID NO.3), probe
5 ' end be marked with fluorescent reporter group, 3' ends are marked with fluorescent quenching group.
Described fluorescent reporter group is preferably FAM, and described fluorescent quenching group is preferably BHQ1.
Third object of the present invention is to provide a kind of transgenic corns MON87403 strain specificities real-time fluorescence PCR inspection
Test agent box, including real-time fluorescence quantitative PCR reaction solution, hot resistant DNA polymerase, detection primer and detection probe, its feature exist
In described detection primer is as follows：
MON87403-F：5’-ATAATAACGCTGCGGACATCTAC-3’；
MON87403-R：5’-GAATGAGTGCTCTGTATCCTCCAC-3’；
Described detection probe is as follows：
MON87403-P：5 '-CCATCATACTCATTGCGATCCACATTTC-3 ', 5 ' ends of probe are marked with fluorescence report
Group is accused, 3' ends are marked with fluorescent quenching group.
Fourth object of the present invention is to provide a kind of transgenic corns MON87403 strain specificities real-time fluorescence PCR inspection
Survey method, it is characterised in that comprise the following steps：
(1) genomic DNA for extracting sample is used as template；
(2) above-mentioned detection primer and detection probe is added, is polymerize with real-time fluorescence quantitative PCR reaction solution and heat-resistant dna
Enzyme is mixed to form amplification reaction system；
(3) amplification reaction system is subjected to real-time fluorescence PCR reaction on fluorescence PCP instrument, after reaction terminates, according to amplification
Whether curve judgement sample is transgenic corns MON87403 strains.
It is preferred that, the amplification reaction system of described step (2) is：25 μ L, including the μ L of Premix Ex TaqTM 12.5,
ROX Reference Dye II 0.2 μ L, 10 μm of ol/L detection primers MON87403-F and MON87403-R each 0.4 μ L, 10 μ
The μ L of mol/L detection probes MON87403-P 0.8, DNA profiling 2 μ L and ddH2O 8.7μL；The real-time fluorescence of described step (3)
PCR reacts, and its response procedures is 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, fluorescence signal is collected in 60 DEG C.
It is preferred that, whether described step (3) is transgenic corns MON87403 strains according to amplification curve judgement sample
Standard be：If amplification curve has Representative fluorescence amplification curve, it is transgenic corns MON87403 strains to illustrate sample；Such as
Fruit amplification curve is without Representative fluorescence amplification curve, then it is not transgenic corns MON87403 strains to illustrate sample.
The 5th purpose of the present invention is to provide a kind of transgenic corns MON87403 strain specificities real-time fluorescence PCR and determined
Quantity measuring method, it is characterised in that comprise the following steps：
(1) the genomic DNA progress gradient dilution for extracting transgenic corns MON87403 strains is dense for use as different startings
The template of degree, is separately added into above-mentioned detection primer and detection probe, poly- with real-time fluorescence quantitative PCR reaction solution and heat-resistant dna
Synthase is mixed to form amplification reaction system, and real-time fluorescence PCR reaction is carried out on fluorescence PCP instrument；
(2) the corresponding Ct values of each initial concentration template, the common logarithm of the Ct values and starting template amount are obtained after reacting
(lg) it is linear, the standard curve of transgenic corns MON87403 strains in the range of linearity is obtained accordingly；
(3) extract the genomic DNA of testing sample, add with the above-mentioned detection primer of same system in step (1) and
Detection probe, amplification reaction system is mixed to form with real-time fluorescence quantitative PCR reaction solution and hot resistant DNA polymerase, in fluorescence PCP
Real-time fluorescence PCR reaction is carried out on instrument, Ct values are obtained after reaction, the transgenic corns of step (2) acquisition are substituted into
The standard curve of MON87403 strains, calculates and obtains containing for transgenic corns MON87403 strain genomic DNAs in measuring samples
Measure (copy number or quality).
The present invention has advantages below and beneficial effect：
1. the present invention breaks the transgenic product tradeing mutual compensation that external other countries and area are set；
2. the present invention makes up and perfect China's transgenic product quantitative measurement technology system.The technology of offer is used to turn base
Because the detection of product can be better protection consumer right to know and right to choose, meet state supervision department to transgenic product
Identification and mark demand.
3. the present invention is directed to transgenic corns MON87403 strain specificities primers and TaqMan probe, set up
Transgenic corns MON87403 real-time fluorescent polyase chain reactions (polymerase chain reaction, PCR) detection side
Method.Using the detection primer and detection probe and the real-time fluorescence PCR system of foundation, quantitatively detected down according to the method for the present invention
32 copies are limited to, the calibration curve equation of the transgenic corns MON87403 strains of foundation is：Y=-3.4802x+39.91, R2
=0.99, amplification efficiency：94% (between 90%~110%), shows that the transgenic corns MON87403 strains of the present invention are special
Property real-time fluorescence PCR detection method template amount 32-640000copies repeated experiments show the present invention method standard
Deviation (SD) and relative standard deviation (RSD) are all in tolerance interval, and specificity is good, sensitivity is high, stability is strong.
Brief description of the drawings：
Fig. 1 is amplification figure when annealing temperature is 58 DEG C.
Fig. 2 is amplification figure when annealing temperature is 60 DEG C.
Fig. 3 is transgenic corns MON87403 event-specific detection method specificity experiments results；1 is HMG-
MON87403 plasmids；2~14 are respectively：Transgene rape MON88302, transgene rape DP-073496-4, transgene cotton
MON88913, genetically engineered soybean A2704-12, genetically engineered soybean GTS 40-30-2, transgenic corns MON810, transgenic corns
BT11, transgenic corns MIR162, transgenic corns NK603, transgenic beet H7-1, non-transgenic corn, negative control and
Blank control.
Fig. 4 is transgene cotton MON87403 event-specific detection sensitivity test amplification figures；1~9 is respectively
320000th, 32000,16000,3200,1600,320,160,32 and 16copies/ μ L DNAs, 10 and 11 be respectively feminine gender
Control and blank control.
Fig. 5 is the standard curve of real-time PCR detection MON87403 strains, and LOG (copies) is Log10
(copies)。
Embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Main material：Transgene rape MON88302, transgene rape DP-073496-4, transgene cotton MON88913,
Genetically engineered soybean A2704-12, genetically engineered soybean GTS 40-30-2, transgenic corns MON810, transgenic corn BT 11, turn base
Because corn MIR162, transgenic corns NK603, transgenic beet H7-1, non-transgenic corn are that this laboratory is purchased and preserved,
Corn endogenous gene is from corn high speed swimming GFP (in high mobility group proteins, HMG, corn
State singly copies, GenBank:) and the dual-gene positive plasmid of transgenic corns MON87403 strain specificity fragments AJ131373.1
(dual-gene positive plasmid is by endogenous gene HMG genetic fragment 150bp sequences (as shown in SEQ ID NO.4) and MON87403
The common 350bp sequences (as shown in SEQ ID NO.6) gram of strain specificity fragment 200bp sequences (as shown in SEQ ID NO.5)
Obtained recombinant plasmid is built on the grand PUC57 carriers to AmpR resistances, recombinant plasmid is transferred to -70 DEG C of guarantors after recipient bacterium DH5a
Deposit.Hereinafter referred to as HMG-MON87403 plasmids, restriction enzyme site BamHI) built for this laboratory.
Corn endogenous gene from corn high speed swimming GFP (high mobility group proteins,
HMG, the single copy of corn China, GenBank:AJ131373.1) (bibliography：Mazzara M,FotiN,Savini C,Van
Den Eede G.report on the verification of the performance of a MON87403 event-
specific method on maize line MON87403 using real-time PCR- Validation Report
And Protocol.EUR24237EN.2009.JRC56609, DOI10.2788/59036) primer HMG-F/R and probe HMG-P
For detecting whether corn sample genomic DNA successfully extracts and whether be adapted for real-time fluorescent PCR amplification；Its sequence is believed
Breath refers to table 1.
Main agents：Primex Ex Taq (2 ×) for qPCR, Dalian is precious biological；DNA extraction kit, Beijing Tiangeng
Company；Primer and probe is synthesized by Shan Jing biotech firms, and the working solution for being diluted to final concentration of 10 μM is used.
Key instrument and equipment：
ABI7500, ABI7500FAST real-time fluorescence quantitative PCR instrument, Applied biosystems；The droplets of QX 200
Formula digital pcr system, Bio Rad Laboratories；Nanodrop2000c micro-spectrophotometers, Thermo companies of the U.S.；Grinder,
German IKA.
Crop material sample gene group DNA uses conventional method extraction and purification.
Embodiment 1：The foundation and optimization of real-time fluorescence PCR detection method
The end of box gene 5 ' being transferred to according to transgenic corns MON87403 strains (turns base with Maize genome adjoining region sequence
Because of corn MON87403 strain specificity fragments, as shown in SEQ ID NO.5), using the Software for Design of Primer Primer 5.0
Primer and probe.The primer of design, probe are determined to the reason of primer and probe through being compared on NCBI websites using BLAST databases
By specificity；Corn endogenous gene HMG is used for the detection of corn source sample DNA and the relative quantification of transgene component.Specifically
Primed probe information is shown in Table 1.
Primer, the probe of the real-time fluorescence PCR of table 1
Annealing temperature and primed probe proportioning are optimized：
The amplification reaction system of A groups is：25 μ L, including μ L, the ROX Reference Dye of Premix Ex TaqTM 12.5
II 0.2 μ L, 10 μm of ol/L detection primers each 0.5 μ L of MON87403-F and MON87403-R, 10 μm of ol/L detection probes
μ L, 32000copies/ μ L DNA profilings (transgenic corns MON87403 strains genomic DNA) the 2 μ L of MON87403-P 1 and
ddH2O 8.3μL；
The amplification reaction system of B groups is：25 μ L, including μ L, the ROX Reference Dye of Premix Ex TaqTM 12.5
II 0.2 μ L, 10 μm of ol/L detection primers each 0.4 μ L of MON87403-F and MON87403-R, 10 μm of ol/L detection probes
μ L, 32000copies/ μ L DNA profilings (transgenic corns MON87403 strains genomic DNA) the 2 μ L of MON87403-P 0.8 and
ddH2O 8.7μL；
The amplification reaction system of C groups is：25 μ L, including μ L, the ROX Reference Dye of Premix Ex TaqTM 12.5
II 0.2 μ L, 10 μm of ol/L detection primers each 0.2 μ L of MON87403-F and MON87403-R, 10 μm of ol/L detection probes
μ L, 32000copies/ μ L DNA profilings (transgenic corns MON87403 strains genomic DNA) the 2 μ L of MON87403-P 4 and
ddH2O 5.9μL；
Response procedures when annealing temperature is 58 DEG C are 95 DEG C of 30s；95 DEG C of 5s, 58 DEG C of 34s, 40 circulations, in 58 DEG C of receipts
Collect fluorescence signal；
Response procedures when annealing temperature is 60 DEG C are 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, in 60 DEG C of receipts
Collect fluorescence signal.
As a result show：Amplification figure when annealing temperature is 58 DEG C is as shown in figure 1, be respectively from left to right A, B and C in figure
The amplification curve of group, A and B group Ct values are approached, and the amplification reaction system that selection B groups are this experiment is considered based on economy；
Amplification figure when annealing temperature is 60 DEG C is as shown in Fig. 2 be respectively from left to right the amplification of A, B and C group in figure
Curve, B and C group Ct values are approached, and tri- groups of Ct values of A, B and C and annealing temperature Ct values at 58 DEG C are approached.
In summary consider, based on economy, the optimal reaction temperature of enzyme and the versatility of amplification platform application are (especially right
In multiple), it is considered to 60 DEG C are selected as annealing temperature, the amplification reaction system that B groups are this experiment.
Embodiment 2：Real time fluorescent PCR method specific test
Extract transgene rape MON88302, transgene rape DP-073496-4, transgene cotton MON88913, turn base
Because soybean A2704-12, genetically engineered soybean GTS 40-30-2, transgenic corns MON810, transgenic corn BT 11, transgenosis are beautiful
Rice MIR162, transgenic corns NK603, transgenic beet H7-1, the genomic DNA of non-transgenic corn are template, positive right
According to for HMG-MON87403 plasmids, negative control is non-transgenic rice DNA.To the real-time fluorescence PCR detection method of foundation
Specificity is tested.
Amplification reaction system is：25 μ L, including μ L, the ROX Reference Dye II of Premix Ex TaqTM 12.5
0.2 μ L, 10 μm of ol/L detection primers MON87403-F and MON87403-R each 0.4 μ L, 10 μm of ol/L detection probes MON87403-
The μ L of P 0.8, DNA profiling 2 μ L and ddH2O 8.7μL；Response procedures are 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, in
60 DEG C of collection fluorescence signals.
As a result (Fig. 3) is shown, using transgenic corns MON87403 strain specificity primer MON87403-F/R and probe
When MON87403-P carries out real-time fluorescence PCR, the DNA profiling of only positive HMG-MON87403 plasmids has Representative fluorescence expansion
Increase curve, the reaction using other crop materials DNA as template is without Representative fluorescence amplification curve.Show the detection side of the present invention
Method specificity is good.
Embodiment 3：Sensitivity test, repeatability test and standard curve are set up
Add TE buffer solutions to be diluted to buffer solution respectively the HMG-MON87403 plasmid DNA solutions of extraction to be diluted to
320000th, 32000,16000,3200,1600,320,160,32 and 16copies/ μ L carry out transgenic corns as DNA profiling
MON87403 real-time PCR detections, real-time fluorescence PCR detecting reaction system and response procedures be the same as Example 2, enter line sensitivity
Test：
Test result shows (Fig. 4) that 9 gradients can have typical amplification curve in the range of 320000-16copies/ μ L,
Its minimal detectable concentration is 16copies/ μ L.Amplification figure be respectively from left to right buffer solution be diluted to 320000,32000,
16000th, 3200,1600,320,160,32 and 16copies/ μ L amplification curves.
Add TE buffer solutions to be diluted to 320000 respectively the HMG-MON87403 plasmid DNA solutions of extraction, 32000,
16000th, 3200,1600,320,160,32 and 16copies/ μ L carry out transgenic corns MON87403 strains as DNA profiling
Real-time PCR detection, carries out range of linearity test and repeatability test, and each sample carries out 3 repetitions and tested, and water is sky
White control, real-time fluorescence PCR detecting reaction system and response procedures be the same as Example 2：
The Ct values of test result are as shown in table 2；According to Ct Value Datas in table 2 and 9 in the range of 32-640000copies
The logarithm value of concentration sets up standard curve (Fig. 5) with gained Ct values, and equation of linear regression is：Y=-3.4802x+39.91, R2=
0.99, amplification efficiency：94% (between 90%~110%), shows the transgenic corns MON87403 strain specificities of the present invention
Real-time fluorescence PCR detection method linear dependence in the range of template amount 32-640000copies is good, and amplification efficiency is high, symbol
Close ENGL related requests [Definition of Minimum Performance Requirements for Analytical
Methods of GMO Testing]；And in template amount in the 32-640000copies range of linearity, the SD of its Ct value is situated between
In 0.07-0.30, RSD is between 0.32%-0.90%, when showing the minimum template amount 32copies in linear scope, its SD and
RSD is respectively less than 25%, so the quantitative Monitoring lower-cut of the present invention is 32copies.
The sensitivity and repeatability test of the real time fluorescent PCR method of table 2
The end of box gene 5 ' and Maize genome adjacent area sequence that the present invention is transferred to for transgenic corns MON87403 strains
Row design primer and probe, the transgenic corns MON87403 strain specificity real-time fluorescence PCR detection methods of foundation can be to turning
Gene corn MON87403 progress strain specificities are quick, high-throughout qualitative and precisely quantitatively detect, satisfaction is detected, supervision department
The demand that door is identified to it.
Sequence table
<110>Huangpu Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China (PRC)
<120>Transgenic corns MON87403 strain specificities real-time fluorescent PCR testing primer, probe, method and kit
<160> 6
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
ataataacgc tgcggacatc tac 23
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
gaatgagtgc tctgtatcct ccac 24
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence
<400> 3
ccatcatact cattgcgatc cacatttc 28
<210> 4
<211> 150
<212> DNA
<213>Corn
<400> 4
cctgagcgag tcggtaagct ccatcttctg tactaaagta gtagttgatt ggactagaaa 60
tctcgtgctg attaattgtt ttacgcgtgc gtttgtgtgg attgtaggac aaggctccct 120
atgtagccaa ggctaacaag ctcaagctcg 150
<210> 5
<211> 200
<212> DNA
<213>Transgenic corns MON87403
<400> 5
taatttgtcg ttttatcaaa atgtactttc attttataat aacgctgcgg acatctacat 60
ttttgaattg aaaaaaaatt ggtaattact ctttcttttt ctccatattg accatcatac 120
tcattgcgat ccacatttcc ctacatggtg gaggatacag agcactcatt ccggagtata 180
atcttgtctt gtgttgccac 200
<210> 6
<211> 350
<212> DNA
<213>HMG-MON87403 plasmids
<400> 6
cctgagcgag tcggtaagct ccatcttctg tactaaagta gtagttgatt ggactagaaa 60
tctcgtgctg attaattgtt ttacgcgtgc gtttgtgtgg attgtaggac aaggctccct 120
atgtagccaa ggctaacaag ctcaagctcg taatttgtcg ttttatcaaa atgtactttc 180
attttataat aacgctgcgg acatctacat ttttgaattg aaaaaaaatt ggtaattact 240
ctttcttttt ctccatattg accatcatac tcattgcgat ccacatttcc ctacatggtg 300
gaggatacag agcactcatt ccggagtata atcttgtctt gtgttgccac 350
Claims (8)
1. a kind of transgenic corns MON87403 strain specificity real-time fluorescent PCR testing primers, it is characterised in that described inspection
Survey primer as follows：
MON87403-F：5’-ATAATAACGCTGCGGACATCTAC-3’；
MON87403-R：5’-GAATGAGTGCTCTGTATCCTCCAC-3’.
2. a kind of transgenic corns MON87403 strain specificities real-time PCR detection probe, it is characterised in that described inspection
Probing pin is as follows：
MON87403-P：5 '-CCATCATACTCATTGCGATCCACATTTC-3 ', 5 ' ends of probe are marked with fluorescence report base
Group, 3 ' ends are marked with fluorescent quenching group.
3. transgenic corns MON87403 strain specificities real-time PCR detection probe according to claim 2, it is special
Levy and be, described fluorescent reporter group is FAM, described fluorescent quenching group is BHQ1.
4. a kind of transgenic corns MON87403 strain specificity real-time fluorescence PCR assay kits, including real time fluorescent quantitative
PCR reaction solutions, hot resistant DNA polymerase, detection primer and detection probe, it is characterised in that described detection primer is as follows：
MON87403-F：5’-ATAATAACGCTGCGGACATCTAC-3’；
MON87403-R：5’-GAATGAGTGCTCTGTATCCTCCAC-3’；
Described detection probe is as follows：
MON87403-P：5 '-CCATCATACTCATTGCGATCCACATTTC-3 ', 5 ' ends of probe are marked with fluorescence report base
Group, 3' ends are marked with fluorescent quenching group.
5. a kind of transgenic corns MON87403 strain specificity real-time fluorescence PCR detection methods, it is characterised in that including following
Step：
(1) genomic DNA for extracting sample is used as template；
(2) detection primer described in claim 1 and the detection probe described in claim 2 are added, with real-time fluorescence quantitative PCR
Reaction solution and hot resistant DNA polymerase are mixed to form amplification reaction system；
(3) amplification reaction system is subjected to real-time fluorescence PCR reaction on fluorescence PCP instrument, after reaction terminates, according to amplification curve
Whether judgement sample is transgenic corns MON87403 strains.
6. transgenic corns MON87403 strain specificity real-time fluorescence PCR detection methods according to claim 5, it is special
Levy and be, the amplification reaction system of described step (2) is：25 μ L, including Premix Ex TaqTM 12.5 μ L, ROX
Reference Dye II 0.2 μ L, 10 μm of ol/L detection primers MON87403-F and MON87403-R each 0.4 μ L, 10 μm of ol/L
The μ L of detection probe MON87403-P 0.8, DNA profiling 2 μ L and ddH2O 8.7μL；The real-time fluorescence PCR of described step (3) is anti-
Should, its response procedures is 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, fluorescence signal is collected in 60 DEG C.
7. transgenic corns MON87403 strain specificity real-time fluorescence PCR detection methods according to claim 5, it is special
Levy and be, described step (3) according to amplification curve judgement sample whether be transgenic corns MON87403 strains standard
For：If amplification curve has Representative fluorescence amplification curve, it is transgenic corns MON87403 strains to illustrate sample；If amplification
Curve is without Representative fluorescence amplification curve, then it is not transgenic corns MON87403 strains to illustrate sample.
8. a kind of transgenic corns MON87403 strain specificity real-time fluorescent PCR quantitative detection methods, it is characterised in that including
Following steps：
(1) genomic DNA for extracting transgenic corns MON87403 strains carries out gradient dilution for use as different initial concentrations
Template, is separately added into the detection probe described in detection primer and the claim 2 described in claim 1, with real time fluorescent quantitative
PCR reaction solutions and hot resistant DNA polymerase are mixed to form amplification reaction system, real-time fluorescence PCR are carried out on fluorescent PCR instrument anti-
Should；
(2) the corresponding Ct values of each initial concentration template are obtained after reacting, the common logarithm (lg) of the Ct values and starting template amount is in
Linear relationship, obtains the standard curve of transgenic corns MON87403 strains in the range of linearity accordingly；
(3) genomic DNA for extracting testing sample is template, in addition and step (1) described in the claim 1 of same system
Detection probe described in detection primer and claim 2, is mixed with real-time fluorescence quantitative PCR reaction solution and hot resistant DNA polymerase
Amplification reaction system is formed, real-time fluorescence PCR reaction is carried out on fluorescence PCP instrument, Ct values are obtained after reaction, step is substituted into
(2) standard curve of the transgenic corns MON87403 strains obtained, calculating obtains transgenic corns in measuring samples
The content of MON87403 strain genomic DNAs.
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| JP2021532744A (en) * | 2018-07-30 | 2021-12-02 | モンサント テクノロジー エルエルシー | Genetically modified events in maize MON95379 and its detection and usage |
| CN114015682A (en) * | 2021-11-16 | 2022-02-08 | 四川省农业科学院生物技术核技术研究所 | Specific probe, primer, kit and method for identifying nucleic acid sample |
| CN116064888A (en) * | 2022-07-14 | 2023-05-05 | 中国检验检疫科学研究院 | Primer, probe, kit and method for quantitative detection of transgenic corn Mon87403 strain |
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Application publication date: 20170908 |