CN107254526A - Transgenic corns MON87411 strain specificities real-time fluorescent PCR testing primer, probe, method and kit - Google Patents
Transgenic corns MON87411 strain specificities real-time fluorescent PCR testing primer, probe, method and kit Download PDFInfo
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- CN107254526A CN107254526A CN201710482322.8A CN201710482322A CN107254526A CN 107254526 A CN107254526 A CN 107254526A CN 201710482322 A CN201710482322 A CN 201710482322A CN 107254526 A CN107254526 A CN 107254526A
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses transgenic corns MON87411 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 MON87411 strains, the transgenic corns MON87411 strain specificity real-time fluorescence PCR detection methods of foundation, with specific good, sensitivity is high, repeatable good the advantages of, amplification efficiency is 98.14%, and minimum quantitative Monitoring lower-cut is 24copies.Effective the problem of solve quantitative measurement technology accurate without transgenic corns MON87411 strain specificities in the prior art, this method can be applied to pass in and out the detection of transgenic corns MON87411 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 MON87411 strain specificities are real-time
Fluorescent PCR detecting primer, probe, method and kit.
Background technology：
MON87411 is anti-corn rootworm (the corn rootworm (CRW), Diabrotica developed by Monsanto Company
Spp.) and resistance glyphosate herbicide corn strain, it is transferred to PV- through agriculture bacillus mediated by corn sterile line LH244 strains
ZMIR10871 plasmids and develop, its T-DNA gene containing Snf7, cry3Bb1 genes and cp4epsps expression casettes.Wherein
Snf7 box genes have and western corn rootworm (western corn rootworm, WCR；Diabrotica virgifera
Virgifera the inverted repeats that Snf7 genes (DvSnf7)) match, it forms Snf7 gene phases in WCR containing west
The 240bp of matching double-stranded RNA transcription thing, CRW death is caused by lowering Snf7 genes；Cry3Bb1 produces related egg
It is white to suppress WCR larvas；Cp4epsps genes provide tolerance glyphosate herbicidal ability.
MON87411 was listed in 2014, 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), Japanese (food and feed and plantation), South Korea's (food and feed), Brazilian (food and feeding
Material and plant), TaiWan, China (food) and the U.S. (food, feed and plantation) 8 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 MON87411 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
The rice transgenic corns MON87411 strain specificities real-time fluorescent PCR testing primer of MON87411 strains, probe, method and
Kit.
First purpose of the present invention is to provide a kind of transgenic corns MON87411 strain specificities real-time fluorescence PCR inspection
Survey primer, it is characterised in that described detection primer is as follows：
MON87411-F：5’-CAATAGGTTCATTTAAAGTGATGGA-3’；(as shown in SEQ ID NO.1)；
MON87411-R：5 '-TTCTTTTTCTCCATATTGACCATC-3 ' (as shown in SEQ ID NO.2).
Second object of the present invention is to provide a kind of transgenic corns MON87411 strain specificities real-time fluorescence PCR inspection
Probing pin, it is characterised in that described detection probe is as follows：
MON87411-P：5 '-CATGTAGATTTCCCGGACATGAAGCC-3 ' (as shown in SEQ ID NO.3), probe
5 ' ends are marked with fluorescent reporter group, and 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 MON87411 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：
MON87411-F：5’-CAATAGGTTCATTTAAAGTGATGGA-3’；(as shown in SEQ ID NO.1)；
MON87411-R：5 '-TTCTTTTTCTCCATATTGACCATC-3 ' (as shown in SEQ ID NO.2)；
Described detection probe is as follows：
MON87411-P：5 '-CATGTAGATTTCCCGGACATGAAGCC-3 ' (as shown in SEQ ID NO.3), probe
5 ' ends are marked with fluorescent reporter group, and 3' ends are marked with fluorescent quenching group.
Fourth object of the present invention is to provide a kind of transgenic corns MON87411 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 MON87411 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 MON87411-F and MON87411-R each 0.4 μ L, 10 μ
The μ L of mol/L detection probes MON87411-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, 58 DEG C of 34s, 40 circulations, fluorescence signal is collected in 58 DEG C.
It is preferred that, whether described step (3) is transgenic corns MON87411 strains according to amplification curve judgement sample
Standard be：If amplification curve has Representative fluorescence amplification curve, it is transgenic corns MON87411 strains to illustrate sample；Such as
Fruit amplification curve is without Representative fluorescence amplification curve, then it is not transgenic corns MON87411 strains to illustrate sample.
The 5th purpose of the present invention is to provide a kind of transgenic corns MON87411 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 MON87411 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 MON87411 strains in the range of linearity is obtained accordingly；
(3) genomic DNA for extracting testing sample is template, adds the above-mentioned detection with same system in step (1)
Primer and detection probe, amplification reaction system is mixed to form with real-time fluorescence quantitative PCR reaction solution and hot resistant DNA polymerase,
Real-time fluorescence PCR reaction is carried out on fluorescence PCP instrument, Ct values are obtained after reaction, the transgenic corns of step (2) acquisition are substituted into
The standard curve of MON87411 strains, calculates and obtains containing for transgenic corns MON87411 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 MON87411 strain specificities primers and TaqMan probe, set up
Transgenic corns MON87411 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
24 copies are limited to, the calibration curve equation of the transgenic corns MON87411 strains of foundation is：Y=-3.37x+39.53, R2=
0.99, amplification efficiency E are 98.14% (between 90%~110%), the standard deviation of the method for the repeated experiment display present invention
(SD) and relative standard deviation (RSD) is all in tolerance interval, 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 MON87411 event-specific detection method specificity experiments results；1 is HMG-
MON87411 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, transgenosis are beautiful
Rice BT11, transgenic corns MIR162, transgenic corns NK603, transgenic beet H7-1, non-transgenic corn, negative control
And blank control.
Fig. 4 is transgenic corns MON87411 event-specific detection sensitivity test amplification figures；1~9 is respectively：
240000th, 24000,12000,2400,1200,240,120,24 and 12copies/ 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 MON87411 strains, and Quantity represents DNA profiling amount (copy
Number).
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 MON87411 strain specificity fragments AJ131373.1
(dual-gene positive plasmid is that endogenous gene HMG genetic fragment 150bp sequences (as shown in SEQ ID NO.4) and transgenosis is beautiful
Common 360bp sequences (such as SEQ ID of rice MON87411 strain specificity fragment 210bp sequences (as shown in SEQ ID NO.5)
Shown in NO.6) recombinant plasmid for building and obtaining on the PUC57 carriers of AmpR resistances is cloned into, recombinant plasmid is transferred to recipient bacterium
- 70 DEG C of preservations after DH5a.Hereinafter referred to as HMG-MON87411 plasmids) 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 MON87411event-
specific method on maize line MON87411using real-time PCR-Validation Report
And Protocol.EUR24237 EN.2009.JRC56609, DOI10.2788/59036) primer HMG-F/R and probe HMG-
P is used to detect whether corn sample genomic DNA successfully extracts and whether be adapted for real-time fluorescent PCR amplification；Its sequence
Information 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 MON87411 strains (turns base with Maize genome adjoining region sequence
Because of corn MON87411 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 MON87411-F and MON87411-R, 10 μm of ol/L detection probes
μ L, 24000copies/ μ L DNA profilings (transgenic corns MON87411 strains genomic DNA) the 2 μ L of MON87411-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 MON87411-F and MON87411-R, 10 μm of ol/L detection probes
MON87411-P 0.8 μ L, 24000copies/ μ L DNA profilings (genomic DNA of transgenic corns MON87411 strains) 2 μ
L 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 MON87411-F and MON87411-R, 10 μm of ol/L detection probes
μ L, 24000copies/ μ L DNA profilings (transgenic corns MON87411 strains genomic DNA) the 2 μ L of MON87411-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：The 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 group in figure
Amplification curve, 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；
The amplification figure when annealing temperature is 60 DEG C is as shown in Fig. 2 be respectively from left to right the amplification song of A, B and C group in figure
Line, B and C group Ct values are approached, but three groups of Ct values than 58 DEG C of annealing temperature when it is high.
In summary consider, it is this experiment to consider 58 DEG C of selection as annealing temperature, B groups based on economy and amplification efficiency
Amplification reaction system.
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-MON87411 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 MON88701-F and MON88701-R each 0.4 μ L, 10 μm of μ of ol/L detection probes MON88701-P 0.8
L, DNA profiling 2 μ L and ddH2O 8.7μL；Response procedures are 95 DEG C of 30s；95 DEG C of 5s, 58 DEG C of 34s, 40 circulations, in 58 DEG C
Collect fluorescence signal.
As a result (Fig. 3) is shown, using transgenic corns MON87411 strain specificity primer MON87411-F/R and probe
When MON87411-P carries out real-time fluorescence PCR, the DNA profiling of only positive HMG-MON87411 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 240000 respectively the HMG-MON87411 plasmid DNA solutions of extraction, 24000,
12000th, 2400,1200,240,120,24 and 12copies/ μ L are real-time as DNA profiling progress transgenic corns MON87411
Fluorescent PCR detects that real-time fluorescence PCR detecting reaction system and response procedures be the same as Example 2 carry out sensitivity test：
Test result shows (Fig. 4) that 9 concentration gradients can have typical case's amplification bent in the range of 240000-12copies/ μ L
Line, its minimal detectable concentration is 12copies/ μ L.Amplification figure from left to right be respectively 240000,24000,12000,2400,
1200th, 240,120,24 and 12copies/ μ L amplification curves.
Add TE buffer solutions to be diluted to 240000 respectively the HMG-MON87411 plasmid DNA solutions of extraction, 24000,
12000th, 2400,1200,240,120,24 and 12copies/ μ L carry out transgenic corns MON87411 product as DNA profiling
It is real-time PCR detection, carries out range of linearity test and repeatability test, each sample carries out 3 repetitions and tested, and water is
Blank 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 24-480000copies
The logarithm value of concentration sets up standard curve (Fig. 5) with gained Ct values, and equation of linear regression is：Y=-3.37x+39.53, R2:
0.99, amplification efficiency is 98.14% (between 90%~110%), shows that the transgenic corns MON87411 strains of the present invention are special
Different in nature real-time fluorescence PCR detection method linear dependence in the range of template amount 24-480000copies is good, amplification efficiency
Height, meets ENGL related requests [Definition of Minimum Performance Requirements for
Analytical Methods of GMO Testing]；And in the range of linearity that template amount is 24-480000copies, its
The SD of Ct values is between 0.02-0.61, and RSD shows the minimum template amount in linear scope between 0.05%-1.64%
During 24copies, its SD and RSD are respectively less than 25%, so the quantitative Monitoring lower-cut of the present invention is 24copies.
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 MON87411 strains
Row design primer and probe, the transgenic corns MON87411 strain specificity real-time fluorescence PCR detection methods of foundation can be to turning
Gene corn MON87411 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 MON87411 strain specificities real-time fluorescent PCR testing primer, probe, method and kit
<160> 6
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
caataggttc atttaaagtg atgga 25
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
ttctttttct ccatattgac catc 24
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence
<400> 3
catgtagatt tcccggacat gaagcc 26
<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> 210
<212> DNA
<213>Transgenic corns MON87411
<400> 5
tttcactgtc tacatgtatg tattttatga ctagacaata ggttcattta aagtgatgga 60
ttatttatta aaaggaaaat aaaaaggcaa aacactaatg aatagttaag tggcttcatg 120
tccgggaaat ctacatggat cagcaatgag tatgatggtc aatatggaga aaaagaaaga 180
gtaattacca attttttttc aattcaaaaa 210
<210> 6
<211> 360
<212> DNA
<213>HMG-MON87411 plasmids
<400> 6
cctgagcgag tcggtaagct ccatcttctg tactaaagta gtagttgatt ggactagaaa 60
tctcgtgctg attaattgtt ttacgcgtgc gtttgtgtgg attgtaggac aaggctccct 120
atgtagccaa ggctaacaag ctcaagctcg tttcactgtc tacatgtatg tattttatga 180
ctagacaata ggttcattta aagtgatgga ttatttatta aaaggaaaat aaaaaggcaa 240
aacactaatg aatagttaag tggcttcatg tccgggaaat ctacatggat cagcaatgag 300
tatgatggtc aatatggaga aaaagaaaga gtaattacca attttttttc aattcaaaaa 360
Claims (8)
1. a kind of transgenic corns MON87411 strain specificity real-time fluorescent PCR testing primers, it is characterised in that described inspection
Survey primer as follows：
MON87411-F：5’-CAATAGGTTCATTTAAAGTGATGGA-3’；
MON87411-R：5’-TTCTTTTTCTCCATATTGACCATC-3’.
2. a kind of transgenic corns MON87411 strain specificities real-time PCR detection probe, it is characterised in that described inspection
Probing pin is as follows：
MON87411-P：5 '-CATGTAGATTTCCCGGACATGAAGCC-3 ', 5 ' ends of probe are marked with fluorescent reporter group,
3' ends are marked with fluorescent quenching group.
3. transgenic corns MON87411 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 MON87411 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：
MON87411-F：5’-CAATAGGTTCATTTAAAGTGATGGA-3’；
MON87411-R：5’-TTCTTTTTCTCCATATTGACCATC-3’；
Described detection probe is as follows：
MON87411-P：5 '-CATGTAGATTTCCCGGACATGAAGCC-3 ', 5 ' ends of probe are marked with fluorescent reporter group,
3' ends are marked with fluorescent quenching group.
5. a kind of transgenic corns MON87411 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 MON87411 strains.
6. transgenic corns MON87411 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 MON87411-F and MON87411-R each 0.4 μ L, 10 μm of ol/L
The μ L of detection probe MON87411-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, 58 DEG C of 34s, 40 circulations, fluorescence signal is collected in 58 DEG C.
7. transgenic corns MON87411 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 MON87411 strains standard
For：If amplification curve has Representative fluorescence amplification curve, it is transgenic corns MON87411 strains to illustrate sample；If amplification
Curve is without Representative fluorescence amplification curve, then it is not transgenic corns MON87411 strains to illustrate sample.
8. a kind of transgenic corns MON87411 strain specificity real-time fluorescent PCR quantitative detection methods, it is characterised in that including
Following steps：
(1) genomic DNA for extracting transgenic corns MON87411 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 MON87411 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 MON87411 strains obtained, calculating obtains transgenic corns in measuring samples
The content of MON87411 strain genomic DNAs.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710482322.8A CN107254526A (en) | 2017-06-22 | 2017-06-22 | Transgenic corns MON87411 strain specificities real-time fluorescent PCR testing primer, probe, method and kit |
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| 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 |
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Application publication date: 20171017 |