CN104195260A - Novel genetically modified corn strain MIR604 detection kit and detection method of kit - Google Patents
Novel genetically modified corn strain MIR604 detection kit and detection method of kit Download PDFInfo
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- CN104195260A CN104195260A CN201410485406.3A CN201410485406A CN104195260A CN 104195260 A CN104195260 A CN 104195260A CN 201410485406 A CN201410485406 A CN 201410485406A CN 104195260 A CN104195260 A CN 104195260A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a novel genetically modified corn strain MIR604 detection kit. High-efficiency and fast detection of MIR604 is achieved by cleverly designing a specific primer and a competitive primer with marks aiming at the genetically modified corn strain MIR604, and applying a PCR (polymerase chain reaction) amplification and test strip joint detection technology. The method has the advantages of being strong in specificity, high in sensitivity, good in stability, convenient and fast to use, simple in principle and operation, free of a special instrument, and suitable for being applied and popularized by inspection and quarantine departments, and cost and time are saved.
Description
Technical field
The present invention relates to transgenic corns strain detection field, a kind of method and test kit thereof that utilizes pcr amplification and nucleic acid test strip associated detection technique rapid detection transgenic corns strain MIR604 is provided, has been applicable to Check and Examination of Port quarantine mechanism and applies the detection to transgenic corns strain MIR604.
Background technology
Be accompanied by the fast development of transgenic technology, genetically modified crops kind is more and more, cultivated area is cumulative year after year also, the crop species that transgenosis relates to is various, comprise soybean, corn, wheat, cotton, tomato, potato etc., and each transgenic strain has unique genome on position and border sequence, China takes examination and approval system to import transgenic corns product at present, corn strain is without permission prohibited from entering China, and setting up transgenic product strain authentication method has actual demand.
Because the gel imaging of regular-PCR method detects, real-time fluorescence PCR detection method all needs the high special inspecting equipment of price, expensive plant and instrument cost has limited the popularization of PCR detection method.
This research of the present invention is by designing dexterously Auele Specific Primer and the competitive primer for the tape label of transgenic corns strain MIR604, apply pcr amplification and nucleic acid test strip associated detection technique, realized the efficient rapid detection of transgenic corns strain MIR604.The method high specificity, highly sensitive and good stability, for improve MIR604 strain accuracy, shorten detection time and there is important effect.This detection method is convenient and swift, and the cost-saving and time, principle and simple to operate, does not need specific apparatus, is applicable to inspection and quarantine department and uses and promote, and the rapid detection of other corn strains is also had to important reference simultaneously.
Summary of the invention
The technical issues that need to address of the present invention are to provide for detection of the Auele Specific Primer of MIR604 and competitive primer.
Another problem that the present invention need to solve is to provide the PCR detection kit that comprises above-mentioned primer.
The present invention for detection of the PCR primer sequence of MIR604 is:
Upstream primer 604-F-bio: 5 ' biotin-TCCGCAATGTGTTATTAAGAGT-3 '
Downstream primer 604-B-fit:5 ' fitc-TATTATAATCCGAAACGGAGCA-3 '
Competition primer 604-F-comp:5 '-GGCCTTAATAACACATTGCGGA-3 '.
MIR604 detection kit of the present invention, comprises following composition:
(1) MIR604DNA template and negative control are intended MIR604 DNA profiling
(2) MIR604 upstream and downstream primer and competition primer
Upstream primer 604-F-bio: 5 ' biotin-TCCGCAATGTGTTATTAAGAGT-3 '
Downstream primer 604-B-fit:5 ' fitc-TATTATAATCCGAAACGGAGCA-3 '
Competition primer 604-F-comp:5 '-GGCCTTAATAACACATTGCGGA-3 '.
(3) 10 × ExTaq PCR Buffer, DDH
2o, 2.5mM dNTP, 5U/ μ l ExTaq enzyme
Testing sample DNA is carried out to pcr amplification with aforesaid method, PCR product ELISA test strip, if control line is normal, detection line presents the positive, illustrates that this sample contains MIR604, if present feminine gender, illustrates and in this sample, does not contain MIR604.
Compared with prior art, progress of the present invention is: use instrument simple, without agarose gel electrophoresis, ultraviolet gel imaging, thereby avoid the pollution of ethidium bromide, saved time, high specificity, highly sensitive, good stability, has reduced the input in laboratory, and applicable laboratories is promoted the use of.
Brief description of the drawings
Fig. 1 is transgenic corns strain MIR604 test strip specific detection result, 1 is MON810,2 for MON863,3 for TC1507,4 for ES3272,5 for GA21,6 be 59122,7 for MIR604,8 is for BT11,9 is for BT176,10 is for NK603,11 is for non-transgenic corn gene group DNA, 12 is for DDH
2o, can find out and use this test kit can detect exactly transgenic corns strain MIR604 from Fig. 1.
Embodiment
MIR604 design of primers:
1
TCCGCAATGT GTTATTAAGA GTTGGTGGTA CGGGTACTTT AACTAACGAG GTGTGTCGCG
61 CAGCGCTCCT GCACGGATGT AGCTTTGGAT TGCTGGATAA TGTCTCGCGC AAGCGTCGTA
121 TTTATTTATT TATTTATTAC AGCCTCCACC GCCGTGCG
TG CTCCGTTTCG GATTATAATA
According to the recombinant DNA sequence of transgenosis Bacillus licheniformis, utilize primer-design software to design two groups of primers, probe (underline position), upstream primer 5 ' mark vitamin H (biotin), downstream primer 5 ' mark fluorescent element (fitc), and upstream primer competition primer.
Upstream primer 604-F-bio: 5 ' biotin-TCCGCAATGTGTTATTAAGAGT-3 '
Downstream primer 604-B-fit:5 ' fitc-TATTATAATCCGAAACGGAGCA-3 '
Competition primer 604-F-comp:5 '-GGCCTTAATAACACATTGCGGA-3 '.
This experiment test 10 kinds of transgenic corns strains and a kind of non-transgenic corn, see the following form:
| Numbering | For examination corn kind |
| 1 | MON810 |
| 2 | MON863 |
| 3 | TC1507 |
| 4 | ES3272 |
| 5 | GA21 |
| 6 | 59122 |
| 7 | MIR604 |
| 8 | BT11 |
| 9 | BT176 |
| 10 | NK603 |
| 11 | Non-transgenic corn |
the detection of embodiment 1, transgenic corns strain MIR604
dNA extraction: adopt Qiagen DNeasy plant mini kit test kit, extract corn gene group DNA be dissolved in 100uL TE by step ,-20 DEG C save backup.
pcr amplification: respectively with MON810, MON863, TC1507, ES3272, GA21,59122, BT11, BT176, NK603, the negative contrast of non-transgenic corn gene group DNA, MIR604 genomic dna is as positive control, ddH
2o is blank, carries out pcr amplification.
PCR reaction system:
10×ExTaq Buffer 2.5μl
dNTP（2.5mmol/L） 1μl
Upstream primer (10 μ mol/L) 0.5 μ l
Downstream primer (10 μ mol/L) 0.5 μ l
Competition primer (10 μ mol/L) 4 μ l
Template DNA 1 μ l
ExTaq enzyme (5U/ μ L) 0.25 μ l
ddH
2O 15.25μl
Cumulative volume: 25 μ l
PCR reaction conditions: 94 DEG C of denaturation 1 min, 94 DEG C of sex change 30 s, 57 DEG C of annealing 30 s, 72 DEG C are extended 30 s, 30 circulations, 72 DEG C are extended 5 min.
eLISA test strip: get respectively 4 μ L amplified productions and be added in the sample pad of test strip, then drip 2-3 and drip damping fluid, interpretation record result after 5 min, result is as Fig. 1,1 is MON810,2 for MON863,3 for TC1507,4 for ES3272,5 for GA21,6 be 59122,7 for MIR604,8 is for BT11,9 is for BT176,10 is for NK603,11 is for non-transgenic corn gene group DNA, 12 is for DDH
2o.
Result shows, the control line of Fig. 1 is all normal, only have No. 7 MIR604 to occur specificity red stripes at detection line, all the other transgenic corns strains, non-transgenic corn and blank all occur without band, only in the time that the detection line of MIR604 test strip shows specific band, show to detect and in thing, contain transgenic corns strain MIR604 composition.
Sequence table
SEQUENCE LISTING
<110> Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection & Quarant
<120> new transgenic corns strain MIR604 detection kit and detection method thereof
<141> 2014－08－20
<160> 3
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
tccgcaatgt gttattaaga gt 22
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
tattataatc cgaaacggag ca 22
<210> 3
<211> 22
<212> DNA
<213> artificial sequence
<400> 3
ggccttaata acacattgcg ga 22
Sequence table
SEQUENCE LISTING
<110> Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection & Quarant
<120> new transgenic corns strain MIR604 detection kit and detection method thereof
<160> 3
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
tccgc aatgt gttat taaga gt 22
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
tatta taatc cgaaa cggag ca 22
<210> 3
<211> 22
<212> DNA
<213> artificial sequence
<400> 3
ggcct taata acaca ttgcg ga 22
Claims (3)
1. the Auele Specific Primer and the competitive primer that detect for transgenic corns strain MIR604, is characterized in that, sequence is:
Upstream primer 604-F-bio: 5 ' Biotin-TCCGCAATGTGTTATTAAGAGT-3 '
Downstream primer 604-B-fit:5 ' Fitc-TATTATAATCCGAAACGGAGCA-3 '
Competition primer 604-F-comp:5 '-GGCCTTAATAACACATTGCGGA-3 '.
2. the test kit for detection of transgenic corns strain MIR604 is characterized in that, comprises following composition:
(1) positive control transgenic corns strain MIR604 DNA profiling and negative control non-transgenic maize dna template
(2) MIR604 upstream and downstream primer and competition primer
Upstream primer 604-F-bio:5 ' Biotin-TCCGCAATGTGTTATTAAGAGT-3 '
Downstream primer 604-B-fit:5 ' Fitc-TATTATAATCCGAAACGGAGCA-3 '
Competition primer 604-F-comp:5 '-GGCCTTAATAACACATTGCGGA-3 '
(3) 10 × ExTaq PCR Buffer, ddH
2o, 2.5mM dNTP, 5U/ μ l ExTaq enzyme
(4) the membrane chromatographic test strip of vitamin H (Biotin), fluorescein (Fitc) antigenic mark.
3. a method for quick of transgenic corns strain MIR604, comprises the steps:
(1) PCR reaction system:
10×ExTaq Buffer 2.5μl
dNTP（2.5mmol/L） 1μl
Upstream primer (10 μ mol/L) 0.5 μ l
Downstream primer (10 μ mol/L) 0.5 μ l
Competition primer (10 μ mol/L) 4 μ l
Template DNA 1 μ l
ExTaq enzyme (5U/ μ L) 0.25 μ l
ddH
2O 15.25μl
Cumulative volume: 25 μ l
(2) PCR reaction conditions: 94 DEG C of denaturation 1 min, 94 DEG C of sex change 30 s, 57 DEG C of annealing 30 s, 72 DEG C are extended 30 s, 30 circulations, 72 DEG C are extended 5 min
(3) result of determination: PCR product ELISA test strip, if control line is normal, detection line presents the positive, illustrates that this sample contains transgenic corns strain MIR604, if present feminine gender, illustrates and does not contain transgenic corns strain MIR604 in this sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410485406.3A CN104195260B (en) | 2014-09-22 | 2014-09-22 | A kind of new Transgenic corn lines MIR604 detection kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410485406.3A CN104195260B (en) | 2014-09-22 | 2014-09-22 | A kind of new Transgenic corn lines MIR604 detection kit and detection method thereof |
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| CN104195260A true CN104195260A (en) | 2014-12-10 |
| CN104195260B CN104195260B (en) | 2015-12-30 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952861A (en) * | 2011-08-26 | 2013-03-06 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize |
| CN103205425A (en) * | 2012-01-17 | 2013-07-17 | 北京万达因生物医学技术有限责任公司 | Antisense interference oligonucleotide used for inhibiting primer non-specific amplification |
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2014
- 2014-09-22 CN CN201410485406.3A patent/CN104195260B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952861A (en) * | 2011-08-26 | 2013-03-06 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize |
| CN103205425A (en) * | 2012-01-17 | 2013-07-17 | 北京万达因生物医学技术有限责任公司 | Antisense interference oligonucleotide used for inhibiting primer non-specific amplification |
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| CN104195260B (en) | 2015-12-30 |
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Inventor after: Zhang Yujun Inventor after: Guo Jingze Inventor after: Zhao Jingyuan Inventor after: Liu Peng Inventor after: He Yan Inventor before: Zhang Yujun Inventor before: Guo Jingze Inventor before: Zhao Jinyuan Inventor before: Liu Peng Inventor before: He Yan |
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Granted publication date: 20151230 Termination date: 20160922 |
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