CN1420181A - Probe sequence and kit for real time fluorescence PCR detection of transgenic rapeseed - Google Patents
Probe sequence and kit for real time fluorescence PCR detection of transgenic rapeseed Download PDFInfo
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- CN1420181A CN1420181A CN 02133168 CN02133168A CN1420181A CN 1420181 A CN1420181 A CN 1420181A CN 02133168 CN02133168 CN 02133168 CN 02133168 A CN02133168 A CN 02133168A CN 1420181 A CN1420181 A CN 1420181A
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Abstract
A probe sequence and reagent kit for real-time fluorescent PCR detection of transgenic rapeseed is disclosed. The probe sequence for detecting Barnase gene, the modified CP4-EPSPS gene, the FMV355 promoter of repessed, and the endogenous gene of chloroplast, the maximal, minimal and preferable sequences, and the reagent kit are disclosed for quantitatively and qualitatively detecting the transgenic products. Its advantages are high sensitivity and correctness, high speed, and no cross infection.
Description
(1) technical field:
The present invention relates to the amplification and the detection of gene.
(2) background technology:
Along with the continuous commercialization of transgenic product, its safety issue is paid close attention to by common people always, and broken hair is not given birth to dispute.In order to ensure the healthy of the mankind, eliminate human consumer's misgivings, help international trade and circulation of commodities, with the European Union a lot of countries of representative, after having experienced of short duration dispute, these type of specialty products are all made the security control regulations, so that stringent regulations.The multinational rules of limiting the quantity of accordingly of also having formulated such as Europe, Japan.China Ministry of Agriculture has also put into effect the management method of transgenic product this year.Yet, the human consumer is understood or accept transgenic product, every rules can be implemented, and except giving relevant information and increasing transparency, key issue is to have corresponding science detection method to analyze and differentiate traditional product and transgenic product.Especially in the face of in the domestic and international trade to the requirement of limiting the quantity of of transgenic product, more should have accordingly, quantivative approach is to transgenic product exactly, especially genetically modified food carries out qualitative and quantitative analysis.In the detection method of the transgenic product of using always both at home and abroad at present, mainly be PCR detection method based on nucleic acid.PCR method is divided into qualitative PCR and quantifying PCR method again.Qualitative PCR, be that polymerase chain reaction PCR has been widely used in each research and the field such as clinical diagnosis based on nucleic acid, this method is used the primer of two sections (about more than 17～20 Nucleotide) oligonucleotide as reaction, and complementary action should not take place the sequence of these two sections Oligonucleolide primers.But they can with two chains of the DNA to be measured that is called template on complementation takes place respectively in certain site.Reaction solution is by comprising the reaction buffer that contains magnesium ion, 4 kinds of mononucleotide dNTP, template DNA and primers.Under archaeal dna polymerase catalysis, by variation of temperature, DNA sex change and annealing, and the dna segment between synthetic two complementary sites.Such reaction is carried out repeatedly, and the dna segment that first circulation is produced is increased.Through 25～30 circulations, amplification times reaches 10
6It is numerous and diverse to detect the whole process of transgenic product with this method, operation steps is many, and need the PCR aftertreatment, as sepharose electricity arteries and veins and ethidium bromide staining, UV-light observations or dye detection by polyacrylamide gel electrophoresis or silver not only needs multiple instrument, and waste time and energy, employed staining agent ethidium bromide is harmful again to human body, and these numerous and diverse experimentations provide chance for again pollution and false positive, have a strong impact on the correct judgement to the result.
(3) summary of the invention:
The present invention is according to real-time fluorescence quantitative PCR method principle, promptly be on the basis of qualitative PCR method, added a fluorescently-labeled probe, collect the record fluorescent signal, thereby can detect the initial copy number of testing sample, judge in the Semen Brassicae campestris sample to be measured whether contain the transgenosis composition, thereby determine whether Semen Brassicae campestris sample to be measured and converted products thereof are transgenic product.
Technical scheme of the present invention is: be to detect on the basis at conventional PCR, and the probe of two fluorophors that added a mark.Fluorescence report group (R) is marked at 5 of probe ' end, fluorescent quenching group (Q) is marked at 3 of probe ' end, both can constitute the transmission ofenergy structure, be that the fluorescence that the fluorescence report group is sent can be absorbed by the fluorescent quenching group, when the two is far away apart from change, restraining effect weakens, and the reporter group fluorescent signal strengthens.In the PCR process, probe is with PCR product hybridization since the Taq enzyme have 5 ' to 3 ' 5 prime excision enzyme activity, in the primer extension stage probe is cut off, fluorescent quenching group (Q) restraining effect disappears, the reporter group fluorescent signal strengthens.Follow the increase of PCR product, fluorescent signal strengthens thereupon, the fluorescent signal increased value Δ R of n circulation time
nEqual R
n/ Q
nDeduct R
0/ Q
0, R
n, R
0Reporter group fluorescent value when being respectively n circulation and PCR reaction beginning, Q
n, Q
0Quenching group fluorescent value when being respectively n circulation and PCR reaction beginning.3-15 round-robin fluorescent value of PCR reaction be as the fluorescence background signal, 3-15 round-robin Δ R
n10 times of standard deviation be set to fluorescence threshold (threshold), the cycle index when signal reaches this fluorescence threshold (Ct value) is directly proportional with the original template amount of PCR reaction.
The probe sequence that is used to detect transgenic rapeseed Barnase gene is: 5 '-agacgtcgctccggggaaaa-3 ', the maximal sequence of this probe is: 5 '-ttgcagacgtcgctccggggaaaagcatcggcggaga-3 ', the minmal sequence of this probe is: 5 '-gtcgctccgggg-3 '.
The probe sequence that is used to detect the CP4-EPSPS gene that transgenic rapeseed modifies is: 5 '-tgttccagaagaccgtgctc-3 ', the maximal sequence of this probe is: 5 '-gaagggtgttactg ttccagaagaccgtgctccttctatgatcg-3 ', the minmal sequence of this probe is: 5 '-ccagaagaccgt-3 '.
The probe sequence that is used to detect transgenic plant Semen Brassicae campestris FMV35S promotor is: 5 '-ctccaaaccattagccaaaa-3 ', the maximal sequence of this probe is: 5 '-ggtacagagtct ccaaaccattagccaaaagctacaggagat-3 ', the minmal sequence of this probe is: 5 '-aaaccattagcc-3 '.
The probe sequence that is used to detect the plant chloroplast native gene is: 5 '-gcaatcctgagccaaatcc-3 ', the maximal sequence of this probe is: 5 '-gcaatcctgagccaaatcc-3 ', the minmal sequence of this probe is: 5 '-tcctgagccaaa-3 '.
Be used for the test kit that the transgenic rapeseed real-time fluorescence PCR detects by what above-mentioned probe sequence was made.
The invention has the beneficial effects as follows: by detecting the endogenous reference gene (Endogenous Reference Gene) that crop itself is had, as plant chloroplast, can working sample in total dna profiling amount of genetically modified crops and non-transgenic crop; By detecting the foreign gene that changes in the genetically modified crops, the CP4-EPSPS gene of modifying as: Semen Brassicae campestris Barnase gene, Semen Brassicae campestris etc. can working sample in the dna profiling amounts of genetically modified crops, the typical curve of the endogenous reference gene by setting up genetically modified crops standard reference sample and poor (the Δ Ct) of foreign gene Ct value and transgene component percentage composition can calculate the percentage composition of transgene component in sample and the converted products thereof.The primer and the probe that are used for the transgenic product detection by quantitative also can carry out qualitative detection to transgenic product, have highly sensitive, avoid crossed contamination to cause false-positive advantage.Real-time fluorescence PCR technology of the present invention adopts complete stopped pipe to detect, and need not the PCR aftertreatment, has avoided crossed contamination and false positive.This technology has used dexterously that the DNA of round pcr efficiently increases, the specificity of nucleic acid hybridization and detection technique of fluorescence fast and susceptibility, make present method have simple to operate, time saving and energy saving, reliable results and accurate advantage such as sensitivity.
(4) description of drawings:
Fig. 1 detects principle schematic for real-time fluorescence PCR.
Fig. 2 transgenic product real-time fluorescence detection curve figure.
(a) pcr amplification appears in foreign gene,
(b) pcr amplification appears in endogenous reference gene.
Fig. 3 non-transgenic product real-time fluorescence detection curve figure.
(a) pcr amplification does not appear in foreign gene,
(b) pcr amplification appears in endogenous reference gene.
(5) embodiment:
Embodiment 1
Be used for probe sequence and test kit that the transgenic rapeseed real-time fluorescence PCR detects, detect transgenic rapeseed foreign gene (transgene component).
Detect gene: the Barnase gene.
Primer: 5 '-ctgggtggcatcaaaagggaacc-3 ', 5 '-tccggtctgaatttctgaagcctg-3 '.
Probe: 5 '-agacgtcgctccggggaaaa-3 '.
Test kit: being used to of using that this probe makes detected the test kit of the Barnase gene of transgenic rapeseed.
Embodiment 2
Be used for probe sequence and test kit that the transgenic rapeseed real-time fluorescence PCR detects, detect transgenic rapeseed foreign gene (transgene component).
Detect gene: the CP4-EPSPS gene of modification.
Primer: 5 '-gacttgcgtgttcgttcttc-3 ', 5 '-aacaccgttgagcttgagac-3 '.
Probe: 5 '-tgttccagaagaccgtgctc-3 '.
Test kit: being used to of using that this probe makes detected the test kit of the CP4-EPSPS gene that transgenic rapeseed modifies.
Embodiment 3
Be used for probe sequence and test kit that the transgenic rapeseed real-time fluorescence PCR detects, detect transgenic plant Semen Brassicae campestris foreign genes (transgene component).
Detect gene: the FMV35S promotor.
Primer: 5 '-aagcctcaacaaggtcag-3 ', 5 '-ctgctcgatgttgacaag-3 '.
Probe: 5 '-ctccaaaccattagccaaaa-3 '.
Test kit: being used to of using that this probe makes detected the test kit of the FMV35S promotor of transgenic plant Semen Brassicae campestris.
Embodiment 4
Be used for probe sequence and test kit that the transgenic rapeseed real-time fluorescence PCR detects, detect Semen Brassicae campestris plant chloroplast native gene (transgene component).
Detect gene: plant chloroplast.
Primer: 5 '-cgaaatcggtagacgctacg-3 ', 5 '-ttccattgagtctctgcacct-3 '.
Probe: 5 '-gcaatcctgagccaaatcc-3 '.
Test kit: the test kit of making by probe sequence that is used for transgenic rapeseed plant chloroplast native gene.
Detect key instrument: Bechtop, sterilization pot, ice-making machine, nucleic acid-protein analyser, high speed freezing centrifuge, the desk-type small whizzer, Mini people's whizzer, cryogenic refrigerator, refrigerating refrigerator, water purifior, distilled water device, vortex oscillator, microsyringe (0.5 μ L, 2 μ L, 10 μ L, 20 μ L, 100 μ L, 200 μ L, 1000 μ L) etc.
Detect main agents: 10 * PCR damping fluid, MgCl
2, dNTP (dATP, dUTP, dCTP, dGTP), UNG enzyme (Uracil N-glycosylase), Taq enzyme, primer and probe (pressing probe sequence generate a reagent box) etc.
Real-time fluorescence PCR check the primer and probe sequence are as described in the embodiment 1～4.
The real-time fluorescence PCR reaction system:
Reagent name stock solution concentration adds volume (μ L)
10 * PCR damping fluid 5.0
MgCl
2 25mol/μL 5.0
dNTP 2.5mol/μL 2.0
UNG enzyme 1U/ μ L 0.5
Primer 2 0p mol/ μ L positive 0.25 anti-0.25
Probe 10p mol/ μ L 0.3
Taq enzyme 5U/ μ L 0.25
Dna profiling
a20ng/L～30ng/ μ L 5 μ L
DdH
2O complements to reaction cumulative volume 50 μ L
Annotate 1:
aBe the template amount of raw material, converted products should be looked processing stage increases the template amount.
Annotate 2: different instruments, PCR reaction volume difference, each reagent should be adjusted in proportion.
The reaction parameter of real-time fluorescence quantitative PCR is: 50 ℃/2min (needing this step when using the UNG enzyme), pre-95 ℃/10min of sex change, 40 circulations: 95 ℃/15s, 60 ℃/1min.
Annotate: different instruments should be done reaction parameter suitably to adjust.
The selection of instrument detecting passage: the setting that PCR reaction tubes fluorescent signal is collected, should be consistent with the reporter group of probe institute mark.Concrete method to set up is different because of instrument, should be with reference to the instrument working instructions.
Last machine operation: put order by predefined sample the PCR reaction tubes is put successively, notice before the last machine checking whether each reaction tubes covers tightly, in order to avoid fluorescent substance leakage pollution instrument brings into operation and carries out the real-time fluorescence PCR reaction.
Interpretation of result and calculating:
Qualitative analysis:
The setting of baseline: after real-time fluorescence PCR reaction end and the analysis, invalid baseline scope should be set.No matter adopt any fluorescence channel baseline scope to be chosen in 3-20 circulation; The threshold value setting principle is with the vertex of baseline just above normal negative control amplification curve, and Ct value=40 are as the criterion.
The result judges: the testing sample foreign gene detects Ct value 〉=40, internal reference gene test Ct value 23-30 person, show not detect * * * gene; The testing sample foreign gene detects Ct value 28-34, internal reference gene test Ct value 23-30 person.Show to detect * * * gene.
Quantitative analysis:
With EXCEL or other method data calculated, can create typical curve with the corresponding cycle number Ct value of DNA concentration (x) (Y).For the concentration of sample DNA, can calculate (b is for intersecting point value, and m is a slope) according to regression equation logX=Y-b/m.Δ Ct value=foreign gene Ct value-native gene Ct value.With Δ Ct value substitution equation, can draw the relative content of a certain foreign gene in the sample.
As shown in Figure 2, (b) pcr amplification has appearred in native gene, and (a) pcr amplification has also appearred in foreign gene, shows that this sample detection has gone out the external source transgene component, is transgenic product.
As shown in Figure 3, (b) pcr amplification has appearred in native gene, and (a) pcr amplification does not appear in foreign gene, shows that this sample does not detect the external source transgene component.
Probe prepares the synthetic method manufacturing routinely.
Claims (10)
1, is used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the maximal sequence that is used to detect the probe of transgenic rapeseed Barnase gene is: 5 '-ttgcagacgtcgctccggggaaaagcatcggcggaga-3 ', the minmal sequence of probe is: 5 '-gtcgctccgggg-3 '.
2, according to claim 1ly be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the probe preferred sequence that is used to detect transgenic rapeseed Barnase gene is: 5 '-agacgtcgctccggggaaaa-3 '.
3, be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the maximal sequence of probe of CP4-EPSPS gene that is used to detect the modification of transgenic rapeseed is: 5 '-gaagggtgttactgttccagaagaccgtgctccttctatgatcg-3 ', the minmal sequence of probe is: 5 '-ccagaagaccgt-3 '.
4, according to claim 3ly be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the probe preferred sequence of CP4-EPSPS gene that is used to detect the modification of transgenic rapeseed is: 5 '-tgttccagaagaccgtgctc-3 '.
5, be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the maximal sequence that is used to detect the probe of transgenic plant Semen Brassicae campestris FMV35S promotor is: 5 '-ggtacagagtctccaaaccattagccaaaagctacaggagat-3 ', the minmal sequence of probe is: 5 '-aaaccattagcc-3 '.
6, according to claim 5ly be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the probe preferred sequence that is used to detect transgenic plant Semen Brassicae campestris FMV35S promotor is: 5 '-ctccaaaccattagccaaaa-3 '.
7, be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: be used to detect the maximal sequence 5 '-gcaatcctgagccaaatcc-3 ' of the probe of plant chloroplast native gene, the minmal sequence of probe is: 5 '-tcctgagccaaa-3 '.
8, according to claim 7ly be used for the probe sequence that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: the probe preferred sequence that is used to detect the plant chloroplast native gene is: 5 '-gcaatcctgagccaaatcc-3 '.
9, according to claim 1,2,3,4,5,6, the 7 or 8 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR, it is characterized in that: the fluorescence report group is marked at 5 of probe ' end, the fluorescent quenching group is marked at 3 of probe ' end, and both can constitute the transmission ofenergy structure.
10, be used for the test kit that the transgenic rapeseed real-time fluorescence PCR detects, it is characterized in that: be used for the test kit that the transgenic rapeseed real-time fluorescence PCR detects as what claim 1,2,3,4,5,6,7,8 or 9 probe sequence were made.
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| CN 02133168 CN1420181A (en) | 2002-10-11 | 2002-10-11 | Probe sequence and kit for real time fluorescence PCR detection of transgenic rapeseed |
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| CN 02133168 CN1420181A (en) | 2002-10-11 | 2002-10-11 | Probe sequence and kit for real time fluorescence PCR detection of transgenic rapeseed |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297668C (en) * | 2004-05-20 | 2007-01-31 | 厦门大学 | Transgenic product low-density gene chip detecting method |
| CN100552033C (en) * | 2007-01-24 | 2009-10-21 | 中国农业科学院油料作物研究所 | Flaking sequence of exogenous event inserting vector for transgenic rape Rf 2 and application thereof |
| CN100552032C (en) * | 2007-01-24 | 2009-10-21 | 中国农业科学院油料作物研究所 | Transgene rape Oxy-235 right boundary flanking sequence of exogenous event inserting vector and application thereof |
| CN100558902C (en) * | 2007-01-24 | 2009-11-11 | 中国农业科学院油料作物研究所 | Transgene rape exogenous origin gene integrator incident Rf1 exogenous insertion vector flanking sequence and application thereof |
| CN101020902B (en) * | 2007-01-24 | 2010-09-15 | 中国农业科学院油料作物研究所 | Flanking sequence of exogenous event inserting vector for transgenic rape Ms8 and its application |
| CN103131792A (en) * | 2012-12-19 | 2013-06-05 | 中国农业科学院油料作物研究所 | Barnase gene quantification PCR (polymerase chain reaction) detection method and application thereof |
| CN103757127A (en) * | 2014-02-05 | 2014-04-30 | 黄广平 | Rape transgenosis detecting kit |
| CN104531865A (en) * | 2014-12-24 | 2015-04-22 | 博奥生物集团有限公司 | Kit for detecting mycobacterium tuberculosis drug resistance gene and application of kit |
-
2002
- 2002-10-11 CN CN 02133168 patent/CN1420181A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297668C (en) * | 2004-05-20 | 2007-01-31 | 厦门大学 | Transgenic product low-density gene chip detecting method |
| CN100552033C (en) * | 2007-01-24 | 2009-10-21 | 中国农业科学院油料作物研究所 | Flaking sequence of exogenous event inserting vector for transgenic rape Rf 2 and application thereof |
| CN100552032C (en) * | 2007-01-24 | 2009-10-21 | 中国农业科学院油料作物研究所 | Transgene rape Oxy-235 right boundary flanking sequence of exogenous event inserting vector and application thereof |
| CN100558902C (en) * | 2007-01-24 | 2009-11-11 | 中国农业科学院油料作物研究所 | Transgene rape exogenous origin gene integrator incident Rf1 exogenous insertion vector flanking sequence and application thereof |
| CN101020902B (en) * | 2007-01-24 | 2010-09-15 | 中国农业科学院油料作物研究所 | Flanking sequence of exogenous event inserting vector for transgenic rape Ms8 and its application |
| CN103131792A (en) * | 2012-12-19 | 2013-06-05 | 中国农业科学院油料作物研究所 | Barnase gene quantification PCR (polymerase chain reaction) detection method and application thereof |
| CN103757127A (en) * | 2014-02-05 | 2014-04-30 | 黄广平 | Rape transgenosis detecting kit |
| CN104531865A (en) * | 2014-12-24 | 2015-04-22 | 博奥生物集团有限公司 | Kit for detecting mycobacterium tuberculosis drug resistance gene and application of kit |
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