CN1203187C - Probe sequence and kit for real time fluorescent PCR detection of transgenic corn - Google Patents
Probe sequence and kit for real time fluorescent PCR detection of transgenic corn Download PDFInfo
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- CN1203187C CN1203187C CN 02133170 CN02133170A CN1203187C CN 1203187 C CN1203187 C CN 1203187C CN 02133170 CN02133170 CN 02133170 CN 02133170 A CN02133170 A CN 02133170A CN 1203187 C CN1203187 C CN 1203187C
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Abstract
The present invention relates to a probe sequence and a kit for real-time fluorescent PCR detection on transgenic maize, which provides a probe sequence, the maximal, the minimal and the optimal sequences for detecting corresponding exogenous genes and endogenous genes of different kinds of transgenic maize. The kit made of the probe sequence is used for the quantitative detection and the qualitative detection of transgenic products, and has the advantages of high sensitivity and avoidance of false positives caused by cross contamination. The real-time fluorescent PCR technology of the present invention adopts completely closed tube detection with no need of PCR after-treatment to avoid cross contamination and false positives. The technology slickly uses the DNA high-efficiency amplification of the PCR technology, the specificity of nucleic acid hybridization, the rapidity and sensitivity of the fluorescence detection technology, so the method has the advantages of simple operation, time saving, labor saving, reliable result, accuracy, sensitivity, etc.
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 the polymerase chain reaction 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 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 whether contain the transgenosis composition in the corn sample to be measured, thereby determine whether corn 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.
Be used to detect the transgenic corn BT 11 kind, the link to each other probe sequence of gene fragment of the border of detecting IVS2 and pat gene is: 5 '-cgagggaacacgggagtctg-3 ', the maximal sequence of this probe is: 5 '-tgtcgagatccgagggaacacgggagtctgcccctgagac-3 ', the minmal sequence of this probe is: 5 '-ggaacacgggag-3 '.
Be used to detect transgenic corns MON810 kind, the link to each other probe sequence of gene fragment of the border of detecting Maize genome and CaMV35S gene is: 5 '-gcccagctatctgtcacttt-3 ', the maximal sequence of this probe is: 5 '-ctttgccattgcccagctatctgtcactttattgtgaaga-3 ', the minmal sequence of this probe is: 5 '-agctatctgtca-3 '.
Be used to detect transgenic corns MON810 kind, the link to each other probe sequence of gene fragment of the border of detecting HSP70 and CryIA (b) gene is: 5 '-ccaactgacactatattgct-3 ', the maximal sequence of this probe is: 5 '-ctagtatctaccaactgacactatattgcttctctttaca-3 ', the minmal sequence of this probe is: 5 '-ctgacactatat-3 '.
Be used to detect transgenic corns 176 kinds, the link to each other probe sequence of gene fragment of the border of detecting CDPK and CryIA (b) gene is: 5 '-atggacaacaaccccaacatc-3 ', the maximal sequence of this probe is: 5 '-ggatccaacaatggacaacaaccccaacatcaacgagtgca-3 ', the minmal sequence of this probe is: 5 '-caacaaccccaa-3 '.
The probe sequence of the continuous gene fragment in the border of gene is: 5 '-atctgggaactactcacaca-3 ', the maximal sequence of this probe is: 5 '-taattcccttatctgggaactactcacacattattatagaga-3 ', the minmal sequence of this probe is: 5 '-gggaactactca-3 '.
Be used to detect transgenic corns T14 and/or T25 kind, the link to each other probe sequence of gene fragment of the border of detecting CaMV35S and pat gene is: 5 '-tggctctgtcctaaagttca-3 ', the maximal sequence of this probe is: 5 '-tttgtggctctgtcctaaagttcactgtagacgtc-3 ', the minmal sequence of this probe is: 5 '-tctgtcctaaag-3 '.
The probe sequence of the continuous gene fragment in the border of gene is: 5 '-cgtgcccgtgaacccagctc-3 ', the maximal sequence of this probe is: 5 '-gatcgtgcccgtgaacccagctcgcgaggccga-3 ', the minmal sequence of this probe is: 5 '-cccgtgaaccca-3 '.
Be used to detect transgenic corns GA21 kind, the link to each other probe sequence of gene fragment of the border of detecting OTP and mEPSPS gene is: 5 '-acctgccgccgctgtctatg-3 ', the maximal sequence of this probe is: 5 '-cgctgtcgtacctgccgccgctgtctatggcgcccaccgt-3 ', the minmal sequence of this probe is: 5 '-gccgccgctgtc-3 '.
The probe sequence that is used to detect corn Zein native gene is: 5 '-taagatgccagctgcgattg-3 ', the maximal sequence of this probe is: 5 '-gggtgataaaggtaagatgccagctgcgattgcctgttggatcc-3 ', the minmal sequence of this probe is: 5 '-atgccagctgcg-3 '.
Be used for the test kit that the transgenic corns 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 the alcohol soluble protein gene of corn (Zein), can working sample in the total dna profiling amount of genetically modified crops and non-transgenic crop; By detecting the foreign gene that changes in the genetically modified crops, as: the border of IVS2 and pat gene link to each other gene fragment 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 corns real-time fluorescence PCR detects, detect BT11 corn gene product foreign gene (transgene component).
The border of detecting gene: IVS2 and the pat gene gene fragment that links to each other.
Primer: 5 '-ctgggaggccaaggtatctaat-3 ' 5 '-gctgctgtagctggcctaatct-3 '.
Probe: 5 '-cgagggaacacgggagtctg-3 '.
Test kit: use the test kit that is used to detect the transgenic corn BT 11 kind that this probe is made.
Embodiment 2
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect MON810 corn gene product foreign gene (transgene component).
Detect gene: the border of Maize genome and the CaMV35S gene gene fragment that links to each other.
Primer: 5 '-tcgaaggacgaaggactctaacg-3 ', 5 '-tccatctttgggaccactgtcg-3 '.
Probe: 5 '-gcccagctatctgtcacttt-3 '.
Test kit: being used to of using that this probe makes the border of detecting the Maizegenome of transgenic corns MON810 and the CaMV35S gene test kit of gene fragment that links to each other.
Embodiment 3
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect MON810 corn gene product foreign gene (transgene component).
The border of detecting gene: HSP70 and CryIA (b) the gene gene fragment that links to each other.
Primer: 5 '-agtttcctttttgttgctctcct-3 ', 5 '-gatgtttgggttgttgtccat-3 '.
Probe: 5 '-ccaactgacactatattgct-3 '.
Test kit: being used to of using that this probe makes the border of detecting the HSP70 of transgenic corns MON810 and CryIA (b) the gene test kit of gene fragment that links to each other.
Embodiment 4
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect 176 corn gene product foreign genes (transgene component).
The border of detecting gene: CDPK and CryIA (b) the gene gene fragment that links to each other.
Primer: 5 '-ctctcgccgttcatgttcgt-3 ', 5 '-ggtcaggctcaggctgatgt-3 '.
Probe: 5 '-atggacaacaaccccaacatc-3 '.
Test kit: use the test kit that is used to detect transgenic corns 176 that this probe is made.
Embodiment 5
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect T14/T25 corn gene product foreign gene (transgene component).
The border of the gene gene fragment that links to each other.
Primer: 5 '-atggtggatggcatgatgttg-3 ', 5 '-tgagcgaaaccctataagaaccc-3 '.
Probe: 5 '-atctgggaactactcacaca-3 '.
The test kit of the continuous gene fragment in the border of gene.
Embodiment 6
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect T14/T25 corn gene product foreign gene (transgene component).
The border of detecting gene: CaMV35S and the pat gene gene fragment that links to each other.
Primer: 5 '-ccttcgcaagacccttcctctata-3,5 '-agatcatcaatccactcttgtggtg-3 '.
Probe: 5 '-tggctctgtcctaaagttca-3 '.
Test kit: being used to of using that this probe makes the border of detecting the CaMV35S of transgenic corns T14/T25 and the pat gene test kit of gene fragment that links to each other.
Embodiment 7
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect CBH-351 corn gene product foreign gene (transgene component).
The border of the gene gene fragment that links to each other.
Primer: 5 '-tactacatcgaccgcatcga-3 ', 5 '-cctaattcccttatctggga-3 '.
Probe: 5 '-cgtgcccgtgaacccagctc-3 '.
Test kit: use the test kit that is used to detect transgenic corns CBH-351 that this probe is made.
Embodiment 8
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, detect GA21 corn gene product foreign gene (transgene component).
The border of detecting gene: OTP and the mEPSPS gene gene fragment that links to each other.
Primer: 5 '-acggtggaagagttcaatgtatg-3 ', 5 '-tctccttgatgggctgca-3 '.
Probe: 5 '-acctgccgccgctgtctatg-3 '.
Test kit: use the test kit that is used to detect transgenic corns GA21 that this probe is made.
Embodiment 9
Be used for probe sequence and test kit that the transgenic corns real-time fluorescence PCR detects, be used to detect the corn native gene.
Detect gene: zein.
Primer: 5 '-cctatagcttcccttcttcc-3 ', 5 '-tgctgtaatagggctgatga-3 '.
Probe: 5 '-taagatgccagctgcgattg-3 '.
Test kit: use the test kit that is used to detect corn and/or transgenic corns native gene that this probe is made.
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 1～9.
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/ μ L～30ng/ μ L 5 μ L
DdH
2O complements to reaction cumulative volume 50L
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 to notice checking whether each reaction tubes covers tightly before order is put machine successively with the PCR reaction tubes by predefined sample, 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 (25)
1, is used for the probe sequence that the transgenic corns real-time fluorescence PCR detects, it is characterized in that: be used to detect the transgenic corn BT 11 kind, the link to each other maximal sequence of probe of gene fragment of the border of detecting IVS2 and pat gene is: 5 '-tgtcgagatccgagggaacacgggagtctgcccctgagac-3 ', and the minmal sequence of probe is: 5 '-ggaacacgggag-3 '; Be used to detect transgenic corns MON810 kind, the link to each other maximal sequence of probe of gene fragment of the border of detecting Maize genome and CaMV35S gene is: 5 '-ctttgccattgcccagctatctgtcactttattgtgaaga-3 ', and the minmal sequence of probe is: 5 '-agctatctgtca-3 '; Be used to detect transgenic corns 176 kinds, the link to each other maximal sequence of probe of gene fragment of the border of detecting CDPK and CryIA (b) gene is: 5 '-ggatccaacaatggacaacaaccccaacatcaacgagtgca-3 ', and the minmal sequence of probe is: 5 '-caacaaccccaa-3 '; The maximal sequence of the probe of the continuous gene fragment in the border of gene is: 5 '-taattcccttatctgggaactactcacacattattatagaga-3 ', and the minmal sequence of probe is: 5 '-gggaactactca-3 '; The maximal sequence of the probe of the continuous gene fragment in the border of gene is: 5 '-gatcgtgcccgtgaacccagctcgcgaggccga-3 ', and the minmal sequence of probe is: 5 '-cccgtgaaccca-3 '; Be used to detect transgenic corns GA21 kind, the link to each other maximal sequence of probe of gene fragment of the border of detecting OTP and mEPSPS gene is: 5 '-cgctgtcgtacctgccgccgctgtctatggcgcccaccgt-3 ', the minmal sequence of probe is: 5 '-gccgccgctgtc-3 '.
2, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 1, it is characterized in that: be used to detect transgenic corns MON810 kind, the link to each other maximal sequence of probe of gene fragment of the border of detecting HSP70 and CryIA (b) gene is: 5 '-ctagtatctaccaactgacactatattgcttctctttaca-3 ', the minmal sequence of probe is: 5 '-ctgacactatat-3 '.
3, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 1, it is characterized in that: be used to detect transgenic corns T14 and/or T25 kind, the link to each other maximal sequence of probe of gene fragment of the border of detecting CaMV35S and pat gene is: 5 '-tttgtggctctgtcctaaagttcactgtagacgtc-3 ', the minmal sequence of probe is: 5 '-tctgtcctaaag-3 '.
4, according to claim 1, the 2 or 3 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR, it is characterized in that: be used to detect the transgenic corn BT 11 kind, the link to each other sequence of probe of gene fragment of the border of detecting IVS2 and pat gene is: 5 '-cgagggaacacgggagtctg-3 '.
5, according to claim 1, the 2 or 3 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR, it is characterized in that: be used to detect transgenic corns MON810 kind, the link to each other sequence of probe of gene fragment of the border of detecting Maize genome and CaMV35S gene is: 5 '-gcccagctatctgtcacttt-3 '.
6, according to claim 1, the 2 or 3 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR, it is characterized in that: be used to detect transgenic corns 176 kinds, the link to each other sequence of probe of gene fragment of the border of detecting CDPK and CryIA (b) gene is: 5 '-atggacaacaaccccaacatc-3 '.
The sequence of the probe of the continuous gene fragment in the border of gene is: 5 '-atctgggaactactcacaca-3 '.
The sequence of the probe of the continuous gene fragment in the border of gene is: 5 '-cgtgcccgtgaacccagctc-3 '.
9, according to claim 1, the 2 or 3 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR, it is characterized in that: be used to detect transgenic corns GA21 kind, the link to each other sequence of probe of gene fragment of the border of detecting OTP and mEPSPS gene is: 5 '-acctgccgccgctgtctatg-3 '.
10, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 2, it is characterized in that: be used to detect transgenic corns MON810 kind, the link to each other sequence of probe of gene fragment of the border of detecting HSP70 and CryIA (b) gene is: 5 '-ccaactgacactatattgct-3 '.
11, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 3, it is characterized in that: be used to detect transgenic corns T14 and/or T25 kind, the link to each other sequence of probe of gene fragment of the border of detecting CaMV35S and pat gene is: 5 '-tggctctgtcctaaagttca-3 '.
12, according to claim 1,2,3, the 10 or 11 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR, it is characterized in that: 5 ' end mark fluorescent reporter group of probe, 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
13, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 4 is characterized in that: 5 ' end mark fluorescent reporter group of probe, and 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
14, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 5 is characterized in that: 5 ' end mark fluorescent reporter group of probe, and 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
15, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 6 is characterized in that: 5 ' end mark fluorescent reporter group of probe, and 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
16, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 7 is characterized in that: 5 ' end mark fluorescent reporter group of probe, and 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
17, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 8 is characterized in that: 5 ' end mark fluorescent reporter group of probe, and 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
18, the probe sequence that is used for the detection of transgenic corns real-time fluorescence PCR according to claim 9 is characterized in that: 5 ' end mark fluorescent reporter group of probe, and 3 ' end mark fluorescent quenching group of probe, both can constitute the transmission ofenergy structure.
19, the test kit of making according to claim 1,2,3, the 10 or 11 described probe sequences that are used for the detection of transgenic corns real-time fluorescence PCR that is used for the detection of transgenic corns real-time fluorescence PCR.
20, according to claim 4ly be used for the test kit that the transgenic corns real-time fluorescence PCR detects that is used for that probe sequence that the transgenic corns real-time fluorescence PCR detects makes.
21, according to claim 5ly be used for the test kit that the transgenic corns real-time fluorescence PCR detects that is used for that probe sequence that the transgenic corns real-time fluorescence PCR detects makes.
22, according to claim 6ly be used for the test kit that the transgenic corns real-time fluorescence PCR detects that is used for that probe sequence that the transgenic corns real-time fluorescence PCR detects makes.
23, according to claim 7ly be used for the test kit that the transgenic corns real-time fluorescence PCR detects that is used for that probe sequence that the transgenic corns real-time fluorescence PCR detects makes.
24, according to claim 8ly be used for the test kit that the transgenic corns real-time fluorescence PCR detects that is used for that probe sequence that the transgenic corns real-time fluorescence PCR detects makes.
25, according to claim 9ly be used for the test kit that the transgenic corns real-time fluorescence PCR detects that is used for that probe sequence that the transgenic corns real-time fluorescence PCR detects makes.
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| CN102409094B (en) * | 2011-11-17 | 2013-06-26 | 四川省农业科学院分析测试中心 | Transgenic corn NK603 strain specific quantitative PCR (Polymerase Chain Reaction) accurate detection method |
| CN102409092B (en) * | 2011-11-17 | 2013-06-26 | 四川省农业科学院分析测试中心 | Structure-specific quantitative PCR (polymerase chain reaction) accurate detection method of transgenic maize NK603 |
| CN102965445A (en) * | 2012-12-11 | 2013-03-13 | 福建出入境检验检疫局检验检疫技术中心 | LAMP (loop-mediated isothermal amplification) detection primer and method for transgenic maize GA21 strain |
| CN104894280A (en) * | 2015-06-25 | 2015-09-09 | 蔡先全 | Primer, kit and method for detecting transgenic maize NOS terminator |
| CN105255874B (en) * | 2015-11-09 | 2019-06-14 | 北京出入境检验检疫局检验检疫技术中心 | A kind of kit and detection method of accurate quantification detection Transgenic corn lines T25 |
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