CN110093439A - A method of for detecting the probe, surface plasma resonance biological sensor and detection genetically modified plants of genetically modified plants - Google Patents
A method of for detecting the probe, surface plasma resonance biological sensor and detection genetically modified plants of genetically modified plants Download PDFInfo
- Publication number
- CN110093439A CN110093439A CN201910290711.XA CN201910290711A CN110093439A CN 110093439 A CN110093439 A CN 110093439A CN 201910290711 A CN201910290711 A CN 201910290711A CN 110093439 A CN110093439 A CN 110093439A
- Authority
- CN
- China
- Prior art keywords
- probe
- concentration
- genetically modified
- modified plants
- sensing chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000523 sample Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 241000196324 Embryophyta Species 0.000 claims description 34
- 230000004044 response Effects 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 238000010168 coupling process Methods 0.000 claims description 20
- 230000008878 coupling Effects 0.000 claims description 19
- 238000005859 coupling reaction Methods 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 14
- 238000011069 regeneration method Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 230000008929 regeneration Effects 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- 240000007594 Oryza sativa Species 0.000 claims description 6
- 235000007164 Oryza sativa Nutrition 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 230000001172 regenerating effect Effects 0.000 claims description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000007995 HEPES buffer Substances 0.000 claims description 5
- 235000009566 rice Nutrition 0.000 claims description 5
- 239000004094 surface-active agent Substances 0.000 claims description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 4
- 108010090804 Streptavidin Proteins 0.000 claims description 4
- 244000189799 Asimina triloba Species 0.000 claims description 3
- 235000006264 Asimina triloba Nutrition 0.000 claims description 3
- 235000009467 Carica papaya Nutrition 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 2
- 235000020958 biotin Nutrition 0.000 claims description 2
- 239000011616 biotin Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 13
- 108020004414 DNA Proteins 0.000 description 16
- 238000012360 testing method Methods 0.000 description 11
- 230000009261 transgenic effect Effects 0.000 description 6
- 239000007836 KH2PO4 Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000003869 genetically modified organism Nutrition 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 244000037671 genetically modified crops Species 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000001216 nucleic acid method Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Classifications
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Botany (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This disclosure relates to a kind of probe for detecting genetically modified plants, surface plasma resonance biological sensor and the method for detecting genetically modified plants.The nucleotides sequence of the probe is classified as sequence shown in SEQ ID NO:1.The probe of the disclosure and surface plasma resonance biological sensor containing the probe can specifically detect CaMV35S promoter, and detection sensitivity is high and renewable, be easy to use.Surface plasma resonance biological sensor using the probe and containing the probe sensitive efficiently can specifically detect the genetically modified plants containing CaMV35S promoter and its Related product.
Description
Technical field
This disclosure relates to which biological technology products analyze testing field, and in particular to a kind of for detecting the spy of genetically modified plants
Needle, surface plasma resonance biological sensor and the method for detecting genetically modified plants.
Background technique
As transgenic technology gradually increases in the application of agriculture field and rapid development, the type of transgenic product with quantity
Add, the safety of transgenic product also increasingly by public's extensive concern, detection GMOs technical system also there is an urgent need to
Lasting innovation and development.Currently, the detection of genetically modified organism mainly includes two methods: one is the detection sides based on nucleic acid
Method, including genetic chip and PCR method, and the PCR method reaction time is longer, experimental arrangement is complicated and is " end-point method ", i.e., often
Reagent, consumptive material not reproducible use after the end of the experiment used in one reaction, and cannot be real in entire reaction process
Existing real-time monitoring and regeneration；Another then be the detection method based on protein, though this method is cheap, sensitivity is low,
And it is suitable only for the detection of a few genetically modified crops.
Summary of the invention
Purpose of this disclosure is to provide a kind of probe and detection sensitivity reproducible biosensor again can be improved.
To achieve the goals above, disclosure first aspect provides a kind of for detecting the probe of genetically modified plants, described
The nucleotides sequence of probe is classified as sequence shown in SEQ ID NO:1.
Disclosure second aspect provides a kind of for detecting the surface plasma resonance biological sensor of genetically modified plants, institute
Stating surface plasma resonance biological sensor includes sensing chip, there is described in the disclosure first aspect coupling on the sensing chip
Probe.
Optionally, the censorchip surface is combined with Streptavidin；The end of probe 5 ' is marked with biotin.
Optionally, the coupling includes the following steps: the conjugate solution containing the probe flowing through the chip, makes institute
Probe is stated in conjunction with the chip surface, obtains the surface plasma resonance biological sensor.
Optionally, the conjugate solution also contains HEPES, NaCl, EDTA and Surfactant P20；The probe is in institute
Stating the concentration in conjugate solution is 20~50nM, and the time of the coupling is 180~300s；The coupling amount of the probe is 200
~400RU.
The disclosure third aspect provides a kind of method for detecting genetically modified plants, and the target gene of the detection is
CaMV35S promoter, this method comprises the following steps: S1, extracts the genomic DNA to measuring plants；S2 makes containing the gene
The solution of group DNA flows through the sensing chip, and detects RU value response signal；It is described to be measured if generating RU value response signal
Contain the target gene in plant, it is described to not contain the target base in measuring plants if not generating RU value response signal
Cause；The sensing chip is the sensing chip of sensor described in disclosure second aspect.
Optionally, the solution containing the genome in step S2 is the genomic DNA and PBSM buffer
Mixed solution；The flow velocity that the mixed solution flows through the sensing chip is 10~30 μ L/min, and the time is 120~300s.
Optionally, the pH of the PBSM buffer be 7.0~8.0, the PBSM buffer contain concentration be 125~
The Na that KCl that the NaCl of 150mM, concentration are 2.0~3.0mM, concentration are 10~15mM2HPO4, concentration be 1~5mM
KH2PO4, the polysorbas20 that concentration is 0.0005~0.001 volume % and the MgCl that concentration is 15~20mM2。
Optionally, this method further includes that upon step s 2, the sensing chip is regenerated, the regeneration method packet
It includes: so that regenerative agent is contacted 30~60s with the sensing chip and carry out regeneration treatment；The regenerative agent be concentration be 10mM~
The HCl and/or concentration of 50mM is the NaOH of 10~50mM.
Optionally, described to measuring plants includes at least one of rice, corn and soybean, rape, cotton and pawpaw.
Through the above technical solutions, the probe of the disclosure and the surface plasma resonance biological sensor energy containing the probe
Enough specifically to detect CaMV35S promoter, detection sensitivity is high and renewable, is easy to use.Using the probe and contain this
The surface plasma resonance biological sensor of probe, which sensitive efficiently can specifically be detected, turns base containing CaMV35S promoter
Because of plant.
Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
Attached drawing is and to constitute part of specification for providing further understanding of the disclosure, with following tool
Body embodiment is used to explain the disclosure together, but does not constitute the limitation to the disclosure.In the accompanying drawings:
Fig. 1 is a kind of detection principle diagram of specific embodiment of the surface plasma resonance biological sensor of the disclosure；
Fig. 2 is six sample detection regenerated outcome figures of embodiment of the disclosure 1；
Fig. 3 is the signal response diagram that embodiment of the disclosure 1 detects that the rice genome of gene transgenic containing p35S generates；
Fig. 4 is the specific test result figure of the surface plasma resonance biological sensor of embodiment of the disclosure 1；
Fig. 5 is the sensitivity test result figure of the surface plasma resonance biological sensor of embodiment of the disclosure 1.
Specific embodiment
It is described in detail below in conjunction with specific embodiment of the attached drawing to the disclosure.It should be understood that this place is retouched
The specific embodiment stated is only used for describing and explaining the disclosure, is not limited to the disclosure.
Disclosure first aspect provides a kind of for detecting the probe of genetically modified plants, and the nucleotides sequence of probe is classified as SEQ
Sequence shown in ID NO:1.Wherein, sequence shown in SEQ ID NO:1 is 5 '-GGCAGAGGCATCTTGAACGATAGCC-
3’。
Disclosure second aspect provides a kind of for detecting the surface plasma resonance biological sensor of genetically modified plants, table
Face plasma resonance biological sensor includes sensing chip, and coupling has the probe of disclosure first aspect on sensing chip.
The probe of the disclosure and surface plasma resonance biological sensor containing the probe can be detected specifically
CaMV35S promoter, detection sensitivity is high and renewable, is easy to use.Surface using the probe and containing the probe etc. from
Sub-resonance biosensor sensitive efficiently can specifically detect the genetically modified plants containing CaMV35S promoter.
According to the disclosure, sensing chip is preferably the chip that there is Streptavidin on surface, is for example, purchased from GE
The SA chip of Healthcare；Further, 5 ' ends of probe can mark, in order to which probe and chip are coupled.
According to the disclosure, the structure and testing principle of surface plasma resonance biological sensor can be conventional for this field
, such as can be using the Biacore purchased from GE HealthcareTM(SPR is raw for T200 surface plasma resonance biological sensor
Object sensor), the surface plasma resonance for obtaining the disclosure by the above-mentioned probe for being coupled the disclosure in censorchip surface is raw
Object sensor, testing principle is for example shown in Fig. 1.
According to the disclosure, what the method for coupling can be conventional for this field.In order to guarantee probe in the coupling of chip surface
Effect, it is preferable that coupling may include steps of: the conjugate solution containing probe being flowed through chip, makes probe and chip list
Face combines, and obtains surface plasma resonance biological sensor；Conjugate solution containing probe can buffer for probe and HBS-EP+
The mixed solution of liquid, i.e. conjugate solution can also contain HEPES, NaCl, EDTA and Surfactant P20, in a kind of embodiment party
In formula, conjugate solution can contain 0.1M HEPES, 1.5M NaCl, 30mM EDTA, 0.5 volume %Surfactant P20；
Further, concentration of the probe in mixed solution can be 10~50nM, and preferably 20nM, the time of coupling can be 180
~300s, preferably 300s, the flow velocity flowed through can be 5~20 μ L/min, preferably 5 μ L/min.
Further, in order to improve biosensor detection sensitivity and guarantee to the special of CaMV35S promoter
Property detection, it is preferable that the coupling amount of probe can be 200~400RU, more preferably 300RU.Wherein, the coupling amount of probe can
To be tested using the conventional method of this field, details are not described herein again, such as in the disclosure, and the coupling amount of probe can pass through table
The RU value of face plasma resonance biological sensor characterizes, and RU refers to the response that surface plasma resonance biological sensor detection generates
Signal value unit.
The disclosure third aspect provides a kind of method for detecting genetically modified plants, and the target gene of detection opens for CaMV35S
Mover, this method comprises the following steps: S1, extracts the genomic DNA to measuring plants；S2 makes the solution stream containing genomic DNA
Through sensing chip, and detect RU value response signal；If generating RU value response signal, to contain target gene in measuring plants, if
RU value response signal is not generated, then to not contain target gene in measuring plants；Sensing chip is the sensing of disclosure second aspect
The sensing chip of device.
The method of the detection genetically modified plants of the disclosure can carry out specific inspection to the plant containing CaMV35S promoter
It surveys, detection method is convenient and efficient, high sensitivity and specificity are good.
In disclosed method, CaMV35S promoter can contain p35S target sequence, the nucleotides sequence of p35S target sequence
Column can be sequence shown in SEQ ID NO:2, and wherein sequence shown in SEQ ID NO:2 is 5 '-
GGCTATCGTTCAAGATGCCTCTGCC-3’。
In disclosed method, the solution containing genome in step S2 is genomic DNA and PBSM buffer
Mixed solution, the pH of PBSM buffer can be 7.0~8.0, for example, 7.4, PBSM buffer can be 125 containing concentration~
The Na that KCl that the NaCl of 150mM, concentration are 2.0~3.0mM, concentration are 10~15mM2HPO4, concentration be 1~5mM
KH2PO4, the polysorbas20 that concentration is 0.0005~0.001 volume % and the MgCl that concentration is 15~20mM2；Such as in a kind of implementation
In mode, KCl that NaCl that PBSM buffer can be 135mM containing concentration, concentration are 2.6mM, concentration are 13mM's
Na2HPO4, concentration be 2mM KH2PO4, the polysorbas20 that concentration is 0.0005 volume % and the MgCl that concentration is 15mM2；Further
Ground, in order to improve detection efficiency and accuracy, it is preferable that the mixed solution of genomic DNA and PBSM buffer flows through sensing core
The flow velocity of piece can be 10~30 μ L/min, and further preferably 10 μ L/min, the time flowed through can be 120~300s, into
One step is preferably 300s.
In an embodiment of the present disclosure, in order to avoid the interference of buffer substrate bring, above-mentioned can be contained
The coupling for having the solution of genome to be injected into sensor has in the TCH test channel of probe and the blank channel of non-conjugated probes, and will
The difference of the RU value response signal of the RU value response signal and blank channel of TCH test channel detection is as detection RU value response signal, so
After judged.
In one embodiment, this method can also include upon step s 2, sensing chip being regenerated, regenerated side
Method may include: to make regenerative agent contact 30~60s with sensing chip to carry out regeneration treatment, preferably contact 30s；Regenerative agent can
Think that the HCl that concentration is 10mM~50mM and/or the NaOH that concentration is 10~50mM, regenerative agent flow through the flow velocity of sensing chip
It can be 20~30 μ L/min, further preferably 30 μ L/min, such as it in one embodiment can be with 30uL/min's
The HCl solution of 50mM is injected 30s by flow velocity.
In disclosed method, the genetically modified plants containing CaMV35S promoter can be detected, to measuring plants
It can be the plant containing CaMV35S promoter of conventional kind, such as to measuring plants may include rice, corn and soybean, oil
At least one of dish, cotton and pawpaw.
The disclosure is further illustrated by the following examples, but therefore the disclosure is not any way limited.
In the case where not illustrated in following embodiment and comparative examples of the disclosure, reagent and instrument used
It can be this field conventional commercial product.
Embodiment
Using 5 ' the biotinylated ssDNA probes (p35S probe, SEQ ID NO:1) in end.
Comparative example
Using not fully complementary oligonucleotide, i.e., non-p35S target sequence (non-target), the nucleosides of non-target
Acid sequence is GCGAAACTGTGGAATTGATCAGCGT (SEQ ID NO:3).
Test case
Embodiment middle probe is coupled to respectively on the channel 2 of chip, is prepared into embodiment surface plasma resonance respectively
Then biosensor carries out sample introduction, regeneration and sensitivity and specificity test respectively, the specific method is as follows:
Coupling: biotinylated ssDNA probe (p35S probe, SEQ ID NO:1) is held to be coupled to chip 20nM 5 '
Channel 2 on, mobile phase that coupling process uses is HBS-EP+1 × buffer (by HBS-EP+10 × buffer solution dilution 10
It obtains again, pH value is 7.4 after dilution, wherein including 0.1M HEPES, 1.5M NaCl, 30mM EDTA, 0.5% (v/v)
Surfactant P20), sample introduction flow velocity is 5uL/min, time 300s, and coupling amount reaches about 300RU, obtain the table of embodiment
Face plasma resonance biological sensor.
Sample introduction and regeneration: (1 is logical after the p35S target for being 100nM by concentration, continuous six 1,2 channels of injection and regeneration
Road is reference channel), the variation of more each sensor response, as a result as shown in Figure 2；The mobile phase used during this for
PBSM buffer (pH 7.4), wherein including 135mM NaCl, 2.6mM KCl, 13mM Na2HPO4、2mM KH2PO4、
0.0005% (v/v) polysorbas20, and 15mM MgCl2.Flow velocity is 10uL/min, sample injection time 300s during sample introduction.It adopts
The 50mM HCl solution for being 30uL/min with flow velocity injection 30s carries out regeneration treatment, and table 1 is that 50mM HCl regeneration cycle stablizes knot
Close response.
The response of 1 50mM HCl regeneration cycle stable bond of table
Sample detection:
The non-transgenic oryza sativa genomic dna of extraction is injected separately into the 1 of sensor, 2 channels, and (1 channel is logical for reference
Road), 1,2 channels do not generate signal response.
The transgenic paddy rice genomic DNA comprising p35S element of extraction is injected separately into the 1 of sensor, 2 channels, 2 is logical
Road generates signal response, as shown in Figure 3.1 channel is not coupled any ssDNA probe that can be complementary with target analytes, for ginseng
Than channel, the influence of buffer solution background is excluded in experiment and proves the signal intensity generated on sense channel and non-targeted point
Analysis object causes in conjunction with the original Streptavidin of chip surface, therefore, often reduces the signal response generated on 1 channel.
Specificity and sensitivity technique:
Specificity: the specificity in order to determine DNA interaction, with the not fully complementary oligonucleotide of 100nM 25bp
(non-p35S target sequence non-target, SEQ ID NO:3) makees negative control, and no response signal generates, and 100nM p35S target
Sequence (p35S target) then generates response signal, it was demonstrated that sensor specificity is good.Experimental result is as shown in Figure 4.
Sensitivity: test concentrations are the combining response signal of the p35S target sequence of 1000~0.01nM, the sample of each concentration
Product sample introduction 3 times, research finds that the p35S target sequence of 0.1nM can produce the response signal of about 1RU, and the p35S target sequence of 0.01nM
Response signal is not generated then, illustrates that the detection of the sensor is limited to 0.1nM, spr sensor sensitivity test result such as Fig. 5 institute
Show.
By embodiment and comparative example Comparative result it is found that compared with other nucleotide sequences, the disclosure has SEQ ID
The probe of nucleotide sequence shown in NO:1 and surface plasma resonance biological sensor containing the probe by with p35S target
Sequence complementation identification, can specifically detect the genetically modified plants containing CaMV35S promoter, and detection sensitivity is high, can be again
It is raw and easy to use.
The preferred embodiment of the disclosure is described in detail in conjunction with attached drawing above, still, the disclosure is not limited to above-mentioned reality
The detail in mode is applied, in the range of the technology design of the disclosure, a variety of letters can be carried out to the technical solution of the disclosure
Monotropic type, these simple variants belong to the protection scope of the disclosure.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, it can be combined in any appropriate way.In order to avoid unnecessary repetition, the disclosure to it is various can
No further explanation will be given for the combination of energy.
In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally
Disclosed thought equally should be considered as disclosure disclosure of that.
Sequence table
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>a kind of probe for detecting genetically modified plants, surface plasma resonance biological sensor and detection transgenosis are planted
The method of object
<130> 11645CAAS-B
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggcagaggca tcttgaacga tagcc 25
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggctatcgtt caagatgcct ctgcc 25
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gcgaaactgt ggaattgatc agcgt 25
Claims (10)
1. a kind of for detecting the probe of genetically modified plants, which is characterized in that the nucleotides sequence of the probe is classified as SEQ ID
Sequence shown in NO:1.
2. a kind of for detecting the surface plasma resonance biological sensor of genetically modified plants, which is characterized in that described surface etc.
Ion resonance biosensor includes sensing chip, on the sensing chip coupling have the right to require 1 described in probe.
3. sensor according to claim 2, wherein the censorchip surface is combined with Streptavidin；The spy
The end of needle 5 ' is marked with biotin.
4. sensor according to claim 2, wherein the coupling includes the following steps: that the idol of the probe will be contained
Connection solution flows through the chip, makes the probe in conjunction with the chip surface, obtains the surface plasma resonance biological and passes
Sensor.
5. sensor according to claim 4, wherein the conjugate solution also contain HEPES, NaCl, EDTA and
Surfactant P20, concentration of the probe in the conjugate solution are 20~50nM, time of the coupling is 180~
300s；The coupling amount of the probe is 200~400RU.
6. a kind of method for detecting genetically modified plants, which is characterized in that the target gene of the detection is CaMV35S promoter,
This method comprises the following steps:
S1 extracts the genomic DNA to measuring plants；
S2 makes the solution containing the genomic DNA flow through the sensing chip, and detects RU value response signal；If generating RU
Be worth response signal, then it is described to contain the target gene, if not generating RU value response signal, the plant to be measured in measuring plants
The target gene is not contained in object；The sensing chip is the sensing of sensor described in any one of claim 2~5
Chip.
7. according to the method described in claim 6, wherein, the solution containing the genome in step S2 is the base
Because of the mixed solution of group DNA and PBSM buffer；The flow velocity that the mixed solution flows through the sensing chip is 10~30 μ L/
Min, time are 120~300s.
8. according to the method described in claim 7, wherein, the pH of the PBSM buffer is 7.0~8.0, and the PBSM is buffered
Liquid contains the Na that the NaCl that concentration is 125~150mM, the KCl that concentration is 2.0~3.0mM, concentration are 10~15mM2HPO4, it is dense
Degree is the KH of 1~5mM2PO4, the polysorbas20 that concentration is 0.0005~0.001 volume % and the MgCl that concentration is 15~20mM2。
9. according to the method described in claim 6, wherein, this method further includes upon step s 2, again by the sensing chip
Raw, the regeneration method includes: to make regenerative agent contact 30~60s with the sensing chip to carry out regeneration treatment；It is described again
Raw reagent is the HCl that concentration is 10mM~50mM and/or the NaOH that concentration is 10~50mM.
10. according to the method described in claim 6, wherein, described to measuring plants includes rice, corn and soybean, rape, cotton
And at least one of pawpaw.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910290711.XA CN110093439A (en) | 2019-04-11 | 2019-04-11 | A method of for detecting the probe, surface plasma resonance biological sensor and detection genetically modified plants of genetically modified plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910290711.XA CN110093439A (en) | 2019-04-11 | 2019-04-11 | A method of for detecting the probe, surface plasma resonance biological sensor and detection genetically modified plants of genetically modified plants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110093439A true CN110093439A (en) | 2019-08-06 |
Family
ID=67444710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910290711.XA Pending CN110093439A (en) | 2019-04-11 | 2019-04-11 | A method of for detecting the probe, surface plasma resonance biological sensor and detection genetically modified plants of genetically modified plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110093439A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004271514A (en) * | 2003-02-18 | 2004-09-30 | Fuji Photo Film Co Ltd | Biosensor |
| CN1580285A (en) * | 2004-05-20 | 2005-02-16 | 厦门大学 | Transgenic product surface plasma resonance biosensor detecting method |
| CN101935694A (en) * | 2010-03-15 | 2011-01-05 | 山东省农业科学院植物保护研究所 | Genetic chip for detecting transgenic plants and products containing screening gene CaMV35S and application thereof |
| US20160138080A1 (en) * | 2013-03-14 | 2016-05-19 | National Institute Of Advanced Industrial Science And Technology | Methylcytosine detection method |
| CN106018347A (en) * | 2016-05-06 | 2016-10-12 | 中国科学院电子学研究所 | Surface plasma resonance sensing chip and preparation method and application thereof |
-
2019
- 2019-04-11 CN CN201910290711.XA patent/CN110093439A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004271514A (en) * | 2003-02-18 | 2004-09-30 | Fuji Photo Film Co Ltd | Biosensor |
| CN1580285A (en) * | 2004-05-20 | 2005-02-16 | 厦门大学 | Transgenic product surface plasma resonance biosensor detecting method |
| CN101935694A (en) * | 2010-03-15 | 2011-01-05 | 山东省农业科学院植物保护研究所 | Genetic chip for detecting transgenic plants and products containing screening gene CaMV35S and application thereof |
| US20160138080A1 (en) * | 2013-03-14 | 2016-05-19 | National Institute Of Advanced Industrial Science And Technology | Methylcytosine detection method |
| CN106018347A (en) * | 2016-05-06 | 2016-10-12 | 中国科学院电子学研究所 | Surface plasma resonance sensing chip and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 肖守斌: "运用表面等离子共振(SPR)生物传感器检测转基因玉米的研究", 《玉米科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20240003892A1 (en) | Heterogeneous single cell profiling using molecular barcoding | |
| Huang et al. | A novel GMO biosensor for rapid ultrasensitive and simultaneous detection of multiple DNA components in GMO products | |
| Chen et al. | An ultrasensitive signal-on electrochemical aptasensor for ochratoxin A determination based on DNA controlled layer-by-layer assembly of dual gold nanoparticle conjugates | |
| CN103620406B (en) | The potential electrode that aptamers is coated and contrast electrode and carry out the method for biomarker detection with it | |
| Xia et al. | Aptamer-based homogeneous analysis for food control | |
| Wang et al. | Amplified electrochemical detection of mecA gene in methicillin-resistant Staphylococcus aureus based on target recycling amplification and isothermal strand-displacement polymerization reaction | |
| US20030152985A1 (en) | Transient electrical signal based methods and devices for characterizing molecular interaction and/or motion in a sample | |
| CN101046461B (en) | Electrochemical sensor and its preparation process and use | |
| Chen et al. | Ultra-sensitive detection of Pb2+ based on DNAzymes coupling with multi-cycle strand displacement amplification (M-SDA) and nano-graphene oxide | |
| CN102519912B (en) | Method for detecting object to be detected by using surface plasmon resonance (SPR) biosensor | |
| CN104034782A (en) | Potential sensor for detecting dichlorotris (1,10-phenanthroline) ruthenium (II) and detection method and application of potential sensor | |
| Liu et al. | Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy | |
| Fu et al. | A homogeneous electrochemical sensor for Hg 2+ determination in environmental water based on the T–Hg 2+–T structure and exonuclease III-assisted recycling amplification | |
| Ou et al. | A highly sensitive DNA-AIEgen-based “turn-on” fluorescence chemosensor for amplification analysis of Hg 2+ ions in real samples and living cells | |
| EP2883961B1 (en) | A nucleic acid analysis method and apparatus | |
| Jiang et al. | A dual functional electrochemical “on–off” switch sensor for the detection of mercury (II) and melamine | |
| Yao et al. | Enzyme-free and label-free detection of Staphylococcus aureus based on target-inhibited fluorescence signal recovery | |
| CN104152449A (en) | MiRNA capture probe, modifying electrode thereof, capture probe complementary strand, capture probe complementary strand modified carbon nanotube-gold magnetic nanoparticle compound | |
| CN105296642A (en) | Isothermal amplification detection kit for pork derived component nucleic acid and detection method | |
| CN104458659B (en) | Surface plasma resonance sensor and preparation and application thereof | |
| Li et al. | Discovery and translation of functional nucleic acids for clinically diagnosing infectious diseases: Opportunities and challenges | |
| Niu et al. | Detection of DNA methyltransferase activity using template-free DNA polymerization amplification based on aggregation-induced emission | |
| CN110093439A (en) | A method of for detecting the probe, surface plasma resonance biological sensor and detection genetically modified plants of genetically modified plants | |
| Xiang et al. | Graphene oxide and molecular beacons-based multiplexed DNA detection by synchronous fluorescence analysis | |
| Dai et al. | Universal DNA biosensing based on instantaneously electrostatic attraction between hexaammineruthenium (III) and DNA molecules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190806 |