CN101812527A - Method for quickly detecting six kinds of genetically modified corns - Google Patents
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Abstract
The invention discloses a method for quickly detecting six kinds of genetically modified corns. The method comprises the following operating steps of: preparing an optical film biosensor chip; selecting a transformant specificity sequence of the genetically modified corns; synthesizing probes; extracting and purifying lattice and sample DNA; synthesizing primers; marking biotin; performing PCR amplification; hybridizing; precipitating; and observing color development. The detection method has the advantages that: the detection sensitivity and accuracy are high, results can be detected when DNA concentration of the sample is 0.01mu M, and the accuracy rate can reach 100.00 percent; detection flux is large, and detection requirements of low, medium and high different fluxes are met; detection speed is high, needed time is short, and the results can be detected within 30 min which is over 10h shorter than that needed by a conventional detection method; detection procedure is simple, the detection results can be directly observed with naked eyes, and experimental procedures of the conventional detection such as fluorescent marking and instrument scanning and the like are saved; and detection cost is low, and investment in reagents and equipment is greatly reduced.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of visible detection method of optical thin film biologic sensor chip, be applied to the rapid detection of 6 kinds of transgenic corns.
Background technology
At present, it is the important content that genetically modified organism detects that transgenic corns detects, and has great importance in agricultural-food and food inspection.The transgenic corns detection method has multiple, mainly contains PCR (polymerase chain reaction) detection method, multi-PCR detection method, PCR method for detecting specificity of transformant, gene chip detection method etc.
The PCR detection method is a most frequently used detection method, and the single target gene order fragment by increasing specific reaches the existence that detects this target gene from the DNA mixture of complexity.But shortcoming is: this technology can only detect the single target gene, and situation is comparatively complicated when detecting more goal gene, and detection efficiency is lower.
Multi-PCR detection method is the detection method that grows up on PCR detection method basis, can detect a plurality of target genes simultaneously, detects flux and improves.But shortcoming is: this method increases many during to primer at the same time, is subjected to the restriction of amplified reaction complicacy, is easy to generate non-specific band (false positive), departs from legitimate reading, and accuracy of detection is lower.
PCR method for detecting specificity of transformant, it also is the detection method that on PCR detection method basis, grows up, by with a certain portion gene sequence of foreign DNA and DNA of plants joining region as the transformant specific fragment, because this fragment has unique identification (high specific), therefore can obtain to increase more accurately, reduce effectively and detect error.But shortcoming is: this technology has the higher requirement of ratio to the design primer.
The gene chip detection method is a kind of microarray detection processes.This method can accurately detect a plurality of target genes simultaneously, and it is bigger to detect flux, and detection speed is fast.By probe with a plurality of target genes, with the array format point sample in chip surface, use the DNA of DNA (passed through isotropic substance or fluorescence etc. and carried out the mark processing) and probe in the sample to carry out complementation then and hybridize, read label information again, can obtain the detected result of high information quantity.But shortcoming is: the laser scanner of this Technology Need costliness reads the fluorescent mark data, and it is higher to detect cost, popularizes and is subjected to certain limitation.
Summary of the invention
The invention provides the method for 6 kinds of transgenic corns of a kind of applied optics thin film bio sensor chip rapid detection, be the detection method of applied optics thin film bio sensor chip, this method be on the basis of specificity of transformant round pcr and microarray technology and optical thin film biologic sensor chip (biochip) technology, develop to the walk abreast novel method of rapid detection of 6 kinds of transgenic corns.
The ultimate principle of described detection method: the optical thin film biologic sensor chip is a kind of refraction principle based on light, and the variation of chip surface thickness is converted into the refracted light wavelength change, changes the thin film chip of the visual colour-change of naked eyes again into.Foreign gene at 6 kinds of transgenic corns (Bt11, Event176, GA21, MON810, NK603, T25) DNA inserts the district, seek the specificity of transformant sequence of transgenic corns, design corresponding probe and primer with this, with the probe dot matrix on the optical thin film biologic sensor chip, extract the DNA of detected sample, with the target gene in the biotin labeled primer amplification testing sample; With mark the pcr amplification product of vitamin H and the probe on the chip hybridize, combine with having connected anti-biotin-HRP (oxidasic biotin antibody of horseradish and vitamin H), with TMB (tetramethyl benzidine,) carry out precipitin reaction with the horseradish oxydase, because throw out has changed the thickness of chip surface, thereby changed the reflected light wavelength of chip surface, cause chip surface change in color (color becomes Lan Se or purple by gold), with the naked eye just can be observed, therefore obtain detected result.The present invention is the method for 6 kinds of transgenic corns of a kind of applied optics thin film bio sensor chip rapid detection, comprises that the optical thin film biologic sensor chip is prepared, the specificity of transformant sequence selection of transgenic corns and probe and design of primers, probe is synthetic and dot matrix, sample DNA extraction and purifying, primer is synthetic and biotin labeling, pcr amplification, hybridization, precipitin reaction and colour developing are observed operation steps.
The invention has the advantages that:
(1) detection sensitivity and accuracy height, DNA concentration just can detect the result in the sample when 0.01 μ M, and accuracy rate can reach 100.00%;
(2) the detection flux is big, is applicable to the detection requirement of basic, normal, high different flux;
(3) detection speed is fast, and used time is short, only needs a 30min just can detect the result, lacks more than the 10h than the conventional sense method time spent;
(4) trace routine is simple, and the experimental arrangement of conventional sense such as fluorescent mark and instrument scanning has been removed in directly visual inspection of detected result from;
(5) the detection cost is low, has greatly reduced reagent and equipment input;
(6) application prospect is wide, can be widely used in the detection of genetically modified organism composition, promotes popularizing of microarray assay technology, specificity of transformant detection technique and optical thin film biologic sensor chip technology.
Description of drawings
Fig. 1 is the specificity of transformant sequence select location figure of 6 kinds of transgenic corns of the present invention;
Fig. 2 A, 2B are with the principle schematic of probe point sample on sensor chip among the present invention;
Fig. 3 A, 3B are the principle schematic of hybridizing and detecting on the sensor chip among the present invention;
Fig. 4 is a detection method operational flowchart of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
The method of 6 kinds of transgenic corns of rapid detection provided by the invention, realization flow specifically realize as shown in Figure 4 as follows:
Step 1, optical thin film biologic sensor chip are prepared: the optical thin film biologic sensor chip of Ding Gouing from the market, shown in Fig. 2 A, this sensor chip is a silicon material optical thin film biologic sensor chip, and specification is long 5～20mm, wide 5～20mm, thick 0.3～0.6mm.Chip is divided into three layers, is followed successively by silicon supporting layer 1, reflection layer 2, surface attachment layer 3 from top to bottom, is coated with reflection layer 2 and surface attachment layer 3 on the described silicon supporting layer 1 successively.Described silicon supporting layer 1 thickness is about
Light reflex layer 2 plays the support effect with surface attachment layer 3; Reflection layer 2 materials are silicon nitride (Si
3N
4), its thickness is about
The reflected light color is golden yellow during its white light.In detection, precipitin reaction has changed surface attachment layer 3 thickness at the throw out of chip surface, thereby has changed the reflected light color, and this just reflects macroscopic color signal with the result of chemical precipitation reaction, reaches the purpose of detection.Surface attachment layer 3 is PPL (phenylalanine and Methionin is with the mixture of equal proportion), and its thickness is about
Hydrazine group-NH-NH2 is rich on the surface, is used for fixing probe.
The specificity of transformant sequence selection of step 2, transgenic corns and probe and design of primers: as shown in Figure 1, exogenous gene sequence on 6 kinds of transgenic corns (Bt11, Event176, GA21, MON810, NK603, the T25) dna sequence dna that in gene pool and United States Patent (USP), finds and the dna sequence dna on the on position, and the gene order that is comprised.Inserting the dna fragmentation that the district chooses 100～300bp length, make this fragment both comprise the exogenous gene sequence that part changes over to, comprise the peculiar gene order of this corn variety of part again, promptly obtained the specificity of transformant sequence of this kind transgenic corns kind, the numeral on band arrow line segment and top thereof is represented the position and the fragment length of the specificity of transformant sequence of selected this kind transgenic corns kind respectively.According to this specificity of transformant fragment design primer; On this specificity of transformant sequence, choose the sequences Design probe of 40bp length, this probe be high specific or uniqueness.For ease of the hybridization of probe, in 20 adenyl-deoxyribonucleotide sequences of 5 ' end connection of probe; For with probe stationary on chip, carry out at 5 ' of probe more aldehyde group modified, concrete probe and design of primers such as following table 1:
Concrete probe and design of primers table on the table 1 specificity of transformant sequence fragment
| Kind | Goal gene | Primer, probe sequence | Fragment length (bp) | The fragment source |
| ??Bt11 | ??IVS2/PAT | ??F：5’cttctgggaggccaaggtatct3’??R：5’biotin-gctgctgtagctggcctaatct3’??P：5’ALD-aaaaaaaaaaaaaaaaaaaaccatccc??atttgtgatctttgtcagtagatatgatacaac3’ | ??192 | Gene pool AY629236 |
| ??T25 | ??CaMV35S/PAT | ??F：5’agatcatcaatccactcttgtggtg3’??R：5’biotin-ccttcgcaagacccttcctctata3’??P：5’ALD-aaaaaaaaaaaaaaaaaaaagccatat??cagctgctgtagctggcctaatctcaactggtc3’ | ??231 | Gene pool BD378188 |
| ??Mon810 | ??Maize?genome??/CaMV35S | ??F：5’tcgaaggacgaaggactctaacg3’??R：5’biotin-tccatctttgggaccactgtcg3’??P：5’ALD-aaaaaaaaaaaaaaaaaaaaccattgc??ccagctatctgtcactttattgtgaagatagtg3’ | ??170 | United States Patent (USP) U.S.Pat. 6713259 |
| ??Event | ??CDPK/CryIA(b) | ??F：5’ctctcgccgttcatgtccgt3’ | ??211 | U.S. Pat |
| ??176 | ??R：5’biotin-ggtcaggctcaggctgatgt3’??P：5’ALD-aaaaaaaaaaaaaaaaaaaatccaaca??atggacaacaaccccaacatcaacgagtgcat3’ | ??Pat.??5625136 |
| Kind | Goal gene | Primer, probe sequence | Fragment length (bp) | The fragment source |
| ??NK603 | ??ctp2/EPSPS | ??F：5’atgaatgacctcgagtaagcttgttaa3’??R：5’biotin-aagagataacaggatccactcaaaca??ct3’??P：5’ALD-aaaaaaaaaaaaaaaaaaaaaaggga??tatcaagcttggtaccacgcgacacacttccactct3’ | ??108 | U.S. Pat .Pat. 6825400 |
| ??GA21 | ??OTP/m-EPSPS | ??F：5’acggtggaagagttcaatgtatg3’??R：5’biotin-tctccttgatgggctgca3’??P：5’ALD-aaaaaaaaaaaaaaaaaaaaccgtgat??gatggcctcgtcggccaccgccgtcgctccg3’ | ??270 | U.S. Pat Pat. 4940835 |
Annotate: F: forward aligning primer
R: the reverse sequence primer that has biotin labeling (biotin) at 5 ' end
P: have the sequence of 20 dATP (adenyl-deoxyribonucleotide) at 5 ' end, and carried out the probe of aldehyde group modified (ALD).
Step 4, sample DNA extract and purifying: extract and purify DNA with Tiangen nucleic acid extraction purification kit; Measure the dna content (judging whether extraction DNA is successful) that is extracted with NanoDrop ND 1000 (ultraviolet-visible pectrophotometer); Quality with DNA in the agarose gel electrophoresis method for detecting measure sample.
Step 6, pcr amplification: the DNA that extracts in the step 4 is carried out pcr amplification with synthetic forward aligning primer in the step 5 and reverse sequence primer, its PCR reaction conditions is 25 μ l mixtures, comprise 1 * PCR Buffer, 2mM MgCl (magnesium chloride), 0.1mM dNTP, 0.2 μ M primer, the dna profiling of 1U rTaq (polysaccharase) and 100ng.Be reflected among the PTC-225 Peltier Thermal Cycler (PCR instrument) and carry out.94 ℃ of sex change 5min of elder generation before the amplification; Carry out 40 amplification cycles afterwards, the amplification cycles formula is set at 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 30s; Circulate back 72 ℃ and extend 10min; Measure the amount and size (whether the DNA fragment specific in order to pcr amplification in the Preliminary detection sample is consistent with design) of pcr amplification product again with agarose gel electrophoresis; 4 ℃ of preservations.
Step 7, hybridization: as shown in Figure 3A, get 90 μ l HB (mixed solution of 5 * SSC and 5mg/ml ATC) and be added on the sensor chip 45 ℃ of incubation 5min; Get 2 μ l pcr amplification products and 8 μ l ddH
2Behind the O mixing, 95 ℃ of sex change 3min; Pcr amplification product after the sex change is added in the HB damping fluid on the sensor chip, makes that having biotin labeled PCR product combines mixing, 45 ℃ of incubation 10min with probe complementation on the sensor chip; Clean chip 3 times with 0.1 * SSC, 100 μ l under the room temperature, dry up.
Step 8, precipitin reaction and colour developing are observed: shown in Fig. 3 B, get 100 μ l anti-biotin-HRP (connected the oxidasic biotin antibody of horseradish, be diluted in HB damping fluid at 1: 1000) and be added on the sensor chip, room temperature is placed 5min; Under the room temperature, clean chip 3 times, dry up with 0.1 * SSC, 100 μ l; Get 100 μ l TMB (tetramethyl benzidine) and be added on the sensor chip, the room temperature lucifuge is placed 5min; With 100 μ l ddH
2 O rinsing chip 3 times dries up; Observe chip surface probe color, obtain detected result.
As shown in Figure 3, in test sample, have the specificity of transformant fragment, must combine, realize hybridization with the probe fragment complementary pairing, not can because of cleaning by flush away; With probe hybridization bonded specificity of transformant fragment owing to be by the primer synthetic, be marked with vitamin H on it, vitamin H combines with biotin antibody, biotin antibody has been carried out precipitin reaction with TMB again, can demonstrate related color change this moment on sensor chip, then thinking has this transformed variety in the test sample; If there is not the specificity of transformant fragment in the test sample, just can not combine, just not hybridization with the probe fragment complementary pairing, will be cleaned, just do not have precipitin reaction, just do not have any variation on the sensor chip, then think in the test sample not this transgene component.
Claims (5)
1. the method for 6 kinds of transgenic corns of a rapid detection is characterized in that following steps:
Step 1, optical thin film biologic sensor chip are prepared;
Described optical thin film biologic sensor chip is a silicon material optical thin film biologic sensor chip, and chip is divided into three layers, is followed successively by silicon supporting layer, reflection layer, surface attachment layer from top to bottom;
The specificity of transformant sequence selection of step 2, transgenic corns, probe and design of primers;
Exogenous gene sequence on 6 kinds of transgenic corns kind dna sequence dnas and the dna sequence dna on the on position, and on the gene order that is comprised, choose the dna fragmentation of 100～300bp length at connecting zone, make this fragment both comprise the exogenous gene sequence that part changes over to, comprise the peculiar gene order of this corn variety of part again, promptly obtained the specificity of transformant sequence of this kind transgenic corns kind; The transgenic corns kind is respectively Bt11, Event176, GA21, MON810, NK603, T25 in described 6;
Step 3, the synthetic dot matrix that reaches of probe;
According to designed probe sequence in the step 2, connect 20 dATP at probe 5 ' end, carry out aldehyde group modifiedly then at 5 ' end, the concentration and probe concentration of diluting with the 0.1M phosphate buffered saline buffer of PH7.8 is 1 μ M, with pipettor or point sample instrument with the probe dot matrix on chip; The room temperature moist environment is placed more than the 2h down, cleans gently 3 times with 0.1 * SDS, uses ddH again
2O washes 3 times, dries up standby;
Step 4, sample DNA extract and purifying;
Extract and purify DNA with Tiangen nucleic acid extraction purification kit;
Step 5, the synthetic biotin labeling that reaches of primer;
The primer sequence of design in two set by step, the forward and the reverse sequence primer of synthetic 6 kinds of transgenic corns respectively, wherein the reverse sequence primer carries out biotin labeling at 5 ' end;
Step 6, pcr amplification;
With synthetic forward aligning primer and reverse sequence primer the DNA that extracts is carried out pcr amplification, its PCR reaction conditions is 25 μ l mixtures, comprises 1 * PCR Buffer, 2mM MgCl, 0.1mM dNTP, 0.2 μ M primer, the dna profiling of 1U rTaq and 100ng; Be reflected among the PTC-225 Peltier Thermal Cycler and carry out; 94 ℃ of sex change 5min of elder generation before the amplification; Carry out 40 amplification cycles afterwards, the amplification cycles formula is set at 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 30s; Circulate back 72 ℃ and extend 10min; 4 ℃ of preservations;
Step 7, hybridization;
Get 90 μ l HB and be added on the chip 45 ℃ of incubation 5min; Get 2 μ l PCR products and 8 μ l ddH
2Behind the O mixing, 95 ℃ of sex change 3min; PCR product after the sex change is added in the HB damping fluid on the chip, and mixing is hatched 10min for 45 ℃; Clean chip 3 times with 0.1 * SSC, 100 μ l under the room temperature, dry up;
Detected result is observed in step 8, precipitin reaction and colour developing;
Get 100 μ l anti-biotin-HRP and be added on the sensor chip, room temperature is placed 5min; Under the room temperature, clean chip 3 times, dry up with 0.1 * SSC100 μ l; Get 100 μ l TMB and be added on the sensor chip, the room temperature lucifuge is placed 5min; With 100 μ l ddH2O rinsing chips 3 times, dry up; Observe chip surface probe color, obtain detected result.
2. the method for 6 kinds of transgenic corns of rapid detection according to claim 1 is characterized in that: described silicon supporting layer thickness is
Light reflex layer and surface attachment layer play the support effect; Be coated with reflection layer and surface attachment layer on the silicon supporting layer successively; Described reflection layer material is a silicon nitride, and thickness is
The surface attachment layer is PPL, and thickness is
Amino is rich on the surface, is used for fixing probe.
3. the method for 6 kinds of transgenic corns of rapid detection according to claim 1, it is characterized in that: described probe and primer design are as follows:
Annotate: F: forward aligning primer;
R: have biotin labeled reverse sequence primer at 5 ' end;
P: have the sequence of 20 dATP at 5 ' end, and carried out aldehyde group modified probe.
4. the method for 6 kinds of transgenic corns of rapid detection according to claim 1 is characterized in that: the probe dot matrix described in the step 3, and designed probe sampling point repeat number on chip, the point sample amount is every 40～250nl.
5. the method for 6 kinds of transgenic corns of rapid detection according to claim 1 is characterized in that: the anti-biotin-HRP described in the step 8 forms in the HB damping fluid with 1: 1000 dilution proportion for having connected the oxidasic biotin antibody of horseradish.
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Cited By (10)
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| CN102191326A (en) * | 2011-04-18 | 2011-09-21 | 南京工业大学 | Nucleic acid hybridization membrane strip for detecting exogenous gene and application thereof |
| CN102409094A (en) * | 2011-11-17 | 2012-04-11 | 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 |
| CN102409092A (en) * | 2011-11-17 | 2012-04-11 | 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 |
| CN102952860A (en) * | 2011-08-26 | 2013-03-06 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11 |
| CN102965442A (en) * | 2012-12-04 | 2013-03-13 | 浙江省检验检疫科学技术研究院 | Detection method and detection chip of transgenosis components |
| 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 |
| CN104561366A (en) * | 2015-02-06 | 2015-04-29 | 曹际娟 | Method for high-throughput detection of transgenic corn by virtue of micro-fluidic chip |
| CN104726580A (en) * | 2015-03-17 | 2015-06-24 | 苏州华麦生物科技有限公司 | Probe method based constant temperature detection primer group, detection kit and detection method of genetically modified maize MON810 |
| CN109337959A (en) * | 2018-10-19 | 2019-02-15 | 浙江省农业科学院 | A kind of RPA detection primer of transgenic corns MON810 and probe combinations, kit and detection method |
| CN112813189A (en) * | 2021-03-12 | 2021-05-18 | 浙江经贸职业技术学院 | Method for rapidly identifying transgenic corn strain by utilizing quadruple real-time fluorescent PCR (polymerase chain reaction) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102191326A (en) * | 2011-04-18 | 2011-09-21 | 南京工业大学 | Nucleic acid hybridization membrane strip for detecting exogenous gene and application thereof |
| CN102952860A (en) * | 2011-08-26 | 2013-03-06 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11 |
| CN102952860B (en) * | 2011-08-26 | 2014-12-31 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11 |
| CN102409094A (en) * | 2011-11-17 | 2012-04-11 | 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 |
| CN102409092A (en) * | 2011-11-17 | 2012-04-11 | 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 |
| CN102965442A (en) * | 2012-12-04 | 2013-03-13 | 浙江省检验检疫科学技术研究院 | Detection method and detection chip of transgenosis components |
| 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 |
| CN104561366A (en) * | 2015-02-06 | 2015-04-29 | 曹际娟 | Method for high-throughput detection of transgenic corn by virtue of micro-fluidic chip |
| CN104726580A (en) * | 2015-03-17 | 2015-06-24 | 苏州华麦生物科技有限公司 | Probe method based constant temperature detection primer group, detection kit and detection method of genetically modified maize MON810 |
| CN109337959A (en) * | 2018-10-19 | 2019-02-15 | 浙江省农业科学院 | A kind of RPA detection primer of transgenic corns MON810 and probe combinations, kit and detection method |
| CN112813189A (en) * | 2021-03-12 | 2021-05-18 | 浙江经贸职业技术学院 | Method for rapidly identifying transgenic corn strain by utilizing quadruple real-time fluorescent PCR (polymerase chain reaction) |
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