CN102758002A - Method of using gene chip technology to identify and detect 16 animal-derived compositions - Google Patents
Method of using gene chip technology to identify and detect 16 animal-derived compositions Download PDFInfo
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Abstract
The invention discloses an oligonucleotide probe for detection of 16 animal-derived compositions, a gene chip preparation and operation method and a detection method of a gene chip made of the oligonucleotide probe for detection of 16 animal-derived compositions (including cattle, goats, sheep, deer, buffalos, donkeys, pigs, dogs, rabbits, chickens, ducks, gooses, quails, turkeys, ostriches and tilapias). Different animal-derived compositions in foods and feeds (including additives) containing animal-derived compositions can be rapidly and stably detected by the method.
Description
One, technical field
The present invention relates to 16 kinds of animal oligonucleotide probes and gene chip and detection method thereof, the biology techniques field.
Background technology
At present, in order to confirm the true composition of food and feed, developed the method for much animal derived materials being differentiated.External the derived component of ox, sheep, pig, chicken has been carried out the research report, carried out the research report domestic ox, sheep, pig, chicken, donkey, horse, deer are belonged to etc.These methods comprise methods such as physical method, chemical process, immunology and molecular biology; Detection method is complicated, detection efficiency is not high but these methods exist; A detection method can only be directed against a target; Can not detect the problem of other target compositions, the demand of incompatibility present feed and the large-scale high throughput testing of meat product, the present invention promptly is to these problems and the articles for use and the method for the system that develops.
Two, summary of the invention
1. the objective of the invention is to the problem that above-mentioned detection method is complicated, detection efficiency is not high the oligonucleotide probe that provides a series of efficient, special animal derived materials to detect.
2. another object of the present invention provides the gene chip of 16 kinds of oligonucleotide probes.
3. another object of the present invention provides the application of 16 kinds of oligonucleotide probes, method for preparing gene chip thereof and detection heterology compositions, method thereof.
The present invention has adopted some technical schemes to achieve these goals:
The invention discloses and be used for the probe that 16 kinds of animal derived materials detect; Said probe is made up of one group of oligonucleotide probe; Said probe comprises at least one among the oligonucleotide probe 1-16, and said oligonucleotide probe 1-16 contains sequence shown in the Seq ID No.1-16 respectively;
Seq?ID?No.1：CAATCTCCATGAGTTGGTAGTTTCG
Seq?ID?No.2：AACAACTCAACCACAAAGGGAT
Seq?ID?No.3：CACTCCTTATGAGTTAACAGTTTCGG
Seq?ID?No.4：CAAGTCGAATCACACAATCGTT
Seq?ID?No.5：CCAAGGGATAACAACATCCTTT
Seq?ID?No.6：CACAAACCTAACCTTCAGGGAC
Seq?ID?No.7：AACAACTCAACCACAAAGGGAT
Seq?ID?No.8：CCTACAAGGCATAACATAACACCA
Seq?ID?No.9：CGAATGATTTTAGCCTAGACCC
Seq?ID?No.10：CACCCACACATAAACCCCTG
Seq?ID?No.11：CCGCGAACCTAAGACTAAACC
Seq?ID?No.12：GCGAATCCTCAATCAACCC
Seq?ID?No.13：GGCACATCCACACTAACACCC
Seq?ID?No.14：GGACCCACCCACATAAAACG
Seq?ID?No.15：TCCCACCCAAACCTACTAAGG
Seq?ID?No.16：GCCCTGTTCTAATGTCTTTGGT
The invention also discloses a kind of gene chip that 16 kinds of animal derived materials detect that is used for; Said gene chip comprises solid phase carrier and is fixed on 16 kinds of animal oligonucleotide probes on the solid phase carrier; Said probe comprises described probe of claim 1 and positive position probe; Said positive position probe has positive probe and the said probe that sequence shown in the Seq ID No.17 is Seq ID No.17:5 '-GGGTGGGATCAATTTGG-3 ' and also comprises a negative probe, and it is the negative probe of Seq ID No.18:5 '-CCAAATTGATCCCACCC-3 ' that said negative probe has preface shown in the Seq ID No.18.
Said solid phase carrier is sheet glass, silicon chip, polypropylene screen, nitrocellulose filter, nylon membrane, optimizes aldehyde radical sheet glass solid phase carrier in the embodiment of the invention.
The invention also discloses a kind of detection method of gene chip of animal derived materials, said detection method comprises the steps:
(1) DNA of extraction detected sample;
(2) be template with the said DNA of step (1), carry out pcr amplification;
(3) pcr amplification product with step (2) is a sample, adopts the said gene chip of claim 2-5 item, carries out gene chip hybridization;
(4) gene chip of accomplishing after hybridizing in the step (3) is carried out signal scanning and data processing.
Said pcr amplification comprises the universal primer of a pair of sample DNA that is used to increase; It is Seq ID No.19:5 '-AAGACGAGAAGACCCTTGGACTTTA-3 ' that the upstream primer of said universal primer has sequence shown in the Seq ID No.19, and it is Seq ID No.20:5 '-GATTGCGCTGTTATCCCTAGGGTA-3 ' that downstream primer has sequence shown in the Seq ID No.20.
Because adopt above technical scheme, beneficial effect of the present invention is:
Adopt gene chip of the present invention and detection method; Can detect 16 kinds of animal derived materials such as ox, goat, sheep, deer, buffalo, donkey, pig, dog, rabbit, chicken, duck, goose, quail, turkey, ostrich, tilapias simultaneously; High specificity, highly sensitive; And easy and simple to handle, the above-mentioned animal derived materials that is specially adapted to import and export in feed and the food detects.
Description of drawings
Fig. 1 is the gene chip sample applying figure of one embodiment of the invention;
Fig. 2 is the pcr amplification electrophoresis result of one embodiment of the invention; The amplification sample of swimming lane 1-17 is respectively ox, goat, sheep, deer, buffalo, donkey, pig, dog, rabbit, chicken, duck, goose, quail, turkey, ostrich, tilapia and blank; Swimming lane M is marker, and each stripe size is 234-262bp;
Fig. 3 is animal derived materials gene chip specific detection result in the embodiment of the invention, and the test sample of 1-16 is respectively beef powder, goat digested tankage, sheep digested tankage, venison powder, buffalo digested tankage, donkey digested tankage, pork powder, dog meats powder, cony meat powder, chicken powder, duck powdered meat, goose powder, quail digested tankage, turkey digested tankage, ostrich digested tankage, tilapia digested tankage among the figure;
Fig. 4 is the gene chip sensitivity detected result of beef sample in the embodiment of the invention, and the sample DNA concentration that 1-5 is corresponding among the figure is respectively 10
-1Ng/ μ L, 10
-2Ng/ μ L, 10
-3Ng/ μ L, 10
-4Ng/ μ L, 10
-5Ng/ μ L;
Fig. 5 is the repeatable gene chip detected result of animal derived materials in the embodiment of the invention, and 1A, 1B, 2A, 2B are respectively among the figure: ox, ox, goat, goat; 1C, 1D, 2C, 2D are respectively: ox, ox, goat, goat.
Embodiment
Mammals and bird mtDNA are relatively independent in heredity; Be double-stranded superhelix ring molecule, have genome little (about 16kb), do not have Tumor-necrosis factor glycoproteins, contain a large amount of Mitochondrial Genome Overview characteristics such as (about 1000～2300 copies of mammalian cell) in the interior inorganization specificity of biont, each cell.During the molecule of animal derived materials differentiate to detect in livestock and poultry meat product and feed, compare with the nucleic acid DNA molecule, that mtDNA has is highly sensitive, tolerance range good, quick, degraded is little (mtDNA keeps more complete in the course of processing), stablize advantages such as easy to operate.Therefore, the present invention quotes mtDNA universal primer in the pertinent literature, and this universal primer all has good suitability to above-mentioned 16 species, amplification when can realize 16 species; On this basis, the present invention is directed to each species and designed specific probe, institute's designed probe can specificly detect the sequence of target species, and the sequence of other species is not had detection signal.
Based on above-mentioned universal primer and specific probe, the present invention has developed and can be used for gene chip and the detection method thereof that above-mentioned 16 kinds of animal derived materials detect.Said gene chip can be realized several or whole species of above-mentioned 16 species are detected simultaneously as required.Said gene chip detection method; At first adopt universal primer that test sample is carried out the fluorescent mark amplification; Solved in present many targets detection method; Particularly many to mutual interferential problem between the primer in the multiplex PCR, the fluorescent mark that is adopted can be a signal mark commonly used during gene chips such as Cy3 or Cy5 detect; Adopt the gene chip that is fixed with specific probe that pcr amplification product is hybridized detection then, thereby the specific recognition that realizes different targets detect; Adopt gene chip scanning instrument that hybridization signal is carried out scanning analysis at last, judge the species that whether contain in the test sample corresponding to specific probe according to having or not of signal with strong and weak.
Below through specific embodiment and combine accompanying drawing that the present invention is done further explain.Following examples and accompanying drawing are only further explained the present invention, should not be construed as limitation of the present invention.
The preparation of embodiment 1 gene chip
One, the design of primer and probe
According to target dna sequence dna homology analysis result; Choose each target specific DNA sequences; Between the universal primer amplification region, filter out all dna fragmentations that are fit to designing probe; Utilize primer and probe analysis software PrimerPremier 5.0 (this software can provide the various parameters of primer and probe, and primer and probe is made effectively evaluating a plurality of aspect essential) to design and analyze, guarantee the science and the practicality that design.For guaranteeing under a hybridization temperature, can to obtain crossbreeding effect preferably at all probes on the chip, general principle of design should strict be followed in the design of primer and probe.
Candidate sequence is delivered to GenBank carries out the Blast compare of analysis, through the screening, on the Choice Theory specificity preferably oligonucleotide sequence do detection probes.The Quality Control probe comprises positive locating point probe and Cy5 mark complementary strand, negative Quality Control probe.The Quality Control probe does not all have homology with the gene order of animal mtDNAs such as ox, sheep, deer, pig, chicken, tilapia.
Two, chip point sample
(1) dilution of probe is dissolved into (40 μ mol/L) after the best point sample concentration with probe with 1 * TE, and mixing is centrifugal, and room temperature was placed 1 hour, treats to dissolve fully the back mixing.
(2) processing of probe and sampling liquid will be diluted behind good probe and the 50%DMSO equal-volume mixing as probe point sample mixed solution, and mixed solution is transferred in 384 orifice plates by the point sample plan, can place 4 ℃ to treat point sample usefulness.
(3) spot sample mode is according to the temperature and humidity controlled variable of envrionment temperature and humidity situation appropriateness adjustment point sample instrument; Through software the point sample parameter being set on point sample instrument is the Cor program; The substrate of treating point sample is placed NANO-PLOTTER 2 (Type:NP2; Serien-Nr2038) on the biochip point sample instrument, on point sample instrument, carry out point sample, each probe repeats point sample 5 times.
Three, point sample aftertreatment
The purpose of point sample aftertreatment mainly be for make probe can with the carrier surface mortise; Simultaneously, also nonspecific absorption does not impact test-results (particularly background) in crossover process to avoid to sealing with the free reactive group of probe bonded on the carrier.
Be transferred to chip in the wet box behind the point sample, the box that will wet is put into 37 ℃ of baking oven hydration 12h, with 0.2%SDS washing lotion rinsing 2min, and rinsed with deionized water 3 times, each 2min.Put into 0.2%NaBH again
4Seal 15min in the confining liquid, 5min is left standstill in the centre; Rinsed with deionized water 3 times, each 2min, 2000r/min, centrifugal 2min is dry, and 4 ℃ keep in Dark Place subsequent use.
Embodiment 2 gene chip detection methods
One, material and instrument
1, material
1.1 sample
Beef, Goral mutton, meat of a sheep, buffalo meat, pork, dog meats, rabbit meat, chicken, duck, goose, quail meat, turkey meat, ostrich meat and tilapia meat are all available from the ShenZhen,GuangDong supermarket, and donkey meat and venison are available from supermarket, Huhehaote, the Inner Mongol.These samples all pass through cultivar identification and sequence verification, and result's proof is the said species of its title.
1.2 main agents
DMSO (worker's biotechnology ltd is given birth in Shanghai)
SDS (worker's biotechnology ltd is given birth in Shanghai)
1 * TE (worker's biotechnology ltd is given birth in Shanghai)
NaBH
4(worker's biotechnology ltd is given birth in Shanghai)
Ultrapure water (ddH
2O)
Blank: 50%DMSO
Point sample damping fluid: 50%DMSO
Washing lotion behind the point sample: 0.2%SDS
Point sample rear enclosed liquid: 0.2%NaBH
4
Probe: (final concentration is 40 μ mol/L)
2, key instrument and testing installation
NANO-PLOTTER 2 chip point sample instruments (Gesim, Germany)
The GenePix4200A chip scanner (Axon, USA)
The AllegraX-25R large capacity refrigerated centrifuge (BECKMAN, Germany)
Milli-Q Advantage ultrapure water system (MILLIPORECHINALTD, USA)
5415R small frozen supercentrifuge (Eppendorf, Germany)
The FD115 baking oven (Binder, Germany)
Aldehyde radical substrate (CapitalBio Corporation)
384 orifice plates (worker's biotechnology ltd is given birth in Shanghai)
The AG135 electronic balance (METTLER TOLEDO, Switzerland)
MS3 basic model shaker (IKA, Germany)
RT15 Power multiple spot heating magnetic stirring apparatus (IKA, Germany)
SW-CJ-LB Bechtop (Suzhou grand auspicious purification Science and Technology Ltd.)
Two, method
1, sample DNA extracts
Adopt cell/tissue-genome test kit extraction method, extract the genomic dna of ox, goat, sheep, deer, buffalo, donkey, pig, dog, rabbit, chicken, duck, goose, quail, turkey, ostrich, tilapia.
2, PCR mark amplification
Adopt the base method of mixing to carry out pcr amplification.Reaction system is seen table 1, and the pcr amplification condition is: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 35 circulations; 72 ℃ are extended 5min.
Table 1 fluorescent mark PCR reaction system
3, molecular hybridization
Get respectively 7 μ L chip hybridization liquid (10 * SSPE, 2%SDS), the positive position probe complementary strand of 6 μ L PCR marked products, Cy5 mark 1 μ L; 95 ℃ of sex change 5min behind the mixing; Behind the ice bath 5min,, add deckglass (noting: bubble is not arranged) immediately with its point sample zone of all transferring to chip; Hybridization adds several dripping in the cabin, to keep humidity, chip is put into the hybridization cabin, and lucifuge insulation 2h in 55 ℃ of thermostat containers is put in sealing hybridization cabin then into.
4, gene chip hybridization and hybridization aftertreatment
Chip behind the chip hybridization is put into 42 ℃ of preheating washing lotion I successively, and (0.3 * SSC, 0.2%SDS) (0.06 * SSC) magnetic agitation is cleaned, each 3min, the centrifugal 2min drying of 2000r/min with washing lotion II.
Attention: scanning at once after (1) suggestion is centrifugal, though can be in the box of lucifuge storage chip, lose fluorescent signal after a few weeks longer possibly.
(2) because SDS can disturb the chip imaging, when from washing fluid I, moving on to chip in second washing tank, should make the amount of the washing fluid I that brings into drop to minimum.
(3) under the low excessively situation of room temperature, because the existence of SDS can cause separating out of crystallisate,, and can suitably improve elution time among elutriant I and the elutriant II so deposition is avoided in attention before use
5, signal detection
The laser confocal scanning appearance is set to the excitation wavelength scanning chip of 635nm, and laser power is 60%, and PMT is set to 60% and scans.Image is preserved with the form of JPEG and TIFF with GenePixs 5.0 software analysis.
Three, the result judges
In order to pass judgment on hybridization signal effectively, according to pertinent literature and combine chip background value size, the positive criterion of native system for the SNR through Gene Pix 4200A scanning identification greater than the positive signal of 3 signal.
Embodiment 3 specific detection
By above-mentioned TP; Extract the DNA of 16 kinds of livestock and poultry such as beef powder, goat digested tankage, sheep digested tankage, venison powder, buffalo digested tankage, donkey digested tankage, pork powder, dog meats powder, cony meat powder, chicken powder, duck powdered meat, goose powder, quail digested tankage, turkey digested tankage, ostrich digested tankage, tilapia digested tankage; Under best fluorescent mark temperature combinations condition, carry out the chip hybridization test, in order to verify the specificity of this method.(Fig. 3)
Embodiment 4 sensitivity detect
This test serves as to detect target gene with the beef powder sample, carries out the chip sensitivity test, and initial template DNA concentration is that 10ng/ μ L carries out ten times of gradient dilutions, and minimum weaker concn is 10
-5Ng mixes method PCR gel electrophoresis result and chip results compares with base, in order to verify the sensitivity of this method.(Fig. 4)
Animal derived materials in embodiment 5 feeds or the food detects
With the gene chip detection method 5 parts of known DNA that contain sheep derived component feed sample are detected; The result all shows the positive; Consistent with conventional PCR method detected result, positive coincidence rate reaches 100% (table 2), explains that this chip detecting method can be used for the detection of feed sample.
Do not know that to eight parts sample (packing is labeled as ox, sheep refrigerated meat) detects, this detected result and conventional PCR detected result consistent (like table 3) explain that this chip detecting method also can be used for the detection of meat product sample.
The PCR of table 2 sheep positive and chip detection deck watch 3 as a result do not know that the PCR of sample and chip detection result compare
Annotate: sample 2#, sample 4#, sample 12#, chocolate feed is raised notes: sample 318
Feed additives is the sheep composition.
Can simple deduction or replace be arranged in methods of the present invention in other concrete enforcements behind this case study on implementation according to the concrete application of molecular biological characteristic in oligonucleotide probe of the present invention, gene chip preparation method or step and detection thereof, all should be regarded as belonging to protection scope of the present invention.
Claims (4)
1. be used for the probe that animal derived materials detects, it is characterized in that: said probe comprises that (annotate: the implicit multiple meaning), said probe 1 to probe 16 contains sequence shown in Seq ID No.1 to the Seq ID No.16 at least one in probe 1 to the probe 16 successively;
Seq?ID?No.1：CAATCTCCATGAGTTGGTAGTTTCG
Seq?ID?No.2：AACAACTCAACCACAAAGGGAT
Seq?ID?No.3：CACTCCTTATGAGTTAACAGTTTCGG
Seq?ID?No.4：CAAGTCGAATCACACAATCGTT
Seq?ID?No.5：CCAAGGGATAACAACATCCTTT
Seq?ID?No.6：CACAAACCTAACCTTCAGGGAC
Seq?ID?No.7：AACAACTCAACCACAAAGGGAT
Seq?ID?No.8：CCTACAAGGCATAACATAACACCA
Seq?ID?No.9：CGAATGATTTTAGCCTAGACCC
Seq?ID?No.10：CACCCACACATAAACCCCTG
Seq?ID?No.11：CCGCGAACCTAAGACTAAACC
Seq?ID?No.12：GCGAATCCTCAATCAACCC
Seq?ID?No.13：GGCACATCCACACTAACACCC
Seq?ID?No.14：GGACCCACCCACATAAAACG
Seq?ID?No.15：TCCCACCCAAACCTACTAAGG
Seq?ID?No.16：GCCCTGTTCTAATGTCTTTGGT
2. one kind is used for the gene chip that animal derived materials detects, and said gene chip comprises solid phase carrier and is fixed on the probe on the solid phase carrier, it is characterized in that: said probe is by the probe that detects for the described animal derived materials of claim 1; Said solid phase carrier is to be selected from least a in aldehyde radical sheet glass, silicon chip, polypropylene screen, nitrocellulose filter, the nylon membrane.
3. the gene chip detection method of an animal derived materials; It is characterized in that: said detection method comprises; Extracting the DNA of detected sample, is template with said DNA, carries out pcr amplification; Be sample then with the pcr amplification product, adopt the described gene chip of claim 2 to carry out gene chip and detect.
4. inspection method according to claim 3; It is characterized in that: the primer that said pcr amplification adopts comprises upstream primer and downstream primer; Said upstream primer contains sequence shown in the Seq IDNo.17, and said downstream primer contains sequence shown in the Seq ID No.18;
Seq?ID?No.17：5’-GGGTGGGATCAATTTGG-3’
Seq?ID?No.18：5’-CCAAATTGATCCCACCC-3’
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| CN103630621A (en) * | 2013-10-28 | 2014-03-12 | 山东东阿阿胶股份有限公司 | Method for detecting sheep-derived ingredients in glue type traditional Chinese medicines and products thereof |
| CN104531890A (en) * | 2015-01-16 | 2015-04-22 | 北京市食品安全监控和风险评估中心 | Kit for detecting ostrich-derived component in food and application of kit |
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| CN105349653B (en) * | 2015-11-20 | 2019-03-29 | 华中农业大学 | Rabbit specific primer, kit and its application in the identification of rabbit derived component |
| CN105349653A (en) * | 2015-11-20 | 2016-02-24 | 华中农业大学 | Rabbit specific primers, kit, and applications thereof in rabbit-source component identification |
| CN106811510A (en) * | 2015-12-01 | 2017-06-09 | 上海市质量监督检验技术研究院 | Animal derived components discrimination method and its application based on high-flux sequence |
| CN107245518A (en) * | 2017-04-07 | 2017-10-13 | 北京市食品安全监控和风险评估中心 | It is a kind of while gene micro-fluid chip and its application of 26 kinds of animal derived materials of detection |
| CN107245518B (en) * | 2017-04-07 | 2021-08-06 | 北京市食品安全监控和风险评估中心 | Gene microfluid chip for simultaneously detecting 26 animal-derived components and application thereof |
| CN108060242B (en) * | 2018-02-13 | 2021-07-16 | 广州海关技术中心 | Dual digital PCR method for quantitative detection of African ostrich-derived components |
| CN108060242A (en) * | 2018-02-13 | 2018-05-22 | 广东出入境检验检疫局检验检疫技术中心 | A kind of dual digital pcr method that African Ostrich derived component quantitatively detects |
| CN108342493A (en) * | 2018-05-08 | 2018-07-31 | 四川华汉三创生物科技有限公司 | One breeding class composition detection kit and method |
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| CN110184330A (en) * | 2019-06-20 | 2019-08-30 | 中国计量大学 | Cross primer isothermal amplification system is synchronous to detect a variety of meat derived component methods |
| CN110184330B (en) * | 2019-06-20 | 2024-01-09 | 中国计量大学 | Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system |
| CN112280869A (en) * | 2020-10-27 | 2021-01-29 | 淮阴师范学院 | Method for detecting marine mammal components in feed by using gene chip technology |
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