CN104611470A - Gene chip for detecting bluetongue virus and detecting method of same - Google Patents
Gene chip for detecting bluetongue virus and detecting method of same Download PDFInfo
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Abstract
The invention discloses a gene chip for detecting bluetongue virus and a detecting method thereof. The gene chip comprises a PCR reaction mixture tube, Taq polymerase, a PCR positive control, a PCR negative control, ddH2O, a detecting chip, dot-type distribution of chip probes, a chip cover plate, a chip rail, hybridization solution, a hybridization positive control, a hybridization box and a detection microarray. PCR primers are analysed and designed through a standard strain genomic sequence, a target gene is cloned and subjected to sequencing analysis, then specific probes are designed, hybridization conditions are optimized, and bluetongue virus can be detected. Therefore a method is established for detecting the bluetongue virus through the microarray chip, which is high in sensitivity, strong in specificity, time-saving and labor-saving and easy for observing results.
Description
Technical field
The invention belongs to biochip field.Specifically, the present invention relates to gene detecting chip and the detection method thereof of blue tongue virus.
Background technology
Bluetongue (Bluetongue disease, BT) is acute, the strong untouchable arthropod borne infection caused by blue tongue rims (Bluetongue virus, BTV).Domestic and the wild ruminants such as BTV main infection sheep, goat, ox, common symptoms is high heat, tongue becomes blue and oral mucosa oedema is hemorrhage etc.Generally, the mortality ratio of this disease is 20%-30%, but for the sheep of some kind, lethality rate up to 90%, can have a strong impact on the development of livestock industry.Blue tongue virus is bitten susceptible animal and is propagated after sucking the malicious blood of band mainly through hematophagous bug (as storehouse midge etc.), and therefore the eruption and prevalence of this disease has certain seasonality, region and periodically.In addition, this virus also carries out vertical transmission by seminal fluid and placenta.Bluetongue is a kind of entomophila sexually transmitted disease of worldwide harm, OIE is classified as category-A epidemic disease, China is classified as a class animal epidemic, mainly be distributed in America, Africa, Australia and Asia, China also in Yunnan, Hubei, Anhui, Sichuan, Shanxi, multiple area such as Guangxi be separated and obtain BTV.In recent years, along with the increase year by year of global warming and international animal trade, bluetongue has in the world and expands pandemic trend, can infect many animals, constitute a serious threat to Animal husbandry production development and human health.
Blue tongue virus belongs to Reoviridae Orbivirus, and virus particle is icosahedral symmetry, without cyst membrane, is made up of the double-stranded RNA of 10 sections, and encode 7 kinds of structural protein and 4 kinds of Nonstructural Proteins are the RNA viruses that molecular weight is up to now maximum.Confirmed that BTV at least exists 24 serotypes at present, between each serotype, cross-protection is low.In addition, blue tongue virus normal with PPR, the polyinfection such as herpetic stomatitis, SVD be malicious, greatly increase the diagnosis difficulty to this disease.The diagnosis of China's bluetongue and the main virulent isolation identification of standard (GB/T18636-2002) method of quarantine, Ag-capture ELISA (Ag-capture ELISA), sizing microneutralization test and plaque and Plaque reduction assay type test etc. are for the detection of cause of disease, and agar immunodiffusion test (AGID) and competitive ELISA (C-ELISA) are for the detection of antibody.Virus Isolation is one of method of the diagnosis BTV that OIE recommends, but the method exists the long limitation of culture cycle.Serology test can be used for the Serotype Identification of viral isolates, also can be used for the detection of BTV specific antibody.BTV nucleic acid detection technique comprises universal detection and somatotype detects, and mainly comprises Nucleic Acid Probe Technique, RT-PCR, quantitative fluorescent PCR, gene chip detecting technique etc.Bluetongue there is no effective methods for the treatment of and vaccine, and early discovery, early process are fixed against detection means fast and accurately.Thus accurately rapid detection can go out BTV, have great importance in the import and export quarantine of animal and byproduct thereof.
Gene chip (Gene chip) is the probe array formed at the solid support through covalent attachment aldehyde radical, amino or poly-lysine by DNA or oligonucleotide probe dense arrangement, under suitable temperature, humidity condition, specific probe is utilized to be hybridized by base pair complementarity with the measuring samples DNA marked through appropriate ways, then by corresponding test set, detect position and the power of hybridization signal, judgement sample kind, completes the work of the aspect such as Viral diagnosis and fundamental research.The method has the advantage of high-throughput, parallelization, milligram ammonia, automatization, low cost.Therefore, this technology is widely used in the numerous areas such as diagnostic detection, Differential expression analysis, order-checking.
Summary of the invention
In order to overcome deficiency of the prior art, laborsaving and be easy to the micro-array chip of observations when the object of the invention is to set up a kind of highly sensitive, high specificity, joint, detect the gene chip of blue tongue virus.
The present invention adopts following technical scheme to achieve these goals: the gene chip for detecting blue tongue virus comprises: PCR reaction mixture pipe, Taq polysaccharase, PCR positive control, PCR negative control, ddH
2the point sample distribution of O, detection chip, chip probe, chip cover plate, chip fence, hybridization solution, hybridization positive control, hybridizing box, adopt gene chip micro-sampling technology, testing sample probe and each special quality control probe are fixed on the aldehyde radical substrate of chemically modified, the microarray of formation.Microarray comprises Quality Control probe region and testing sample probe region, arranges following subregion in wherein said Quality Control probe region:
Point sample position Quality Control subregion P1, comprises at least one point sample position Quality Control probe: SEQ ID NO.2;
Hybridization positive quality control subregion P2, comprises at least one hybridization positive quality control probe: SEQ ID NO.3;
Hybridize negative Quality Control subregion N, comprise spotting buffer;
Described testing sample probe region comprises at least one blue tongue virus probe: SEQ ID NO.1.
Utilize the detection method of said gene chip detection blue tongue virus, concrete steps are as follows:
(1) testing sample preparation: extract according to commercialization RNA the full-length genome that test kit working instructions extract the cell proliferation liquid of tissue to be measured or virus, carry out reverse transcription and prepare cDNA, obtain measuring samples DNA;
(2) pcr amplification: 25 μ L PCR reaction solutions comprise: rTaq enzyme 12.5 μ L, primer Mix 5.4 μ L, testing sample DNA 3 μ L, nuclease free aqua sterilisa 4.1 μ L;
Wherein, described primer Mix contains:
20 μm of ol/L blue tongue virus special upstream primer SEQ ID NO.4 0.2 μ L,
20 μm of ol/L blue tongue virus special downstream primer SEQ ID NO.5 0.2 μ L,
20 μm of ol/L PCR general upstream primer SEQ ID NO.6 4 μ L,
20 μm of ol/L PCR general downstream primer SEQ ID NO.7 1 μ L;
Increase as follows: 94 DEG C of 5min; 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 25sec circulate 12 times; 94 DEG C of 30sec, 50 DEG C of 30sec, 72 DEG C of 25sec circulate 30 times; 72 DEG C of 10min;
(3) hybridize: first preparing hybrid liquid: hybridization buffer 7.9 μ L, pcr amplification product 7 μ L, hybridization positive control 0.1 μ L, mix rear 95 DEG C of sex change 8min, place 5min on ice, then adopt 15 μ L hybridization solutions to hybridize; The every 1000 μ L of hybridization buffer comprise 20 × SSC 500 μ L, 10%SDS 10 μ L and nuclease free aqua sterilisa 490 μ L.
(4) result interpretation: use micro-array chip scanner scanning analysis, removes negative control background, illustrates and contrast, result of determination with chip hybridization.
The advantage of comprehensive gene chip of the present invention, utilizes the stability of oligonucleotide probe, set up a kind of fast, the gene chip detecting technique of precise Identification blue tongue virus, for controlling the propagation of blue tongue virus and the quarantine of animal of passing in and out has vital role.
(1) successfully construct BTV and detect gene chip: the blue tongue virus probe prepared by the present invention can carry out specific binding with corresponding virus goal gene, and hybridization signal is comparatively strong and stable.
(2) the detection gene chip prepared has good Methodological characteristics: the present invention establishes that a species specificity is good, highly sensitive, stability is strong and joint time labour-saving gene chip detection method.
(3) detect the structure of gene chip, for the synchronous detection of polyinfection disease and differential diagnosis provide means fast and efficiently, in order to enter and leave the border from now on, Animal Quarantine and corresponding Blight control provide technical guarantee.To meeting Animal Quarantine requirements of one's work of passing in and out, control the propagation of blue tongue virus, livestock industry safety, the sound development tool of guarantee China are of great significance.
Accompanying drawing explanation
Fig. 1 chip probe distribution schematic diagram;
The hybridization of Fig. 2 blue tongue virus and specific outcome figure;
Fig. 3 blue tongue virus genechip detection sensitivity results figure;
In figure: P1-point sample position Quality Control subregion; P2-hybridizes positive quality control subregion; 1-foot and mouth disease virus A type probe subregion; 2-foot and mouth disease virus Asia I type probe subregion; The universal probe subregion of 3-foot and mouth disease virus; 4-foot and mouth disease virus O type probe subregion; 5-vesicular stomatitis virus probe subregion; 6-swine vesicular disease virus probe subregion; 7-blue tongue virus probe subregion; 8-PPR virus probe subregion; N-hybridizes negative Quality Control subregion;
Hybridization illustrates: P1: positive control fixed by probe, and monitoring chip deposition process, all answers the positive before and after chip hybridization;
P2: hybridization positive control, monitoring chip crossover process, detects any sample and all answers the positive;
N: hybridization negative control, detects any sample and answers feminine gender;
Blue tongue virus cDNA is through the results of hybridization of pcr amplification product: except Quality Control probe meets the requirements, and No. 7 probes are bright;
In Fig. 3, A:2.99 × 10
8copies/uL; B:2.99 × 10
7copies/uL; C:2.99 × 10
6copies/uL; D:2.99 × 10
5copies/uL E:2.99 × 10
4copies/uL; F:2.99 × 10
3copies/uL.
Embodiment
There is provided specific embodiment to set forth technical scheme of the present invention further, but the application of the technology of the present invention is not limited to following examples.
Embodiment 1, the present invention is based on the cell proliferation to blue tongue virus, extracts viral genome.Choose different special conserved sequences and devise 1 oligonucleotide probe, specific binding can be carried out with the PCR primer of corresponding viral cDNA, when PCR carries out genome amplification, general upstream primer 5 ' end utilizes Cy3 fluorochrome to mark, and probe can be hybridized by product therewith at 50 DEG C.Judge results of hybridization according to fluorescent signal position, intensity, detect blue tongue virus with this.Primer Premier 5.0 biosoftware is utilized to design PCR primer SEQ ID NO.4, SEQID NO.5, SEQ ID NO.6 and SEQ ID NO.7 according to the blue tongue virus gene order that Genbank website is announced.Cloned gene sequence, and carry out checking order, compare of analysis, choose conserved sequence utilizing Primer Premier biosoftware and design described 1 specific oligonucleotide probe, SEQ ID NO.1.By above-mentioned primer and probe, detection blue tongue virus that can be quick, special, responsive.
5 ' end of every bar probe is all connected with an amino by 15 thymidylic acids, and concrete nucleotide sequence is as follows:
Blue tongue virus probe SEQ ID NO.1 is:
5'-NH
2-T
15-GATACGATTTCACAACCACCGAGATATGCTCCGAG
Point sample position Quality Control probe SEQ ID NO.2 is:
5'-NH
2-T
15-GCTGCCTCGGCAAGGAGT-TAMRA；
Hybridization positive quality control probe SEQ ID NO.3 is:
5'-NH
2-T
15-GTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATT。
Embodiment 2, see Fig. 1, for the gene chip that blue tongue virus detects, adopts gene chip micro-sampling technology, is fixed on the aldehyde radical substrate of chemically modified by testing sample probe and each special quality control probe, form the microarray that 7 row × 8 arrange.On microarray, probe distribution is followed successively by from top to bottom: point sample position Quality Control subregion P1:1 is capable × and 8 points, hybridization positive quality control subregion P2:1 is capable × 4 points, foot and mouth disease virus A type detection probes subregion 1:1 is capable × 4 points, foot and mouth disease virus Asian type detection probes subregion 2:1 is capable × 4 points, foot and mouth disease virus universal detection probes subregion 3:1 is capable × 4 points, foot and mouth disease virus O type detection probes distinguish 4:1 capable × 4 points, vesicular stomatitis virus detection probes subregion 5:1 is capable × 4 points, swine vesicular disease virus detection probes subregion 6:1 is capable × 4 points, blue tongue virus detection probes subregion 7:1 is capable × 4 points, PPR virus detection probes subregion 8:1 is capable × 4 points, hybridize negative Quality Control subregion N:1 capable × 4 points, point sample position Quality Control subregion P1:1 is capable × 8 points.Often open on chip and have at least one above-mentioned microarray, each microarray can detect a sample.The testing sample probe region of microarray can also be set to only containing blue tongue virus probe.
Hybridize negative Quality Control subregion N, comprise spotting buffer, spotting buffer is the commercially available prod that Bo Aosheng company limited produces.All the other each subregions comprise corresponding detection probes and Quality Control probe.
Fig. 2 and Fig. 3 is the result utilizing gene chip of the present invention and detection method to carry out specificity experiments and sensitivity experiment.The lowest detection limit reaches and can reach 2.99 × 10
3copy/uL.
Embodiment 3, reagent prepare
1) chip washing lotion
As required and practical situation, by following proportions washings I and washings II.
Washing lotion I: SSC final concentration is 2 ×, SDS final concentration is 0.2%.As 500mL washing lotion I=440mL distilled water+50mL 20 × SSC+10mL 10%SDS, or prepare in proportion as required.
Washing lotion II: SSC final concentration is 0.2 ×.As 500mL washing lotion II=495mL distilled water+5mL 20 × SSC, or prepare in proportion as required.
If 10%SDS produces white flock precipitate, please be placed in after mixing is dissolved in 42 DEG C of water-baths and prepare washing lotion.
2) dehydrated alcohol.
3) mixture of ice and water.
Embodiment 4, virus genome RNA extract
Extract test kit working instructions according to corresponding commodity RNA, extract the complete genome DNA of the cell proliferation liquid of tissue to be checked or virus, carry out the cDNA that testing sample is prepared in reverse transcription.
Embodiment 5, pcr amplification full-length cDNA
1, PCR reaction system: in PCR dosing district, at thawed on ice primer Mix and genome cDNA, by following system preparation reaction solution.25 μ L PCR reaction solutions comprise: rTaq enzyme 12.5 μ L, primer Mix5.4 μ L, testing sample DNA 3 μ L, nuclease free aqua sterilisa 4.1 μ L;
Wherein, described primer Mix contains:
20 μm of ol/L blue tongue virus special upstream primer SEQ ID NO.4 0.2 μ L,
20 μm of ol/L blue tongue virus special downstream primer SEQ ID NO.5 0.2 μ L,
20 μm of ol/L PCR general upstream primer SEQ ID NO.6 4 μ L,
20 μm of ol/L PCR general downstream primer SEQ ID NO.7 1 μ L;
Blue tongue virus special upstream primer SEQ ID NO.4:
5′-
AGGTGACACTATAGAATAATGCTATCCGGGCTGATCCRA-3′
Blue tongue virus special downstream primer SEQ ID NO.5:
5′-
GTACGACTCACTATAGGGATCACRTCATCACGAAACGCTT C-3′
PCR general upstream primer SEQ ID NO.6:5 '-cy3-AGGTGACACTATAGAATA-3 ',
PCR general downstream primer SEQ ID NO.7:5 '-GTACGACTCACTATAGGGA-3 '
2, increase: the reaction solution configured is placed in PCR amplification instrument, carries out PCR reaction according to the PCR reaction cycle program of table 1.
Table 1PCR response procedures
PCR primer wants lucifuge to place.Short-term preservation places 4 DEG C of refrigerators.
Embodiment 6, interpretation
1, develop a film: hybridization buffer, 42 DEG C of thawings, presses system preparing hybrid liquid below, hybridization system mixed solution 95 DEG C of sex change 8 minutes, ice bath 5 minutes, for subsequent use.Prepare 15 μ L hybridization solutions: hybridization buffer 7.9 μ L, pcr amplification product 7 μ L, hybridization positive control 0.1 μ L.
Open chip hybridization box, hybridizing box is kept flat on the table, in hybridizing box bottom groove, add about 200 μ L aqua sterilisas.By chip front side upward (fence faces up, and label is towards operator) put between hybridizing box two steady braces; Put chip cover plate, note having one of boss facing to chip, the first contact chip in upper end, then under slowly covering; Then slowly inject the hybridization solution after 15 μ L sex change with pipettor by cover plate well, hybridization solution can form liquid film together by means of between the boss of surface tension of liquid below cover plate and chip surface.Notice that vibrations cover plate or chip be not to avoid destroying liquid film.Cover tightly hybridization lid.Put into 50 DEG C of waters bath with thermostatic control, leave standstill, hybridize 3 hours.
After hybridization, taking out chip is placed in 42 DEG C of preheated washing lotions I, 42 DEG C shake cleaning 4 minutes, use the washing lotion II that 42 DEG C preheated again, 42 DEG C of concussion cleanings 4 minutes, finally use 42 DEG C of preheated clean water once, and centrifugal 2 minutes of the chip 2000rpm after cleaning is to remove the liquid of chip surface, this chip can keep in Dark Place, all effective interscan in 4 hours.
2, scanner uni result interpretation
The chip cleaned uses micro-array chip scanner to carry out scanning analysis under brilliant core LuxScanTM10K-A micro-array chip scanner, removes negative control background, illustrates and contrast, result of determination with chip hybridization.
Embodiment 7, gurry process
Process according to local or national infectivity and potential infectious trash processing specification and to use or without the waste of used reagent and pollution.
Claims (5)
1. for detecting the gene chip of blue tongue virus, comprising: PCR reaction mixture pipe, Taq polysaccharase, PCR positive control, PCR negative control, ddH
2the point sample distribution of O, detection chip, chip probe, chip cover plate, chip fence, hybridization solution, hybridization positive control, hybridizing box, adopt gene chip micro-sampling technology, testing sample probe and each special quality control probe are fixed on the aldehyde radical substrate of chemically modified, the microarray formed, it is characterized in that: described microarray comprises Quality Control probe region and testing sample probe region, in wherein said Quality Control probe region, following subregion be set:
Point sample position Quality Control subregion (P1), comprises at least one point sample position Quality Control probe: SEQ ID NO.2;
Hybridization positive quality control subregion (P2), comprises at least one hybridization positive quality control probe: SEQ ID NO.3;
Hybridize negative Quality Control subregion (N), comprise spotting buffer;
At least one blue tongue virus probe is comprised: SEQ ID NO.1 in described testing sample probe region.
2. according to claim 1 for detecting the gene chip of blue tongue virus, it is characterized in that: often open in described detection chip and comprise at least one microarray according to claim 1, each microarray can detect a sample.
3. according to claim 1 for detecting the gene chip of blue tongue virus, it is characterized in that: 5 ' end of described every bar Quality Control probe and testing sample probe is all connected with an amino by 15 thymidylic acids.
4. utilize the detection method of the non-diseases diagnostic purpose of genechip detection blue tongue virus described in claim 1, comprise the steps:
(1) testing sample preparation: extract according to commercialization RNA the full-length genome that test kit working instructions extract the cell proliferation liquid of tissue to be detected or virus, carry out reverse transcription and prepare cDNA, obtain measuring samples DNA;
(2) pcr amplification: 25 μ L PCR reaction solutions comprise: rTaq enzyme 12.5 μ L, primer Mix 5.4 μ L, testing sample DNA 3 μ L, nuclease free aqua sterilisa 4.1 μ L;
Wherein, described primer Mix contains:
20 μm of ol/L blue tongue virus special upstream primer SEQ ID NO.40.2 μ L,
20 μm of ol/L blue tongue virus special downstream primer SEQ ID NO.50.2 μ L,
20 μm of ol/L PCR general upstream primer SEQ ID NO.64 μ L,
20 μm of ol/L PCR general downstream primer SEQ ID NO.71 μ L;
Increase as follows: 94 DEG C of 5min; 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 25sec circulate 12 times; 94 DEG C of 30sec, 50 DEG C of 30sec, 72 DEG C of 25sec circulate 30 times; 72 DEG C of 10min;
(3) hybridize: first preparing hybrid liquid: hybridization buffer 7.9 μ L, pcr amplification product 7 μ L, hybridization positive control 0.1 μ L, mix rear 95 DEG C of sex change 8min, place 5min on ice, then adopt 15 μ L hybridization solutions to hybridize;
(4) result interpretation: use micro-array chip scanner scanning analysis, removes negative control background, illustrates and contrast, result of determination with chip hybridization.
5. detection method according to claim 4, is characterized in that: the every 1000 μ L of described hybridization buffer comprise 20 × SSC 500 μ L, 10%SDS 10 μ L and nuclease free aqua sterilisa 490 μ L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002565A (en) * | 2015-05-28 | 2015-10-28 | 四川农业大学 | Gene chip of vesicular stomatitis viruses and/or swine vesicular disease viruses and reagent kit |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
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| CN1958808A (en) * | 2006-08-11 | 2007-05-09 | 北京爱普益生物科技有限公司 | Gene chip, detection method, and kit for detecting virus infected by oxen |
| CN202595152U (en) * | 2012-06-06 | 2012-12-12 | 河南出入境检验检疫局检验检疫技术中心 | Glass gene chip for detecting fur carried virus |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1958808A (en) * | 2006-08-11 | 2007-05-09 | 北京爱普益生物科技有限公司 | Gene chip, detection method, and kit for detecting virus infected by oxen |
| CN202595152U (en) * | 2012-06-06 | 2012-12-12 | 河南出入境检验检疫局检验检疫技术中心 | Glass gene chip for detecting fur carried virus |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002565A (en) * | 2015-05-28 | 2015-10-28 | 四川农业大学 | Gene chip of vesicular stomatitis viruses and/or swine vesicular disease viruses and reagent kit |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
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