CN107119330A - A kind of preparation method and application method of the genetic chip for detecting pig parvoviral - Google Patents
A kind of preparation method and application method of the genetic chip for detecting pig parvoviral Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and application method of the genetic chip for detecting pig parvoviral, design and synthesize primer and probe, synthetic probe is dissolved with TE buffer, with gene chip sample applying instrument on aldehyde radical substrate contact point sample, prepare the genetic chip for detecting pig parvoviral.In use, extracting pig parvoviral genomic DNA to be measured, hybridization PCR primer is prepared using asymmetric PCR, and the gene chip hybridization prepared, it is scrubbed with dry after, genetic chip is placed on laser confocal scanner and scanned, analysis result.The present invention is easy to operation, eliminate the hydration process and pre-hybridization step of point sample chip, hybridization is only needed 1 hour, greatly save the time, the purpose of quick detection is reached, higher signal intensity can just be reached using 5 μm/L concentration and probe concentration, testing cost is reduced, available for extensive detection.
Description
Technical field
The present invention relates to biomedical sector, the preparation method of specifically a kind of genetic chip for detecting pig parvoviral and
Application method.
Background technology
Existing pig parvoviral detection method has isolation of virus, serological Identification and molecular Biological Detection etc..Disease
Poison separation identification method is that lesion occurs for cellular morphology according to caused by virus growing multiplication on cell etc..In inverted microscope
Down it was observed that cell there occurs lesion, there are cell aggregation, circle contracting, cell fragment to be attached to the first-class phenomenon of its cell.Serology is examined
Survey method includes blood clotting and hemagglutination-inhibition test, neutralization test, EUSA, wherein blood clotting and hemagglutination-inhibition test
The most frequently used detection method.Molecular biology for detection includes common PCR detections, multiplex PCR detection, quantitative fluorescent PCR
There is the sensitiveness of very strong specificity and height etc. detection method.
Genetic chip has the advantages that high sensitivity, pinpoint accuracy, high information quantity as detection means, is applied to many
In the detection for planting cause of disease, the detection method of the genetic chip for swine disease virus has been set up, but has needed the chip to point sample
Aquation and prehybridization processing are carried out, and the concentration of required hybridization probe is higher, testing cost is high.
The content of the invention
It is an object of the invention to provide a kind of preparation method and application method of the genetic chip for detecting pig parvoviral,
Existing detection device and method can effectively be solved, and time-consuming, and error is big, the problem of putting into big.
To achieve the above object, the present invention provides following technical scheme：
A kind of preparation method for the genetic chip for detecting pig parvoviral, comprises the following steps：
(1) design specific primer and probe：VP2 protein gene sequences using pig parvoviral is target sequences, and design is special
SEQ ID NO in specific primer and probe, sense primer such as sequence table：Shown in 1；SEQ ID NO in anti-sense primer such as sequence table：
Shown in 2；SEQ ID NO in probe such as sequence table：Shown in 3；
(2) above-mentioned specific primer and probe are synthesized, synthesising probing needle is made；
(3) preparation of genetic chip：With TE buffer solution synthesising probing needles, the contact point sample on aldehyde radical substrate is dried
After produce genetic chip.
It is used as further scheme of the invention：In step (2), the concentration of specific primer is 10 μm of mol/L.
It is used as further scheme of the invention：In step (2), the concentration of probe is 5 μm of mol/L-20 μm of mol/L.
It is used as further scheme of the invention：In step (3), the ambient parameter of contact point sample is relative humidity 55-
65%, 15-30 DEG C of temperature.
It is used as further scheme of the invention：In step (3), drying process is to be statically placed in 80 DEG C of oven dryings 2 hours.
A kind of application method for the genetic chip for detecting pig parvoviral, comprises the following steps：
(1) genomic DNA of pig parvoviral bacterium to be measured is extracted；
(2) preparation of hybridization PCR primer：Using asymmetric PCR, in first premixing reaction system in addition to anti-sense primer
All the components, then add to be placed in PCR instrument after anti-sense primer, mixing and carry out amplification in darkroom hybridization PCR primer be made；
Described reaction system is：5 × Buffer is 5.0 μ l, and sense primer is 1.0 μ l, and anti-sense primer is that 2.0 μ l, dNTP are 2.0 μ
L, template DNA is that 1.0 μ l, Prime STAR are 0.3 μ l, ddH2O is 13.7 μ l, and cumulative volume is 25 μ l；Amplification reaction condition is：
94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 circulations, 72 DEG C of extension 5min；
(3) hybridization PCR primer and gene chip hybridization：Hybridization is placed in 98 DEG C of denaturation in PCR instrument with PCR primer
After 10min, ice bath 5min is taken out immediately, takes hybridization to be added on the μ l of PCR primer 40 and the μ l of hybridization buffer 160 in centrifuge tube, mix
Take the mixed liquor to be added to the point sample area of genetic chip after even, genetic chip is put into hybridizing box, be placed in 47 DEG C of water-baths and keep away
Light hybridizes 1h；
(4) washing and drying of genetic chip：In darkroom, the genetic chip that hybridization is finished is placed in glass frame,
Wash liquid is used immediately three times, each 2min, centrifugal drying；
(5) interpretation of result of genetic chip：Genetic chip is placed on laser confocal scanner and scanned, analysis Cy3 is glimmering
The intensity and ratio of optical signal.
It is used as further scheme of the invention：DNTP concentration is 10mmol/ μ l.
It is used as further scheme of the invention：Three washings of described genetic chip washing lotion used respectively is 1 ×
SSC/0.2%SDS, 0.1 × SSC/0.2%SDS, 0.1 × SSC.
It is used as further scheme of the invention：The foundation of described gene chip hybridization result judgement is as follows：
(1) the PCR expanding effects of template DNA are good；
(2) signal intensity median >=1000, with reference to hybrid screens image, hybridization spot is clear；
(3) results of hybridization of detection genetic chip introducing sampling point signal threshold concept SNR when judging, during it is signal intensity
The ratio between place value and noise median, when their ratio is more than or equal to 1.5, show that hybridization signal is good, i.e. SNR >=1.5.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention is easy to operation, eliminates the hydration process and pre-hybridization step of point sample chip, only need 1 are small for hybridization
When, the time is greatlyd save, the purpose of quick detection has been reached, higher letter can just be reached using 5 μm/L concentration and probe concentration
Number intensity, reduces testing cost, available for extensive detection.
Brief description of the drawings
Fig. 1 is pig parvoviral gene chip sample applying schematic diagram.
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all
Belong to the scope of protection of the invention.
The present invention designs and synthesizes primer and probe, and synthetic probe is dissolved with TE buffer, gene chip sample applying is used
Instrument presses pre-set program contact point sample on aldehyde radical substrate, prepares the genetic chip for detecting pig parvoviral.
In use, extracting pig parvoviral genomic DNA to be measured, hybridization PCR primer is prepared using asymmetric PCR, with preparing
Gene chip hybridization, it is scrubbed with dry after, by genetic chip be placed on laser confocal scanner scan, analysis result.
Tests prove that, the method is sensitive reliable.Application effect is good, and relevant information is as follows：
1 materials and methods
1.1 material
1.1.1 reference culture is originated
Pig parvoviral bacterial strain is given by Agricultural University Of He'nan.
1.1.2 chip material
Genetic chip substrate uses aldehyde radical substrate, purchased from rich classical Bioisystech Co., Ltd difficult to understand.
1.1.3 test apparatus
1.1.4 main agents and solution
5 × electrophoresis storing liquid (5 × TBE), 1.5% Ago-Gel, 20 × SSC, 10%SDS, eluent are by this reality
Test room preparation.
1.2 method
1.2.1 the culture of pig parvoviral
According to regular growth cultural method, with the DMEM in high glucose culture medium (pH7.2) containing 10% hyclone for nutrition
Liquid, in 37 DEG C, 5%CO2Culture propagation PK-15 cells 24h in incubator；Digested carefully with 0.25% pancreatin during passage
Born of the same parents, sub-bottle passes inoculation PPV viruses after 3 generations and continues to cultivate.
1.2.2 the extraction of pig parvoviral genomic DNA
Virus to above-mentioned culture uses the Ezup pillar viral DNA extraction agents of Shanghai bioengineering limited company
Box is extracted, and is comprised the following steps that：
(1) sample treatment：0.2ml virus liquid samples are added in 1.5ml centrifuge tubes.By 1.5ml liquid in 4 DEG C of 24000g
60min is centrifuged under refrigerated centrifuge, it is standby that remaining 0.2ml (2) step after 1.3ml is abandoned in shifting.
(2) 0.6ml solution As are added in the 0.2ml samples that the first step is obtained, concussion mixes 30s, and subsequent room temperature is placed
10min。
(3) moved on to the of short duration 500 μ l solution centrifuged are careful with rifle in adsorption column, 2min is placed at room temperature.
(4) 12000rpm centrifuges 1min at room temperature, discards and penetrates liquid, adsorption column is put back in collecting pipe again.
(5) it will be transferred completely into the adsorption column in (3) step, place at room temperature after the of short duration centrifugation of remaining solution
2min。
(6) 12000rpm is centrifuged after 1min at room temperature, is discarded and is penetrated liquid, and adsorption column is put back in collecting pipe.
(7) 0.7ml is added in adsorption column washes post liquid, and 12000rpm room temperatures centrifugation 1min is discarded and penetrated liquid, absorption
Post is put back in collecting pipe.
(8) 12000rpm centrifuges 1min at room temperature, gives up containing the centrifuge tube for penetrating liquid.
(9) after adsorption column is inserted in new 1.5ml centrifuge tube, 30- is added at the middle part of the filter membrane of adsorption column
100 μ l DNA eluents, place 2min at room temperature.
(10) 12000rpm centrifuges 1min at room temperature, and ttom of pipe solution is the DNA solution extracted, can immediately using or put -20
DEG C refrigerator is preserved for a long time.
1.2.3 the design and synthesis of primer
Reference literature, the VP2 protein gene sequences using pig parvoviral (GenBank Serial No. NC-001718) is targets
Sequence, utilizes the Software for Design specific primers of Primer Premier 5.0 and probe.Sense primer：PPV：Sense primer：5’-
CAAACAGATCTCTAGGACTGC-3 ', anti-sense primer：5 '-Cy3-GTGGTTAAGTGTCCATGTTGG-3 ', probe：5’-
NH2-TTTTTTTT-CAGCAATTAGGCCAGCTC-3’.Cy3 fluorescence labelings, 5 ' Amino End Groups of probe are carried out at the end of downstream 5 '
Modification, and plus 8 T between amino and probe.Primer and probe is synthesized by the precious bioengineering Co., Ltd in Dalian.Use TE
Buffer by the concentration that synthetic lyophilized primer is diluted to 10 μM/L be placed in -20 DEG C freeze it is standby.
1.2.4 the preparation of genetic chip
First with aqua sterilisa by synthetic lyophilized probe dilution into 100 μM/L mother liquor, then be diluted to 5 μM/L (5 μ l successively
The μ l spotting buffers of the μ l of probe+45 aqua sterilisas+50), 10 μM/L (the μ l spotting buffers of+40 μ l aqua sterilisas of 10 μ l probes+50), 15
μM/L (the μ l spotting buffers of+35 μ l aqua sterilisas of 15 μ l probes+50), 20 μM/L (the μ l point samples of+30 μ l aqua sterilisas of 20 μ l probes+50
Buffer solution).
100 μ l spotting buffers are added in the A1 holes of 96 hole U-boards and make negative control, A4 holes add 100 μ l containing fluorescence
PCR primer makees positive control, and A2, A3, A5, A6 hole are separately added into 100 5 μM/L of μ l, 10 μM/L, 15 μM/L, 20 μM/L spy
Pin.
Using the AD1500 gene chip sample applyings instrument of Bio-Dot companies of the U.S. by pre-set program in aldehyde radical base
Contact point sample on piece.Point sample ambient parameter is relative humidity 55-65%, and 20-30 DEG C of temperature, each probe points spacing is 1.5mm,
Sample application array is set as six matrixes according to requirement of experiment, and each matrix point sample is 5 × 3, and the genetic chip that point sample is finished is stood
It is standby in 80 DEG C of oven drying 2h.
Gene chip sample applying schematic diagram is shown in Fig. 1.
1.2.5 the preparation and identification of hybridization PCR primer
Reaction condition and parameter are optimized one by one, optium concentration and amplification reaction condition is established, hybridization is needed with product
Using asymmetric PCR.Reaction cumulative volume is 25 μ l, and its composition is as follows：
The all the components in above-mentioned reaction system in addition to anti-sense primer are first pre-mixed during experiment, are finally added in darkroom
PCR instrument is placed in after anti-sense primer, mixing to be expanded, obtained hybridization PCR primer.
Pcr amplification reaction condition is：94 DEG C of pre-degeneration 5min, (94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extensions
30s, 35 circulations) 72 DEG C of extension 5min.
The μ l of hybridization PCR primer 6 and 1 μ l 6 × Lording Buffer are taken, after being well mixed with 10 μ l pipettors, by rifle
Head is carefully deep into well, is careful not to reach bottom hole portion, in order to avoid penetrate well and cause sample excessive, it is slow to release
Samples vertical is set to fall into well.
After sample-adding terminates, electrophoresis groove lid is covered, regulation voltage is 110V, closes Electrophoresis Lab light, lucifuge electrophoresis 30min.
After electrophoresis terminates, the careful gel that takes out is positioned in gel imaging system, is observed and preservation result of taking pictures.
1.2.6 the hybridization of genetic chip
Hybridization is placed in PCR instrument with PCR primer after 98 DEG C of denaturation 10min, ice bath 5min is taken out immediately, takes hybridization to use
The μ l of PCR primer 40 and the μ l of hybridization buffer 160 are added in centrifuge tube, take the mixed liquor to be added to the point sample of genetic chip after mixing
Area, avoids producing bubble, genetic chip is put into hybridizing box as far as possible, is placed in lucifuge in 47 DEG C of insulating boxs and hybridizes 1h.
1.2.7 the washing and drying of genetic chip
The washing of genetic chip should be completed in darkroom.Hybridize the genetic chip finished to be placed in glass frame, immediately with 1
× SSC/0.2%SDS, 0.1 × SSC/0.2%SDS, 0.1 × SSC, which are respectively washed, can be used to scanning point after 2min, centrifugal drying
Analysis.
1.2.8 the scanner uni scanning result analysis of genetic chip
Genetic chip is placed on InnoScan 700A High-performance lasers confocal scanners and scanned, if background is higher
It can again washed once with washing lotion 3 (1 × SSC), image is preserved with TIF forms, JPG format pictures are then saved as again.Use MAPIX
The intensity and ratio of software analysis Cy3 fluorescence signals, with foundation of the three below condition as gene chip hybridization result judgement：
(1) the PCR expanding effects of template DNA are good；
(2) signal intensity median >=1000, with reference to hybrid screens image, hybridization spot clear (readability and hybridization
Condition has much relations)；
(3) sampling point signal threshold concept SNR is introduced when detection gene chip hybridization result judges, it is signal intensity middle position
The ratio between value and noise median, when their ratio is more than or equal to 1.5, show that hybridization signal is good, i.e. SNR >=1.5.
Present invention process is simple, and quick, sensitive detection can be carried out to pig parvoviral, satisfied effect is achieved through experiment
Really, relevant result of the test is as follows：
1st, PCR amplifications
Using pig parvoviral genomic DNA as template, asymmetric PCR amplification is carried out with its specific primer, through agarose
Gel electrophoresis analysis amplified fragments size is about 313bp, identical with estimated purpose fragment, and specificity is preferably.
2nd, gene chip hybridization result
Asymmetric hybridization is placed in PCR instrument after 98 DEG C of denaturation 10min with PCR primer, and ice bath 5min, takes the product immediately
40 μ l and the μ l of hybridization buffer 160, which are added in centrifuge tube, to be sufficiently mixed uniformly, and the mixed liquor is all added into gene chip sample applying
Area, genetic chip is put into hybridizing box, is placed in 47 DEG C of insulating box lucifuge hybridization 1h.The scanned instrument scanning of results of hybridization, it is each miscellaneous
The SNR value of spot is handed over to be all higher than 1.5.
As a result show, higher signal intensity can just be reached using 5 μM/L concentration and probe concentration, detected into so as to reduce
This, available for extensive detection.
The present invention is easy to operation, eliminates the hydration process and pre-hybridization step of point sample chip, only need 1 are small for hybridization
When, the time is greatlyd save, the purpose of quick detection has been reached, higher letter can just be reached using 5 μM/L concentration and probe concentration
Number intensity, reduces testing cost, the investigation work available for large-scale pig farm parvovirus.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, nothing
By from the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended
Claim rather than described above are limited, it is intended that will fall the institute in the implication and scope of the equivalency of claim
Change and include in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It may be appreciated other embodiment.
SEQUENCE LISTING
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<120>A kind of preparation method and application method of the genetic chip for detecting pig parvoviral
<130> 2017
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<170> PatentIn version 3.3
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<211> 21
<212> DNA
<213>It is unknown
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<212> DNA
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cygtggttaa gtgtccatgt tgg 23
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<212> DNA
<213>It is unknown
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, or t
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nhtttttttt cagcaattag gccagctc 28
Claims (9)
1. a kind of preparation method for the genetic chip for detecting pig parvoviral, it is characterised in that comprise the following steps：
(1) design specific primer and probe：VP2 protein gene sequences using pig parvoviral is target sequences, design specificity
SEQ ID NO in primer and probe, sense primer such as sequence table：Shown in 1；SEQ ID NO in anti-sense primer such as sequence table：2 institutes
Show；SEQ ID NO in probe such as sequence table：Shown in 3；
(2) above-mentioned specific primer and probe are synthesized, synthesising probing needle is made；
(3) preparation of genetic chip：With TE buffer solution synthesising probing needles, the contact point sample on aldehyde radical substrate, after drying i.e.
Obtain genetic chip.
2. the preparation method of the genetic chip of detection pig parvoviral according to claim 1, it is characterised in that step
(2) in, the concentration of specific primer is 10 μm of mol/L.
3. the preparation method of the genetic chip of detection pig parvoviral according to claim 1, it is characterised in that step
(2) in, the concentration of probe is 5 μm of mol/L-20 μm of mol/L.
4. the preparation method of the genetic chip of detection pig parvoviral according to claim 1, it is characterised in that step
(3) in, the ambient parameter of contact point sample is relative humidity 55-65%, 15-30 DEG C of temperature.
5. the preparation method of the genetic chip of detection pig parvoviral according to claim 1, it is characterised in that step
(3) in, drying process is to be statically placed in 80 DEG C of oven dryings 2 hours.
6. a kind of application method of the genetic chip of detection pig parvoviral as described in claim 1-5 is any, its feature exists
In comprising the following steps：
(1) genomic DNA of pig parvoviral bacterium to be measured is extracted；
(2) preparation of hybridization PCR primer：Using asymmetric PCR, the institute in first premixing reaction system in addition to anti-sense primer
There is composition, then added in darkroom and the obtained hybridization PCR primer of PCR instrument progress amplification is placed in after anti-sense primer, mixing；It is described
Reaction system be：5 × Buffer is 5.0 μ l, and sense primer is 1.0 μ l, and anti-sense primer is that 2.0 μ l, dNTP are 2.0 μ l, mould
Plate DNA is that 1.0 μ l, Prime STAR are 0.3 μ l, ddH2O is 13.7 μ l, and cumulative volume is 25 μ l；Amplification reaction condition is：94℃
Pre-degeneration 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 circulations, 72 DEG C of extension 5min；
(3) hybridization PCR primer and gene chip hybridization：Hybridization is placed in PCR primer in PCR instrument after 98 DEG C of denaturation 10min,
Ice bath 5min is taken out immediately, takes hybridization to be added on the μ l of PCR primer 40 and the μ l of hybridization buffer 160 in centrifuge tube, this is taken after mixing
Mixed liquor is added to the point sample area of genetic chip, and genetic chip is put into hybridizing box, is placed in lucifuge in 47 DEG C of water-baths and hybridizes 1h；
(4) washing and drying of genetic chip：In darkroom, the genetic chip that hybridization is finished is placed in glass frame, immediately
With wash liquid three times, each 2min, centrifugal drying；
(5) interpretation of result of genetic chip：Genetic chip is placed on laser confocal scanner and scanned, analysis Cy3 fluorescence letters
Number intensity and ratio.
7. the application method of the genetic chip of detection pig parvoviral according to claim 6, it is characterised in that dNTP's
Concentration is 10mmol/ μ l.
8. the application method of the genetic chip of detection pig parvoviral according to claim 6, it is characterised in that described
Three washings of genetic chip washing lotion used respectively is 1 × SSC/0.2%SDS, 0.1 × SSC/0.2%SDS, 0.1 × SSC.
9. the application method of the genetic chip of detection pig parvoviral according to claim 6, it is characterised in that described
The foundation of gene chip hybridization result judgement is as follows：
(1) the PCR expanding effects of template DNA are good；
(2) signal intensity median >=1000, with reference to hybrid screens image, hybridization spot is clear；
(3) sampling point signal threshold concept SNR, and SNR >=1.5 are introduced when the results of hybridization of detection genetic chip judges.
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| CN108359600A (en) * | 2018-01-22 | 2018-08-03 | 中国科学院苏州生物医学工程技术研究所 | A kind of highly sensitive, quick, absolute quantitation DNA fragmentation detecting system and detection method |
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Application publication date: 20170901 |