CN104232790A - Gene chip and kit for detecting foot and mouth disease virus and porcine reproductive and respiratory syndrome virus - Google Patents
Gene chip and kit for detecting foot and mouth disease virus and porcine reproductive and respiratory syndrome virus Download PDFInfo
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Abstract
The invention discloses a gene chip and a kit for detecting foot and mouth disease virus and porcine reproductive and respiratory syndrome virus. The foot and mouth disease virus and the porcine reproductive and respiratory syndrome virus can be accurately and effectively detected through the gene chip and the detection kit and a detection method, and the gene chip and the detection kit and the detection method are strong in specificity, high in sensitivity, short in consumption time, fast to detect, and excellent in application prospects.
Description
Technical field
The present invention relates to a kind of gene chip and the detection method that detect foot and mouth disease virus and porcine reproductive and respiratory syndrome virus.
Background technology
Foot and mouth disease (foot-and-mouth disease, FMD) be that the one artiodactylous that caused by foot and mouth disease virus (FMDV) is acute, hot, hyperinfection epidemic disease, principal character occurs blister at oral mucosa, hoof, breast, skin, the transmissible disease that the class velocity of propagation that ulcer occurs then is exceedingly fast.This sick serious harm Animal husbandry production, affects international trade and national reputation, receives the common concern of national governments, be classified as the zoonosis of legal circular by International Office of Epizootics, and China is classified as first of the great animal epidemic of a class.FMD susceptible animal comprises the domestic and wild artiodactyls of kind more than 70 such as ox, sheep, goat, camel and pig.This disease was once widely current in the world, caused huge financial loss to world's aquaculture.In recent years, once there is the FMD epidemic situation of O type, A type, Asia1 type 3 serotypes in China: a lot of Asia1 type foot and mouth disease epidemic situation occurs for 2005 ~ 2007; 2009 there is A type FMD epidemic situation in Wuhan, Shanghai and Beijing; Within 2009 and 2010, at TaiWan, China and Hong Kong autonomous region, O type FMD epidemic situation occurs in succession, in the Baiyun District of Guangzhou, Guangdong in 2010, O type FMD epidemic situation occurs, 18 of 9 provinces to China mainland at the end of 2010 are local there are O type FMD epidemic situations.In addition, in healthy cows, find that there is the malicious situation of band.The national FMD popularity more complicated such as India, Vietnam, Afghanistan of China's periphery, external strain repeatedly imports China into, causes huge threat to the prevention and control of China FMD.FMD infectivity is strong, endangers huge, diagnoses the prevention and control of FMD significant fast and accurately.
Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) also known as pig blue-ear disease, by porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome, PRRSV), the high degree in contact sexually transmitted disease of pig is caused.Classical pig blue-ear disease and highly pathogenic PRRS can be divided into by the difference of clinical manifestation.Classical pig blue-ear disease with sow breeding difficulty, premature labor, miscarriage, stillborn foetus, mummy tire and piglet respiration syndrome for feature; Highly pathogenic PRRS spreads through sex intercourse with high degree in contact, systemic bleeding, lung's consolidation and sow breeding difficulty are feature, piglet, growing and fattening pigs and Adult Pig all can be fallen ill and death, wherein piglet sickness rate can reach 100%, and mortality ratio can reach more than 50%, and Sow abortion rate can reach more than 30%.Highly pathogenic PRRS is classified as a class animal epidemic by " one, two, three class animal epidemic diseases plant register " of China's newly revision in 2008.At present, this disease has been widely current in all countries and regions of raising pigs in the world, is the disease that harm pig industry is the most serious.PRRSV, since last century, the nineties imported China into, has caused huge loss to pig industry, and highly pathogenic PRRS (HPRRSV) epidemic situation of the particularly outburst second half year in 2006, causes huge financial loss.At present, China's PRRSV epidemic isolates is mainly american type, and classical strain and highly pathogenic mutant strain all extensively exist in swinery.
Foot and mouth disease (FMD) and pig blue-ear disease (porcine reproductive and respiratory syndrome, PRRS) can cause huge financial loss, are emphasis and the top priority of swinery control and prevention of disease to these 3 sick prevention and control.Diagnose timely and accurately morbidity livestock and poultry, carry out animal epidemic active monitoring in a large number, grasping the infection conditions of livestock and poultry pestilence, is prerequisite and the basis of taking science bridle measure, is also the inevitable requirement improving animal epidemic science bridle level; , by monitoring, etiology animals showing positive is purified meanwhile, effectively can reduce wild poison and pollute, promote livestock birds health.At present for the existing a lot of report of pathogeny detection method of FMDV and PRRSV, detect separately each disease, bothersome, effort, adds testing cost.The high-throughput associated detection technique of high in the urgent need to susceptibility in production, high specificity, to improve detection efficiency, effectively reduces labour intensity, saves testing cost.
Wang little Qiang, " for diagnosing the development of four boar epidemic disease virus oligonucleotide chips ", Northwest University, microbiology, 2007 disclose the gene chip simultaneously detecting swine influenza virus, PRV (Pseudorabies virus), foot and mouth disease virus, pig blue-ear disease poison (porcine reproductive and respiratory syndrome virus), the lowest detectable limit of this genechip detection foot and mouth disease virus and porcine reproductive and respiratory syndrome virus is respectively: foot and mouth disease virus 6/mL, porcine reproductive and respiratory syndrome virus 5 .05 × 10
2individual/mL.
Summary of the invention
In order to solve the problem, the invention provides a kind of newly, highly sensitive while detect gene chip and the test kit of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus.
The present invention detects the gene chip of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, and it comprises solid phase carrier and is fixed on the probe on solid phase carrier; Described probe to comprise shown in SEQ ID NO:5 ~ 7 shown in any one or any number of gene fragment and SEQ ID NO:8 ~ 10 any one or any number of gene fragment.
Gene chip, refer to and refer to, by different methods, biomolecules (oligonucleotide, cDNA, genomic DNA, polypeptide, antibody, antigen etc.) is bonded to the biomolecules dot matrix that the solid phase mediators such as silicon chip, sheet glass (pearl), plastic sheet (pearl), gel, nylon membrane are formed, its outstanding feature is microminiaturized, integrated, parallelization and high-throughput.
Wherein, it also comprises positive control probe is the gene fragment shown in SEQ ID NO:12, and negative control probe is the gene fragment shown in SEQ ID NO:11, and position probe is the gene fragment shown in SEQ ID NO:14 that marked fluorescence dye.
Described fluorescence dye is cy3 fluorescence dye or cy5 fluorescence dye.
Described solid phase carrier is aldehyde radical slide.
Described foot and mouth disease virus is A type, O type or Asia1 type foot and mouth disease virus.
Described porcine reproductive and respiratory syndrome virus is american type porcine reproductive and respiratory syndrome virus.
Described american type porcine reproductive and respiratory syndrome virus is american type high-pathogenicity porcine reproductive and respiratory syndrome virus or american type pig blue-ear disease classical strains.
The present invention detects the test kit of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, it is characterized in that: it comprises the reagent of the gene of the gene chip described in claim 1 or 2 and increase foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, wherein, the reagent of amplification foot-and-mouth disease virus gene comprises primer pair shown in SEQ ID NO:1 ~ 2; The reagent of amplification porcine reproductive and respiratory syndrome virus gene comprises primer pair shown in SEQ ID NO:3 ~ 4.
In described primer pair, shown in SEQ ID NO:1,3, shown in upstream primer and SEQ ID NO:2,4, the mol ratio of downstream primer is 1:10
Downstream primer shown in described SEQ ID NO:2,4 is marked with fluorescence dye.
Described fluorescence dye is cy3 fluorescence dye or cy5 fluorescence dye.
Described foot and mouth disease virus is A type, O type or Asia1 type foot and mouth disease virus.
Described porcine reproductive and respiratory syndrome virus is american type PRRS virus.
Described american type PRRS virus is american type high-pathogenicity porcine reproductive and respiratory syndrome virus or american type pig blue-ear disease classical strains.
To the invention provides shown in SEQ ID NO:5 ~ 7 any one or any number of gene fragment, prepare purposes in the gene chip that detect foot and mouth disease virus and porcine reproductive and respiratory syndrome virus with any one or any number of gene fragment SEQ ID the NO:8 ~ 10 Suo Shi.
It is the gene fragment shown in SEQ ID NO:12 that described gene chip also comprises positive control probe, and negative control probe is the gene fragment shown in SEQ ID NO:11, and position probe is the gene fragment shown in SEQ ID NO:14 that marked fluorescence dye.
Present invention also offers the purposes that primer pair shown in SEQ ID NO:1 ~ 2 and SEQ ID NO:3 ~ 4 increases in the reagent of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus gene in preparation simultaneously.
The invention provides primer pair shown in SEQ ID NO:1 ~ 2 and SEQ ID NO:3 ~ 4, prepare purposes in the test kit that detect foot and mouth disease virus and porcine reproductive and respiratory syndrome virus with any one or any number of gene fragment shown in any one or any number of gene fragment, SEQ ID NO:8 ~ 10 SEQ ID the NO:5 ~ 7 Suo Shi; Wherein, 2 pairs of primer pairs are amplifing reagent, and shown in SEQ ID NO:5 ~ 10, gene fragment is detection probes.
In described primer pair, shown in SEQ ID NO:1,3, shown in upstream primer and SEQ ID NO:2,4, the mol ratio of downstream primer is 1:10.
Shown in described SEQ ID NO:2,4, downstream primer is marked with fluorescence dye.
Described fluorescence dye is cy3 fluorescence dye or cy5 fluorescence dye.
Described foot and mouth disease virus is A type, O type or Asia1 type foot and mouth disease virus.
Described porcine reproductive and respiratory syndrome virus is american type PRRS virus.
Described american type PRRS virus is american type high-pathogenicity porcine reproductive and respiratory syndrome virus or american type pig blue-ear disease classical strains.
Gene chip of the present invention and test kit can effectively detect foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, high specificity, highly sensitive, and the order of magnitude of two-strain minimum detectable concentration is 10
2copies/ μ L, consuming time short, detect fast, can grasp rapidly the infection conditions of livestock and poultry pestilence, the generation of effective prevention and control foot and mouth disease (FMD) and pig blue-ear disease, potential applicability in clinical practice is good.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.The technology that all contents recorded based on claims of the present invention realize all belongs to scope of the present invention.
Accompanying drawing explanation
Fig. 1 oligonucleotide microarray probe point sample schematic diagram;
Fig. 2 position probe regulating YIN and YANG contrast concentration and probe concentration screening chip point sample schematic diagram;
Fig. 3 FMDV detection probe concentrations screening chip point sample distribution plan;
Fig. 4 M1: molecular weight Marker DL1000; M2: molecular weight Marker DL2000; 1-3:A, O, Asia1 FMDV RNA amplification product; 4:pMD20-T contrasts; 5:PMD3D amplified production;
The sequencing result of Fig. 5 clone gene fragment;
Fig. 6 RT-PCR amplified production electrophoresis result.The molecular weight Marker of M:100bp; 1,2:CH1-R strain and JXA1-R strain RNA amplification result; 3: negative control; 4,5: plasmid PMDP and PMDHP amplification; 6:pMD-20T vehicle Control;
Fig. 7 clone gene fragment sequence;
The result of Fig. 8 positive control marker thing target gene (PC2) and probe (PC1);
Fig. 9 positive control marker thing (PC2) concentration and probe (PC1) concentration are on the impact of hybridization signal;
Figure 10 substance and multiplex RT-PCR amplification product electrophoresis result.The molecular weight Marker of M:100bp; 1,3,5,7,9: substance RT-PCR amplification; 2,4,6,8,10: multiplex RT-PCR amplification result; 1,2:FMDV; 7,8:CH1-R; 9,10:JXA1-R;
The multiple asymmetric RT-PCR marked product results of hybridization of Figure 11.1：FMDV；2:CH1-R；3：JXA1-R；
Figure 12 FMDV 3D fragment label thing and detection probes results of hybridization at different temperatures;
Classical strain (CH1-R) marker of Figure 13 pig blue-ear disease results of hybridization at different temperatures;
Figure 14 highly pathogenic PRRS variant (JXA1-R) marker results of hybridization at different temperatures;
Figure 15 specific detection result;
The genechip detection result of different extent of dilution plasmid PMD3D (FMDV) of Figure 16.The plasmid PMD3D of 1# ~ 10#:10 × dilution, concentration is respectively 8.34 × 10
9~ 8.34 × 10
0copies/ μ L;
The genechip detection result of different extent of dilution plasmid PMDP (PRRSV) of Figure 17.The plasmid PMDP of 1# ~ 10#:10 × dilution, concentration is respectively 3.76 × 10
9~ 3.76 × 10
0copies/ μ L;
The genechip detection result of different extent of dilution plasmid PMDHP (HPRRSV) of Figure 18.The plasmid PMDHP of 1# ~ 10#:10 × dilution, concentration is respectively 3.65 × 10
9~ 3.65 × 10
0copies/ μ L.
Embodiment
One, experiment material and instrument
Material prepares
1.1 strain
A type, O type, Asia1 type FMDV standard antigen: the foot-and-mouth disease antibody LPB-ELISA test kit that Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences produces; American type high-pathogenicity porcine reproductive and respiratory syndrome virus (HPRRSV, JXA1-R strain), american type pig blue-ear disease classical strains (PRRSV, CH1-R strain), PRV (Pseudorabies virus) (PRV): from commodity attenuated vaccine; The sick virus (BVDV) of pig circular ring virus (PCV-2), pig parvoviral (PPV), Latex agglutination test (JEV), bovine viral diarrhoea/mucous membrane: Sichuan Agricultural University preserves; 20 parts of clinical unstable swinery tissue samples, animal epidemic prevention and control center, Sichuan Province is preserved.
1.2 main agents
RT-PCR one step amplification test kit PrimeScript one step RT-PCR kit, carrier pMD-20T are purchased from TaKaRa company; Sepharose DNA reclaims test kit, plasmid extraction kit TIAN pure Midi Plasmid Kit, and competent cell DH5 α is purchased from Tian Gen company; It is prosperous purchased from Beijing Century unit that RNA, DNA pillar extracts test kit.
Baio aldehyde radical slide glass, Baio2 × spotting buffer, Baio hybridization buffer, all purchased from Baiao Science and Technology Co. Ltd., Shanghai; Washings I (2 × SSC, 0.1%SDS), washings II (0.2 × SSC, 0.1%SDS), washings III (0.2 × SSC), 0.5%NaBH40.1mol/L phosphate buffered saline buffer etc.
1.3 key instrument
Gene chip sample applying instrument (Microgrid II Compact, BioRobotics, UK); Gene chip scanning instrument (GenePix Personal 4100A, Axon Instruments Inc., USA); UV-crosslinked instrument (CL-508, Uvitec, UK); High speed freezing centrifuge (Sigma, Germany); Common gradient PCR instrument (BIO-RAD, USA); Concussion incubator (THZ-92C, Shanghai Bo Xun Industrial Co., Ltd.); Nucleic acid protein detector (Ependorf Biophotometer, Germany); Water isolation type constant incubator (Shanghai Qi Xin scientific instrument company limited).
Embodiment 1 detection method
1, material and instrument
With aforementioned experiment material and instrument.
2, experimental technique
The Design and synthesis of 2.1 PCR primer, detection probes
Log in GenBank and obtain the gene order that FMDV, PRRSV main genotypes (serotype) represent strain, the MegAlign module of DNAStar software is utilized to carry out homology analysis respectively, choose conservative gene as detection target area, rule of thumb, with Oligo7.0 software design amplimer and oligonucleotide probe.When marking sample, downstream primer selects 5` to hold the primer of Cy3 modification; Oligonucleotide probe 5` holds and adds Poly T15 connecting arm and amination.Primer sequence is in table 1, and oligonucleotide probe, in table 2, serves Hai Shenggong synthesis after Blast comparison.
Table 1: primer sequence and position
Table 2: oligonucleotide probe sequence
1.2 nucleic acid extraction
Beijing Century Yuan Heng company RNA pillar extraction test kit and DNA pillar is used to extract the nucleic acid that test kit extracts corresponding virus.
The structure of 2.3 standard plasmids
Respectively with O type FMDV, PRRSV virus CH1-R strain and JXA1-R strain nucleic acid are template, RT-PCR amplification is carried out with Primescript one step RT-PCR kit ver.2 test kit and corresponding primer, by amplified production purifying, be cloned into pMD-20T carrier, with pcr amplification and order-checking qualification positive plasmid.And by positive plasmid called after PMD3D, PMDP and PMDHP respectively.
2.4 chip preparations
With sampling liquid, oligonucleotide detection probes and position probe are diluted to certain concentration, get 8 μ L and add 384 orifice plates by chip design array, chip point sample instrument adopts solid point needle that probe points is formed on aldehyde radical slide, every bar probe points 4 point, various kinds dot center distance is 1100 μm, during point sample, humidity remains on 70%, puts the chip dry overnight at room temperature made.Hydration 1h in chip 42 DEG C of wet boxes, 42 DEG C of dry 20min.Chip is placed in and hatches 10min containing 0.5%NaBH40.1mol/L phosphate buffered saline buffer, to close the aldehyde radical that is not combined with probe, with 0.2%SDS solution washing 3 times, distilled water wash 2 times, the centrifugal 3min of each washing 2min, 800r/min dries, and is saved backup by the chip 4 DEG C handled well.
Chip design is the microarray (see Fig. 1) of 8 × 8, and comprising 6 part: DW is position probe; " blank " is not point sample contrast; " sampling liquid " is sampling liquid contrast; NC is negative control probe; PC1 is positive control probe; Other site is detection probes.
2.4.1 the optimization of position probe point sample concentration
By position probe DW 2 × doubling dilution, again with Baio2 × spotting buffer balanced mix, DW probe final concentration is made to be respectively 50 μMs, 25 μMs, 12.5 μMs, 6.25 μMs, 3.125 μMs and 1.5625 μMs, according to the layout shown in Fig. 2, point sample, in aldehyde radical slide, prepares chip.
2.4.2 positive control probe hybridization kinetics is studied
By positive control probe PC1 doubling dilution, again with Baio2 × spotting buffer balanced mix, PC1 probe dilution is become 50 μMs, 25 μMs, 12.5 μMs and 6.25 μMs, according to the layout shown in Fig. 2, point sample is in aldehyde radical slide, preparation respective chip, makes 2 × doubling dilution with by corresponding positive mark's thing (PC2) of 20 μMs, gets 4 μ L, after adding 10 μ LDEPC water and the mixing of 14 μ L hybridization solutions, hybridize 3h with chip at 44 DEG C, after washing, detect and analyze hybridization signal.
2.4.3 detection probe concentrations screening
For FMDV detection probes, by TF1, TF2 and TF3 doubling dilution, then with Baio2 × spotting buffer balanced mix, FMDV probe is finally diluted to 50 μMs, 25 μMs, 12.5 μMs, 6.25 μMs, according to the layout shown in Fig. 3, point sample, in aldehyde radical slide, prepares respective chip.Take PMD3D as template (8.34 × 10
6copies/ μ L), adopt asymmetric RT-PCR amplification label, mark sample and chip 44 DEG C hybridize 3h, detect and analyze hybridization signal.
2.5 object fragment labels
2.5.1 the determination of asymmetric PCR primer optimal proportions
For FMDV, asymmetric RT-PCR technology is adopted to mark goal gene fragment.(consider with the One Step PrimeScriptTM RT-PCR Kit that TaKaRa produces and finally by single stage method, sample is increased, therefore select based on One step RT-PCR test kit), the ratio of mark downstream primer and upstream primer is optimized.Reaction system is: Buffer III (2x) 12.5 μ L, Enzyme Mix 1 μ L, Cy3 mark downstream primer 1 μ L (10 μMs), upstream primer (0.5 μM) adds 4 μ L/2 μ L/1 μ L/0.5 μ L/0.25 μ L respectively, template 2 μ L (8.34X106copies/ μ L), mends DEPC water to 25 μ L.Loop parameter is: 50 DEG C, 30min, 94 DEG C of 2 min, 1 circulation; 94 DEG C of 25s, 58 DEG C of 25s, 72 DEG C of 25s, 40 circulations; 72 DEG C of 5min.Amplified production and chip are hybridized 3h at 44 DEG C, detects and analyze hybridization signal.
2.5.2 multiplex RT-PCR amplification
Extract FMDV (O type), PRRSV (CH1-R strain and JXA1-R strain) nucleic acid, use corresponding substance RT-PCR and multiplex RT-PCR amplification respectively, whether the brightness observing amplified production electrophoretic band has notable difference, judges whether have interference between primer with this.Reaction system is: with One Step PrimeScriptTM RT-PCR Kit amplification kit, Buffer III (2x) 12.5 μ L, Enzyme Mix1 μ L, unlabelled primers F 1 (1 μM) and F2 (10 μMs) 1 μ L, P1 (1 μM) and P2 (10 μMs) 1 μ L, template 2.5 μ L, mend DEPC water to 25 μ L; Substance RT-PCR only adds corresponding primer, all the other components unchanged.Loop parameter is: 50 DEG C, 30min, 94 DEG C of 2 min, 1 circulation; 94 DEG C of 25s, 58 DEG C of 25s, 72 DEG C of 25s, 40 circulations; 72 DEG C extend 5min.After having increased, get 10 μ L products, with 2% agarose gel electrophoresis analysis.
2.5.3 multiple asymmetric RT-PCR marks sample
Extract FMDV (O type), PRRSV (CH1-R strain and JXA1-R strain) nucleic acid, to increase the nucleic acid samples carried with multiple asymmetric RT-PCR, the ratio of mark downstream primer and upstream primer is 10:1, all the other compositions of reaction system are shown in 2.5.1 and 2.5.2, carry out amplification label respectively to single template and hybrid template.
2.6 chip hybridization
The product, 2 μ L PC2 (0.625 μM), 7 μ LDEPC water and 14 μ L Baio hybridization buffer (2 ×) that are marked by 5 μ L Cy3 mix, 95 DEG C, sex change 5min, rapid ice bath 10min, be added drop-wise to chip spotted area, covered, puts in wet box and hybridizes under certain temperature condition.Chip after hybridization uses the washings I (2 × SSC of 42 DEG C of preheatings successively, 0.1%SDS), washings II (0.2 × SSC, 0.1%SDS), washings III (0.2 × SSC) and the centrifugal 3min of distilled water wash 3min, last 800r/min dry.
2.6.1 the selection of hybridization time
Shown in Fig. 1, prepare gene chip.For FMDV detection probes, with asymmetric RT-PCR method mark sample, template is the PMD3D plasmid of 8.34 × 106copies/ μ L, and with chip hybridization under 44 DEG C of conditions, hybridization time is respectively 1h, 2h, 3h, 4h, 5h and 6h.Post-hybridization washing, detects and analyzes hybridization signal.
2.6.2 hybridization temperature optimization
Shown in Fig. 1, prepare gene chip, detection probes regulating YIN and YANG contrast concentration and probe concentration is 25 μMs, and position probe concentration is 6.25 μMs.With asymmetric RT-PCR method mark sample, template is respectively PMD3D, PMDP, PMDHP plasmid.Mark sample is hybridized respectively under 40 DEG C, 44 DEG C, 48 DEG C and 52 DEG C of temperature condition, detects and analyzes hybridization signal.
2.7 signal detection
Use Genpix4100A chip scanner, to excite dual wavelength 532nm and 635nm scanning collection information; PMT is 700, and scanning resolution is 10 μm; Image is preserved with 16 TIF forms and JPG form.
2.8 data analysis
With GenePixPr05.0 software analysis scanning result, sampling point signal is obtained as diameter using 600nm, automated manner is taked to look for a little, obtain the hybridization signal of chip each point, the information such as total signal strength (Total Intensity), strength of signal median (Median), strength of signal mean value (Mean), preserve with * .txt file layout and export data.Through comparative analysis, the ratio (SNRm) of position and background signal mean value in this test employing 4 some signals, and evaluate results of hybridization in conjunction with scan image hybridization spot sharpness.When strength of signal median >=1000, SNRm value >=2, scan image hybridization spot is clear, and three's result is unanimously for hybridization is positive.When DW and PC1 site fluorescent signal is positive, when blank, sampling liquid and NC site hybridization signal are negative, chip detection result is effective.
3, experimental result
The structure of 3.1 object fragment amplifications and recombinant plasmid
By the 3D Gene Partial fragment of RT-PCR amplification A type, O type, Asia1 type FMDV, all amplify the specific fragment of about 107 bp.Clone builds the recombinant plasmid containing O type FMDV 3D gene fragment, and carry out PCR qualification to recombinant plasmid, stripe size conforms to expected results (Fig. 4); The sequencing result of plasmid is also confirmed (Fig. 5), 107bp object fragment successful clone in pMD-20T carrier, by its called after PMD3D.
With C-NA-PRRSV (CH1-R) and H-NA-PRRSV (JXA1-R) RNA for template, all amplify the specific fragment of about about 135bp with RT-PCR.Identify the plasmid of clone with PCR, amplified band size conforms to expection; Also confirm the sequencing result of plasmid, object fragment successful clone, in pMD-20T carrier, is distinguished called after PMDP and PMDHP (Fig. 6, Fig. 7).
3.2 probe point sample concentration optimization results
3.2.1 the optimization of position probe point sample concentration
Chip is prepared according to the layout of Fig. 2, after 14 μ L DEPC water and the mixing of 14 μ L hybridization solutions, be added to chip point sample district, 44 DEG C of hybridization 3h, by Scanning Detction after the washing of chip hybridization program, detected result shows: the position probe fluorescent signal median of each concentration is between 5650 ~ 65535 (saturated), and SNRm value is between 11.42 ~ 119.59.
Experimental result illustrates, in the inventive method, the point sample concentration of position probe can be 1.56 ~ 25 μMs, is preferably 6.25 μMs
3.2.2 positive control probe results of hybridization
Prepare gene chip according to shown in Fig. 2, the results of hybridization of different dilution positive mark's thing (PC2) is shown in Fig. 8, Fig. 9.From results of hybridization, hybridization signal increases along with the increase of marker concentrations (when signal is state of saturation, hybridization point is white, and signal value is 65535).Not linear between the concentration of marker and hybridization signal, when the concentration of marker is lower than 1.25 × 2
-6μM time, the amplitude that hybridization signal increases along with concentration is less; Concentration is 1.25 × 2
-6μM ~ 1.25 × 2
-1time between μM, the amplitude that hybridization signal increases along with concentration is comparatively large, when concentration higher than 1.25 time, hybridization signal is all state of saturation substantially.
Concentration and probe concentration is different, and its hybridization signal intensities also has certain difference, and the factor such as sensitivity, chip manufacturing cost of comprehensive detection, the point sample concentration of probe is decided to be 25 μMs.When the concentration of marker is 1.25 × 2
-9μM time, the median of hybridization signal is 1358, SNRm is 3.03, and macroscopic spot appears in chip scanning figure, can be judged to the positive, therefore its limit of detection is 1.25 × 2
-9μM.In this test, when carrying out the detection of other mark sample, positive control adds 2 μ L, and concentration is 0.625 μM, and its hybridization signal value is just in state of saturation.
Therefore, in the inventive method, positive control probe concentration is 25 μMs, and marker is 0.625 μM.
3.2.3 detection probe concentrations the selection result
Prepare gene chip according to the layout of Fig. 3, by the PMD3D gene fragment marked at 44 DEG C, hybridization 3h, after washing, scanning analysis hybridization signal, result is as shown in table 3 below.
The FMDV detection probe result of table 3 different concns
Results of hybridization shows, concentration and probe concentration is when 25 μMs and 12.5 μMs, there is stronger hybridization signal, different probe best point sample concentration slightly difference, different probe best point sample concentration slightly difference, all can obtain good crossbreeding effect generally at 12.5 μMs ~ 25 μMs, optimal concentration is 25 μMs.
3.3 sample mark optimum result
3.3.1 asymmetric RT-PCR primer ratio optimization result
Example is labeled as with FMDV object fragment, in 25 μ L reaction systems, the reverse primer (10 μMs) of Cy3 mark adds 1 μ L, when cold upstream primer (0.5 μM) adds 2 μ L, the strongest with the hybridization signal of chip, namely, when the ratio of mark downstream primer and upstream primer is 10:1, the hybridization signal of marked product and chip is the strongest.
Respectively with recombinant plasmid PMDP, PMDHP for template, when being 10:1 according to mark downstream primer and the ratio of upstream primer, the object fragment of mark and gene chip all have very strong hybridization signal.
Therefore, in the inventive method, the ratio of mark downstream primer and upstream primer is 10:1.
3.3.2 multiplex RT-PCR method primer is evaluated
Substance and multiple RT-PCR increase to the RNA of FMDV (O type), PRRSV (CH1-R strain and JXA1-R strain) respectively, and amplified production electrophoresis result is shown in Figure 10.
Detected result shows, multiple RT-PCR can to O type FMDV, PRRSV (CH1-R), and the RNA of HPRRS (JXA1-R) effectively increases, the brightness of its product and corresponding substance RT-PCR no significant difference.
experimental result illustrates, can not mutually disturb between the present invention's 2 pairs of primers, can effectively increase simultaneously 2 kinds of viral genes.
3.3.3 asymmetric multiplex RT-PCR method mark result
With multiple asymmetric RT-PCR labeled rna sample, marked product results of hybridization is shown in Figure 11.
Multiple asymmetric RT-PCR marks substance template and hybrid template, and the marked product probe hybridization spot corresponding on chip is obvious, and results of hybridization is positive, and often kind of template is all placed in the probe specific combination of correspondence.
experimental result illustrates, chip of the present invention can accurately detect different virus simultaneously.
3.4 chip hybridization parameters are determined
3.4.1 hybridization time is on the impact of hybridization signal
With asymmetric RT-PCR method mark PMD3D (8.34X10
6copies/ μ L), with chip hybridization 1h, 2h, 3h, 4h, 5h, 6h under 44 DEG C of conditions, results of hybridization is in table 4.
Table 4 hybridization time is on the impact of hybridization signal
As can be seen from Table 4, hybridization time all can detect hybridization in 1 ~ 6 hour, and 3 probe hybridization signal medians have the trend with slowly increasing between having during hybridization, but change not obvious.Chip hybridization should have the sufficient time, prevents overlong time again, and hybridization solution is vaporized many, and background value increases, and hybridization time is located 3h by this research.
Experimental result illustrates, in the inventive method, hybridization time can be 1 ~ 6h.Be preferably 3h.
3.4.2 goal gene results of hybridization at different temperatures
Shown in Fig. 1, prepare gene chip, detection probes regulating YIN and YANG contrast concentration and probe concentration is 25 μMs, and position probe concentration is 6.25 μMs.Mark sample by the asymmetric RT-PCR method after optimizing, template is about 10 respectively
6pMD3D, PMDP, PMDHP plasmid of copy/μ L.Mark sample is carried out hybridization 3h respectively at 40 DEG C, 44 DEG C, 48 DEG C, 52 DEG C temperature, detects and analyze hybridization signal.
The FMDV 3D gene fragment of mark and the results of hybridization of gene chip are shown in Figure 12:
3D gene fragment and corresponding detection probes TF1 hybridization signal Median value (median) are between 826 ~ 5975, and SNRm value is between 2.16 ~ 15.15; And TF2 hybridization signal Median value is between 7469 ~ 23305, and SNRm value is between 19.60 ~ 57.12; And TF3 hybridization signal Median value is between 13019 ~ 57739, and SNRm value is between 34.04 ~ 130.04; Other detection probes signal Median value is all below 800, and SNRm value is all less than 2.Yin and yang attribute contrast is set up, and 3D gene marker only has positive hybridization with corresponding TF1, TF2 and TF3 probe, and with other detection probes no cross reactions; Along with temperature raises, hybridization signal weakens, and 40 ~ 48 DEG C time, hybridization signal is obvious, and 40 ~ 44 DEG C time, hybridization signal is comparatively strong, and 44 DEG C time, hybridization signal is the strongest.
PRRSV (CH1-R) the Nsp3 gene fragment of mark and the results of hybridization of gene chip are shown in Figure 13.Goal gene fragment and detection probes TP1 hybridization signal Median value (median) are between 1245 ~ 5926, and SNRm value is between 2.77 ~ 12.54; And between TP2 hybridization signal Median value 3632 ~ 42061, SNRm value is between 8.03 ~ 74.94; And TP3 hybridization signal Median value is between 765 ~ 897, between SNRm value 1.54 ~ 1.80; With other detection probes signal Median all below 1000, SNRm value is all less than 2.Detected result shows, yin and yang attribute contrast is set up, and goal gene fragment label thing only has positive hybridization with corresponding TP1 and T P2 probe, and with other detection probes no cross reactions.Along with temperature raises, hybridization signal first increases and weakens afterwards, and 40 ~ 52 DEG C time, hybridization signal all clearly, and 44 ~ 48 DEG C time, hybridization signal is comparatively strong, and 44 DEG C time, hybridization signal is the strongest.
HPRRSV (JXA1-R) the Nsp3 gene fragment of mark and the results of hybridization of gene chip are shown in Figure 14.Goal gene fragment and detection probes TP3 hybridization signal Median value (median) are between 7007 ~ 12872, and SNRm value is between 12.83 ~ 23.58; The hybridization signal Median value of TP1, TP2 and other detection probes is all below 1000, and SNRm value is all less than 2.Detected result shows, yin and yang attribute contrast is set up, and goal gene fragment label thing only has positive hybridization with TP3 probe, and with other detection probes no cross reactions.Along with temperature raises, hybridization signal first increases and weakens afterwards, and 40 ~ 52 DEG C time, hybridization signal all clearly, and 44 ~ 48 DEG C time, hybridization signal is comparatively strong, and 44 DEG C time, hybridization signal is the strongest.
Therefore, adopt the inventive method to detect, temperature can be 40 ~ 48 DEG C, is preferably 44 DEG C.
Meanwhile, as seen from Figure 12, TF1 ~ TF3 all can detect FMDV gene fragment, illustrates that in TF1, TF2, TF3, any one probe all effectively can detect foot and mouth disease virus.
It can also be seen that according to Figure 13 and Figure 14, probe TP1, TP2 can detect american type pig blue-ear disease classical strains PRRSV (CH1-R), but american type high-pathogenicity porcine reproductive and respiratory syndrome virus HPRRSV (JXA1-R) can not be detected, probe TP3 effectively can detect HPRRSV (JXA1-R), but can not detect PRRSV (CH1-R).Therefore, when detecting american type pig blue-ear disease classical strains if only need, gene chip fixes TP1 or/and TP2 probe, when detecting american type high-pathogenicity porcine reproductive and respiratory syndrome virus if only need, gene chip only need fix TP3, if when need detect multiple american type porcine reproductive and respiratory syndrome virus simultaneously, need fixing TP1 or/and TP2 probe, and TP3 probe.
To sum up, the inventive method adopts two pairs of primers to be increased by multiplex RT-PCR method the gene of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus simultaneously, by increasing, the gene fragment obtained detects with the gene chip being fixed with detection probes of the present invention, can accurately detect in measuring samples whether have foot and mouth disease virus and porcine reproductive and respiratory syndrome virus.
Embodiment 2 specific test
One, test method
With multiple asymmetric RT-PCR to pig circular ring virus (PCV-2), pig parvoviral (PPV), Pseudorabies virus (PRV), Latex agglutination test (JEV), Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), the sick virus of bovine viral diarrhoea/mucous membrane (BVDV) nucleic acid-templated amplification label, the genechip detection that amplified production uses embodiment 1 to prepare respectively, to evaluate its specificity.
The method of multiplex RT-PCR amplification is as follows:
Extract measuring samples nucleic acid (using Beijing Century Yuan Heng company RNA pillar extraction test kit and DNA pillar to extract the nucleic acid that test kit extracts corresponding virus), multiplex RT-PCR amplification, reaction system is: with One Step PrimeScriptTM RT-PCR Kit amplification kit, Buffer III (2x) 12.5 μ L, Enzyme Mix 1 μ L, unlabelled primers F 1 (1 μM) and F2 (10 μMs) 1 μ L, P1 (1 μM) and P2 (10 μMs) 1 μ L, template 2.5 μ L, mend DEPC water to 25 μ L; Substance RT-PCR only adds corresponding primer, all the other components unchanged.Loop parameter is: 50 DEG C, 30min, 94 DEG C of 2 min, 1 circulation; 94 DEG C of 25s, 58 DEG C of 25s, 72 DEG C of 25s, 40 circulations; 72 DEG C extend 5min.After having increased, get 10 μ L products, for chip detection.
Genechip detection:
5 μ L preceding product, 2 μ L PC2 (0.625 μM), 7 μ LDEPC water and 14 μ L Baio hybridization buffer (2 ×) are mixed, 95 DEG C, sex change 5min, rapid ice bath 10min, be added drop-wise to chip spotted area, covered, put in wet box and hybridize, hybridization time is 3h, and hybridization temperature is 44 DEG C.Chip after hybridization uses the washings I (2 × SSC of 42 DEG C of preheatings successively, 0.1%SDS), washings II (0.2 × SSC, 0.1%SDS), washings III (0.2 × SSC) and the centrifugal 3min of distilled water wash 3min, last 800r/min dry.
Use Genpix4100A chip scanner, to excite dual wavelength 532nm and 635nm scanning collection information; PMT is 700, and scanning resolution is 10 μm; Image is preserved with 16 TIF forms and JPG form.
With GenePixPr05.0 software analysis scanning result, sampling point signal is obtained as diameter using 600nm, automated manner is taked to look for a little, obtain the hybridization signal of chip each point, the information such as total signal strength (Total Intensity), strength of signal median (Median), strength of signal mean value (Mean), preserve with * .txt file layout and export data.Through comparative analysis, the ratio (SNRm) of position and background signal mean value in this test employing 4 some signals, and evaluate results of hybridization in conjunction with scan image hybridization spot sharpness.When strength of signal median >=1000, SNRm value >=2, scan image hybridization spot is clear, and three's result is unanimously for hybridization is positive.When DW and PC1 site fluorescent signal is positive, when blank, sampling liquid and NC site hybridization signal are negative, chip detection result is effective.
Two, result
Experimental result as shown in figure 15, all negative in conjunction with the detection method pig circular ring virus (PCV-2) of gene chip, pig parvoviral (PPV), Pseudorabies virus (PRV), Latex agglutination test (JEV), Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), the sick virus of bovine viral diarrhoea/mucous membrane (BVDV) detection of nucleic acids result with multiple asymmetric RT-PCR, show the high specificity of gene chip of the present invention and test kit.
Embodiment 3 sensitivity test
One, test method
According to the method (each detect parameters employing optimum value) of embodiment 1, by recombinant plasmid PMD3D (8.34 × 10
9copies/ μ L), PMDP (3.76 × 10
9copies/ μ L), PMDHP (3.65 × 10
9copies/ μ L) make 10 × doubling dilution respectively, with multiple asymmetric RT-PCR mark, marked product respectively with the chip hybridization of preparation, to evaluate the susceptibility detecting Gene Chip system.
The method of multiplex RT-PCR amplification and gene chip detection method are with embodiment 2.
Two, result
Adopt the inventive method to detect plasmid PMD3D, minimal detectable concentration can reach 8.34 × 10
2copies/ μ L, detected result is shown in Figure 16.
Adopt the inventive method to detect plasmid PMDP, minimal detectable concentration can reach 3.76 × 10
2copies/ μ L, detected result is shown in Figure 17.
Adopt the inventive method to detect plasmid PMDHP, minimal detectable concentration can reach 3.65 × 10
2copies/ μ L, detected result is shown in Figure 18.
When adopting the inventive method to detect, the order of magnitude of the minimal detectable concentration of each viral sample is 10
2copies/ μ L, illustrates that the detection sensitivity of gene chip of the present invention and test kit is high.
Embodiment 4 replica test
One, experimental technique
According to the method (each parameter employing optimal values) of embodiment 1, select the same a collection of FMDV-CSFV-PRRSV oligonucleotide chip of structure, carry out replica test with FMDV 3D marker, evaluate its stability.
The method of multiplex RT-PCR amplification and gene chip detection method are with embodiment 2.
Two, experimental result
With the marked product of the asymmetric RT-PCR of PMD3D, hybridize with batch gene chip 10, results of hybridization is in table 5.
Table 5 detects with batch chip repeatability
TF1 probe hybridization signal Median is 2612.9 ± 101.44, and the variation coefficient is 3.88%, SNR value is 7.00 ± 0.22, and the variation coefficient is 3.88%; TF2 probe hybridization signal Median is 19672.6 ± 1705.30, and the variation coefficient is 8.67%, SNR value is 52.39 ± 3.01, and the variation coefficient is 5.74%; TF3 probe hybridization signal Median is 22694.7 ± 1321.55, and the variation coefficient is 5.82%; SNR value is 60.20 ± 3.19, and the variation coefficient is 5.30%; Negative control probe hybridization signal Median is 551.6 ± 36.11, and the variation coefficient is 6.55%, SNR value is 1.51 ± 0.10, and the variation coefficient is 6.51%.
Detected result shows, genechip detection signal stabilization of the present invention, consistence is good.
Embodiment 5 clinical sample detects
One, experimental technique
By the method for the embodiment of the present invention 1,20 parts of clinical unstable swinery samples are detected.
Two, result
Detect 20 parts of clinical samples by the chip detection technology set up, positive 5 parts of positive 7 parts of PRRSV, HPRRSV, does not detect FMDV.
Experimental result illustrates, gene chip of the present invention and test kit effectively can detect foot and mouth disease virus in sample and breath syndrome virus.
To sum up, gene chip of the present invention and test kit can effectively detect foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, and high specificity, susceptibility are high, consuming time short, detect fast, have a good application prospect.
Claims (10)
1. detect a gene chip for foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, it is characterized in that: it comprises solid phase carrier and is fixed on the probe on solid phase carrier; Described probe to comprise shown in SEQ ID NO:5 ~ 7 shown in any one or any number of gene fragment and SEQ ID NO:8 ~ 10 any one or any number of gene fragment.
2. gene chip according to claim 1, it is characterized in that: it also comprises positive control probe, negative control probe and/or position probe, wherein, positive control probe is the gene fragment shown in SEQ ID NO:12, negative control probe is the gene fragment shown in SEQ ID NO:11, and position probe is the gene fragment shown in SEQ ID NO:14 that marked fluorescence dye.
3. one kind is detected the test kit of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, it is characterized in that: it comprises the reagent of the gene of the gene chip described in claim 1 or 2 and increase foot and mouth disease virus and porcine reproductive and respiratory syndrome virus, wherein, the reagent of amplification foot-and-mouth disease virus gene comprises primer pair shown in SEQ ID NO:1 ~ 2; The reagent of amplification porcine reproductive and respiratory syndrome virus gene comprises primer pair shown in SEQ ID NO:3 ~ 4.
4. test kit according to claim 3, is characterized in that: in described primer pair, and shown in SEQ ID NO:1,3, shown in upstream primer and SEQ ID NO:2,4, the mol ratio of downstream primer is 1:10.
5. test kit according to claim 4, is characterized in that: shown in described SEQ ID NO:2,4, downstream primer is marked with fluorescence dye.
Any one or any number of gene fragment shown in 6.SEQ ID NO:5 ~ 7, preparing the purposes in the gene chip detecting foot and mouth disease virus and porcine reproductive and respiratory syndrome virus with any one or any number of gene fragment SEQ ID the NO:8 ~ 10 Suo Shi.
7. purposes according to claim 6, it is characterized in that: described gene chip also comprises positive control probe, negative control probe and/or position probe, wherein, positive control probe is the gene fragment shown in SEQ ID NO:12, negative control probe is the gene fragment shown in SEQ ID NO:11, and position probe is the gene fragment shown in SEQ ID NO:14 that marked fluorescence dye.
The purposes that primer pair shown in 8.SEQ ID NO:1 ~ 2 and SEQ ID NO:3 ~ 4 increases in the reagent of foot and mouth disease virus and porcine reproductive and respiratory syndrome virus gene in preparation simultaneously.
Primer pair shown in 9.SEQ ID NO:1 ~ 2 and SEQ ID NO:3 ~ 4, is preparing the purposes in the test kit detecting foot and mouth disease virus and porcine reproductive and respiratory syndrome virus with any one or any number of gene fragment shown in any one or any number of gene fragment and SEQ ID NO:8 ~ 10 SEQ ID the NO:5 ~ 7 Suo Shi; Wherein, two pairs of primer pairs are amplifing reagent, and shown in SEQ ID NO:5 ~ 10, gene fragment is detection probes.
10. purposes according to claim 9, is characterized in that: in described primer pair, and shown in SEQ ID NO:1,3, shown in upstream primer and SEQ ID NO:2,4, the mol ratio of downstream primer is 1:10.
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