CN102888467A

CN102888467A - Gene chip for detecting porcine respiratory disease complex virus and method for detecting porcine respiratory disease complex virus

Info

Publication number: CN102888467A
Application number: CN2012103381218A
Authority: CN
Inventors: 杨小燕; 黄翠琴; 戴爱玲; 黄其春; 尹会方
Original assignee: Longyan University
Current assignee: Longyan University
Priority date: 2012-10-20
Filing date: 2012-10-20
Publication date: 2013-01-23

Abstract

The invention relates to a gene chip for a detecting porcine respiratory disease complex virus and a method for detecting the porcine respiratory disease complex virus, wherein the gene chip comprises a solid phase carrier, and an oligonucleotide probe and a quality control oligonucleotide probe which are fixed on the carrier. The oligonucleotide probe comprises DNA sequences selected from an NPS2 gene of porcine reproductive and respiratory syndrome virus, gene among circovirus II type genome sequence 990-1500bp, gE gene and pK gene of pseudorabies, HA, NA and M genes of swine influenza virus H1N1, and HA and NA genes of swine influenza virus H3N2. After a to-be-detected sample genomic DNA is amplified and labeled by utilizing a designed primer, the gene chip is used for hybridization, and the porcine respiratory disease complex virus can be detected according to the hybridization signals. According to the gene chip and the detection technology of the gene chip, four samples and five viruses can be simultaneously detected on the same one chip, and the diagnosis efficiency can be greatly improved.

Description

Detect gene chip and the detection method thereof of porcine respiratory disease syndrome virus

Technical field

The present invention relates to a kind of gene chip and detection method, particularly a kind of gene chip and detection method thereof that detects the porcine respiratory disease syndrome virus.

Background technology

PRDC (porcine respiratory disease complex) (PRDC) is a disease problem that multiple performance is arranged, and has become one of disease the most outstanding in the swine disease.PRDC is the result who produces synergistic effect and additive effect behind multiple virus and bacterium while or the first postoperative infection, often at first caused by virus, and common secondary conditionality pathogenic bacterial infection.Because PRDC (porcine respiratory disease complex) is a kind of multi-factor disease, the cause of disease that separate on different pig farms even same pig farm is all different, diagnosis for this disease, traditional method has the PCR detection method of serology detection, single virus etc., the length consuming time that these technology have, the expense that has is high, and detects when all can not carry out multiple pathogens, therefore be difficult to reach quick, accurate, high-throughout purpose, caused the difficulty of its diagnosis and control.

In July, 1997, Affymetrics, the Thomas of Inc. (Santa Clara, CA) company, the 6th, 228, No. 575 United States Patent (USP)s of the people such as R. invention disclose the method for with biochip technology microorganism being carried out deciding kind and phenotype analytical.Since nineteen nineties, biochip technology is set up as a kind of brand-new foranalysis of nucleic acids detection technique, and the progressively development along with carrying out of the Human Genome Project.Development along with this biotechnology of gene chip, utilize high-throughput, hypersensitivity, repeatability and the narrow spectrum characteristics of chip, can detect simultaneously a plurality of virogenes on same chip, so that it is compared with other diagnostic techniques, diagnosis speed and accuracy significantly improve.In recent years, existing many about the report that utilizes the genechip detection pathogenic micro-organism both at home and abroad, for example, the people such as calendar year 2001 Li utilize chip technology to analyze human influenza virus A and viral B and their hypotype in " clinical microbiology magazine ", and think and compare with the diagnostic method of PCR-based that biochip technology is more effective diagnostic method.The people such as Baxi in 2006 are used for chip technology diagnosis and the evaluation of foot and mouth disease in " animal doctor's magazine ", think that this technology is the unique method that can identify simultaneously and diagnose many cause of diseases.Domestic scholars also successively successfully are used for biochip technology the detection of the various viruses of humans and animals and bacterium aspect etc., the people such as Chen Hongjun had delivered the foundation of swine influenza virus typing gene chip and the research of Preliminary Applications in " journal of animal science and veterinary medicine " in 2007, the people such as Xiao Guosheng have delivered gene chip and have been used for the detection of pathogenic micro-organism and the research of somatotype in " Chinese animal doctor's magazine ", and the people such as Yang Lin are also respectively at " Chinese animal doctor's magazine ", disclosing Pestivirus suis in " Chinese Animal Quarantine " detects with the screening of oligonucleotide chip probe and the pig breeding research with the Establishment and optimization of dyspnoea syndrome virus gene chip detecting technique.Therefore, for very complicated and loaded down with trivial details this difficult problem of the diagnosis of PRDC, the diagnosis that utilizes chip technology to carry out PRDC is a kind of comparatively effective means.Have not yet to see the applying gene chip and carry out the report that the porcine respiratory disease syndrome virus detects.

Summary of the invention

The object of the present invention is to provide a kind of gene chip and detection method thereof that detects the porcine respiratory disease syndrome virus.A kind of multi-factor disease for PRDC (porcine respiratory disease complex), the defective that traditional method such as serology detect, the PCR detection method of single virus etc. all is difficult to accurately, diagnoses timely, the present invention adopts biochip technology, foundation can detect gene chip and the detection method thereof of several different virus infection that cause PRDC (porcine respiratory disease complex) simultaneously, improve this sick diagnosis efficiency, thereby solve its clinically problem of difficult diagnosis.

The technical solution adopted in the present invention is: a kind of gene chip that detects the porcine respiratory disease syndrome virus, comprise solid phase carrier and the oligonucleotide probe and the quality control oligonucleotide probe that are fixed on this solid phase carrier, this above-mentioned oligonucleotide probe comprises one or more sequences of choosing from following sequence:

(1) the NPS2 gene of porcine reproductive and respiratory syndrome virus, gene between PCV-II II type genome sequence 990～1500bp, the gE gene of pseudoabies and pK gene, the HA of swine influenza virus H1N1, NA and M gene, the dna sequence dna of choosing in the HA of swine influenza virus H3N2, the NA gene;

The complementary dna sequence of the dna sequence dna of (2) choosing in described (1);

The complementary RNA sequence of the dna sequence dna of (3) choosing in described (1) or (2).

Described NPS2 gene from porcine reproductive and respiratory syndrome virus, gene between PCV-II II type genome sequence 990～1500bp, the gE gene of pseudoabies and pK gene, the HA of swine influenza virus H1N1, NA and M gene, the dna fragmentation of choosing in the HA of swine influenza virus H3N2, the NA gene have one or more dna sequence dnas in the dna sequence dna shown in SEQ ID NO:1-SEQ ID NO:14.

Described quality control oligonucleotide probe comprises fixedly positive control probe of hybridization positive control probe, hybridization negative control probe, chip.

Described hybridization positive control probe comprises the Y5 gene, has the dna sequence dna shown in the SEQ ID NO:15.

Described hybridization negative control probe comprises the tetK gene, has the dna sequence dna shown in the SEQ ID NO:16.

Described chip fixedly positive control probe has the dna sequence dna shown in the SEQ ID NO:17.

This gene chip is used at least a detection of porcine reproductive and respiratory syndrome virus, PCV-II II type, pseudoabies, swine influenza virus H1N1, swine influenza virus H3N2.

Another technical scheme of the present invention is: a kind of using method that detects the gene chip of porcine respiratory disease syndrome virus is characterized in that may further comprise the steps:

(1) according to the NPS2 gene of porcine reproductive and respiratory syndrome virus, gene between PCV-II II type genome sequence 990～1500bp, the gE gene of pseudoabies and pK gene, the HA of swine influenza virus H1N1, NA and M gene, the dna sequence dna of choosing in the HA of swine influenza virus H3N2, the NA gene design and prepare the primer for pcr amplification;

(2) prepare according to a conventional method genomic dna or the cDNA of testing sample, use in the step (1) primer to treat to survey sample gene group DNA and carry out pcr amplification and purification of target sequence;

(3) target sequence in the markers step (2);

(4) with the target sequence behind the mark and gene chip hybridization claimed in claim 1;

(5) obtain hybridization signal and Analysis and Identification results of hybridization with biochip scanner.

The applied detection primer of above-mentioned steps (1) comprises at least one pair of in the dna sequence dna shown in SEQ ID NO:18-SEQ ID NO:31.

Use interpretation software CapitalBio Pig Virus Detection Analysis System Analysis and Identification results of hybridization in the above-mentioned steps (5).

Above-mentioned interpretation software CapitalBio Pig Virus Detection Analysis System follows these steps to the Analysis and Identification results of hybridization:

(1) probe is described: title, symbol abbreviation and yin and yang attribute threshold value to probe arrange;

(2) the dot matrix mode chart is set;

(3) chip parameter setting;

(4) the data analysis algorithm arranges;

(5) the results list formatting;

(6) sample message setting;

(7) report style setting;

(8) click keys is carried out interpretation of result

The judgment rule of identifying of above-mentioned interpretation software CapitalBio Pig Virus Detection Analysis System is: exist arbitrary positively in the probe of same virus if detect, then there is this virus in explanation; Have arbitrary gray area that is in the probe of same virus if detect, then illustrate doubtful should virus.

The invention has the advantages that: the present invention introduces biochip technology the detection of porcine respiratory disease syndrome virus, high-throughput, hypersensitivity, repeatability and the specificity of effectively having used gene chip to have be good, can be in single test simultaneously thousands of genes of parallel analysis, can on same chip, detect simultaneously the advantage such as a plurality of virogenes, so that it is compared with other diagnostic techniques, diagnosis speed and accuracy significantly improve.For very complicated and loaded down with trivial details this difficult problem of the diagnosis of PRDC (porcine respiratory disease complex), the diagnosis that utilizes chip technology to carry out PRDC is undoubtedly a kind of comparatively effective means.Gene chip and the detection technique thereof of several PRDC (porcine respiratory disease complex) viruses of this detection of setting up, can on a chip, detect simultaneously 4 duplicate samples, 5 kinds of viruses, greatly improved diagnosis efficiency, can solve the investigation and analysis for molecular epidemiology, have Important Academic meaning and potential application foreground.

Description of drawings:

Fig. 1 is the profile synoptic diagram of an embodiment of gene chip of the present invention

Fig. 2 is dot matrix distribution plan on the micro-array chip of finally determining in the one embodiment of the invention

Embodiment:

For above and other purpose of the present invention, feature and advantage can be become apparent, the below is especially exemplified by preferred embodiment, and the cooperation Figure of description, does following detailed description, and these embodiment are in order to illustrate rather than limit in any form the present invention.

Embodiment one: the design of probe and preparation:

1. the sequence of oligonucleotide probe obtains:

Download the NPS2 gene of porcine reproductive and respiratory syndrome virus (PRRSV) from Genbank public data storehouse, gene between PCV-II II type (PCV-2) genome sequence 990～1500bp, the gE gene of pseudoabies (PRV) and pK gene, the HA of swine influenza virus H1N1, NA and M gene, the HA of swine influenza virus H3N2, NA gene order;

2. the probe design of oligonucleotide probe:

Above-mentioned sequence is introduced in Primer Premire 5.0 softwares, extract virulent ORF sequence, carrying out overall situation connection joins, obtain the feature section that certain specifies virus according to the connection figure, at character zone design specific probe, the specificity site is as far as possible in the middle of probe or slightly near probe 3 ' end, setup parameter, the Tm value is as far as possible between 50 ℃ to 70 ℃, discard and contain hairpin structure, primer dimer, the probe of mispairing structure is compared the database of the probe after having designed and NCBI, carries out specific check, abandon the problematic probe of specificity, treat and select the performance of probe to estimate, classification, and adjust suitable Tm value.

3. the probe design of oligonucleotide probe screening:

Utilize hybrid experiment to carry out the probe screening by the method that embodiment four provides, finally obtain for the preparation of required special, the sensitive probe of gene chip of the present invention, as shown in table 1 below:

Table 1 is for detection of the oligonucleotide probe of porcine respiratory disease syndrome virus

4.

5. quality control oligonucleotide probe design:

Adopted following Quality Control probe (table 2) when chip design, these probes have all carried out the BLAST comparison in NCBI, and known organism is without obvious homology.

Table 2 is for detection of the quality control oligonucleotide probe of porcine respiratory disease syndrome virus

The probe numbering	SEQ ID	Effect	Probe sequence
				NO.15	NO：15	The hybridization positive control	CTCATGCCCATGCCGATGC
NO.16	NO：16	The hybridization negative control	GTTGCTTCTGGAATGAGTTTGCT
				NO.17	NO：17	Chip is positive control fixedly	GTCACATGCGATGGATCGAGCTCCTTTATCATCGTTCCCACCTTAATGCA

6. probe is synthetic:

To entrust probe Synesis Company (handsome Bioisystech Co., Ltd) synthetic, for subsequent use after probe sequence 5 ' 15～20 T of end prolongation in table 1 and the table 2 and the amination.

Embodiment two: the design of primer and preparation

1. retrieval:

2. design primer:

Above-mentioned sequence is imported in Primer Premire 5.0 softwares, and setup parameter Tm value is 50 ℃～70 ℃, length 20bp ± 2bp, then working procedure.

3. Primer selection:

Choosing the Tm value from Output rusults is 50 ℃～70 ℃, length 20bp ± 2bp, and comprise probe sequence used in the gene chip at interior primer.Indivedual probe manual regulation, primer suitably strengthens or shortens several bases, makes it include probe, meets Tm value and other parameters.In preferred embodiment of the present invention, chosen the primer that not only comprises probe shown in the above-mentioned table 1 but also be fit to multiplex PCR, as shown in table 3:

Table 3 is for detection of the primer sequence of the pcr amplification of porcine respiratory disease syndrome virus DNA

The primer numbering	SEQ ID	Detect virogene	Primer sequence
				P-1	NO：18	H1	F:GGAACRTGTTACCCAGGRSA
P-2	NO：19	H1	R:TCTTGATCCCTCACTTTGGG
				P-3	NO：20	N1	F:TCGCTTGGTCAGCAAGTGC
P-4	NO：21	N1	R:CAGTCCATCCGTTCGGATCC
				P-5	NO：22	H3	F:TGGACAATAGTDAADCCGGG
P-6	NO：23	H3	R:CCCATTGATTTGRTCGATKG
				P-7	NO：24	N2	F:AGCAAGTGTGCATAGCATGG
P-8	NO：25	N2	R:TTCTGGGTGTGTCTCCAACA
				P-9	NO：26	PRRSV	F:CCAADAGTTCAVCCTCGAAA
P-10	NO：27	PRRSV	R:AGACAAATCYAGRGGCTCGT
				P-11	NO：28	PCV2	F:CTCAGGGACAACGGAGTGAC
P-12	NO：29	PCV2	R:GTTACCGCTGGAGAAGGAAA
				P-13	NO：30	PRV-PK	F:ATCCCCGACGAGATCCTGTA
P-14	NO：31	PRV-PK	R:CGCATGGTCAGGTAGCTGTA

4. primer is synthetic:

Entrust primer Synesis Company (handsome Bioisystech Co., Ltd) synthetic, for subsequent use primer sequence in the table 3.

Embodiment three: gene chip preparation---chip point sample

1. dissolving probe: probe synthetic in above-described embodiment one is dissolved among 50% DMSO, with centrifugal fast behind the vibrator mixing, with the liquid on the tube wall from lower.After at room temperature placing 1 hour, get and survey OD after 1 μ L dilutes with 50% DMSO, measure the nucleic acid concentration of this probe, this probe dilution being become final concentration is 40 μ M again.

2. splice: add 5 μ L, 30～60 μ M oligonucleotide aqueous solution in 384 orifice plates, add 5 μ L, 2 * gene chip sampling liquid, and fully mix with pipettor.

3. point sample: utilize automatic point sample instrument with the probe points in above-mentioned 384 orifice plates to slide, form the microarray that designs.What utilize in the present embodiment is that the brilliant core micro-array chip point sample system that Boao Biological Co., Ltd produces finishes.Concrete operation step is as follows: take the aldehyde radical slide of cleaning as solid phase carrier, be put on the Stage microscope of chip point sample instrument, above-mentioned 384 orifice plates that added probe are put into the sample table place, then start control software, set corresponding parameter, in the point sample district of above-mentioned slide, four dot matrix designs are identical on the slide by arrangement mode point shown in Figure 2 for beginning point sample, sample.The probe that five kinds of viruses are respectively arranged arranges nucleic acid fixedly positive control, the experiment positive and negative control simultaneously.

The situation of arranging of the probe in the point sample district as seen from Figure 2, each probe points is the probe of the probe numbering shown in corresponding table 1 and the table 2.

4. the quality control of chip:

Chip after preparation is finished scans under the sweep parameter of brilliant core micro-array chip scanner at PMT/Power=90/900, in same chip, and same pin mark sample, the spot diameter deviation is less than 20%.Dot matrix is neat, without obviously connecting a phenomenon, proves that namely chip is qualified.

Embodiment four: utilize the above-mentioned gene chip rapid detection porcine respiratory disease syndrome virus for preparing

1. viral nucleic acid extracts: wherein the DNA of PCV II and PRV virus is according to traditional method for extracting.And H1N1, H3N2 and PRRSV are RNA viruses, need carry out first reverse transcription and become cDNA; The RNA that extracts is carried out reverse transcription by the table 4 corresponding material of adding and condition:

Table 4 reverse transcription reaction system

2.PCR amplification:

(1) reference culture PCR:H1N1, H3N2, PRRSV, PCV II and five kinds of standard virus strains of PRV design special primer according to virus sequence, and the cDNA that generates take reverse transcription or DNA carry out pcr amplification, system such as table 5 as template:

Table 5 PCR reaction system

Reacted constituent	Volume
		10× PCR Buffer	2μl
25mM MgCl ₂	2μl
		10mM dNTP mix	1.6μl
Primer-F(5uM)	0.2μl
		Primer-R(5uM)	0.5μl
Template	3 ml
		LA-TaqE(5U/Ul)	0.2 ml
H ₂O	10.5 ml

Above-mentioned reaction tubes is put into the PCR instrument, pcr amplification program such as the table 6 of setting

Table 6 pcr amplification program

Step	Temperature	Time	Cycle number
				1	94℃	10min
2	94℃	30sec
				3	Tm	30sec
4	72℃	30sec	2,3,4 40cycles
				5	72℃	5min

(2) testing sample multiplex PCR:

The DNA that gets testing sample adds mixing in three pipes that multi-PRC reaction liquid is housed, and each testing sample is three pipe PCR.The prescription of PCR reaction mixture is as shown in table 7 below.

Table 7 multi-PRC reaction mixture formula

Reacted constituent	Volume
		10× PCR Buffer	2μl
25mM MgCl ₂	2μl
		10mM dNTP mix	1.6μl
P5, P9, P11, P13 upstream primer (5uM)	Each 0.2 μ l
		P6, P10, P12, P14 downstream primer (5uM)	Each 0.5 μ l
Template	4 ml
		LA-TaqE(5U/Ul)	0.2 ml
H ₂O	12.4 ml

Above-mentioned 3 reaction tubess are put into the PCR instrument, loop parameter such as the table 8 of setting:

Table 8 multi-PRC reaction loop parameter

Step	Temperature	Time	Cycle number
				1	95℃	5min
2	95℃	30sec
				3	50	45sec
4	72℃	1min	2,3,4 35cycles
				5	72℃	5min

Carry out primer to the single mass system PCR of P1 and P2, P3 and P4 and P7 and P8 according to the pcr amplification program of the PCR reaction system shown in the table 5 and table 6 in addition, each testing sample is three pipe PCR.

3.PCR mark:

The PCR product that step 2 is obtained carries out labeled reactant, the system of reaction and condition such as table 9:

Table 9 labeled reactant

4. hybridization

(1) preparing hybrid system (15 μ l): with the marked product that obtains of step 3 by table 10 preparing hybrid system:

Table 10 hybridization system

Composition	The application of sample amount	Final concentration
			20*SSC	2.25μl	3×SSC
10%SDS	0.3μl	0.2%
			Methane amide	3.75μl	25%
50×Denhardt`s	1.5μl	5×
			The hybridization positive control	0.3μl
The PCR product	7μl

Annotate: each dot matrix 15 μ l.Chip totally 4 dot matrix, can hybridize four increments this.

(2) with above four part of 15 μ l hybridization solution with the pipettor mixing after the centrifugal 30s of 3000rpm, 95 ° of C thermally denatures are 3 minutes on the PCR instrument, ice bath quenching 1min.With pipettor four parts of hybridization solutions are injected respectively four apertures on the cover plate.

(3) after four dot matrix on the affirmation hybridization solution covering chip, cover tightly the hybridization lid, put into 42 ° of C thermostat water baths, hybridized 2 hours, sample and probe are fully reacted.

5. chip cleans

After hybridization finishes, use the chip cleaning apparatus to chip clean, drying.

6. chip scanning

Chip behind the cleaning-drying is put into brilliant core scanner to be scanned.

7. interpretation of result:

The said gene chip scanning obtains a pictures, and wherein the brightness of each probe points is representing the results of hybridization of this point, and for the less chip of probe points, detected result can be judged by naked eyes.Yet, make with the naked eye judgement neither objective also unrealistic for the more chip of data point.Therefore, for objective, accurately the structure of detection chip is analyzed, the invention provides a computer interpretation software---CapitalBio Pig Virus Detection Analysis System.This software follows these steps to the Analysis and Identification results of hybridization:

(2) the dot matrix mode chart is set;

(3) chip parameter setting;

(4) the data analysis algorithm arranges;

(5) the results list formatting;

(6) sample message setting;

(7) report style setting;

(8) click keys is carried out interpretation of result;

Wherein this software identifies that judgment rule is: exist arbitrary positively in the probe of same virus if detect, then there is this virus in explanation; Have arbitrary gray area that is in the probe of same virus if detect, then illustrate doubtful should virus.

Description according to technical scheme of the present invention and preferred embodiment thereof; any those skilled in the art; without departing from the spirit and scope of the present invention; can make various possible being equal to and change or replacement, and all these changes or replacement all should belong to the protection domain of claim of the present invention.

Sequence table

＜110〉the yellow emerald green qin of the Sheng Ke of Longyan School institute

＜120〉a kind of gene chip and detection method thereof that detects the porcine respiratory disease syndrome virus

<160> 31

<210> 1

<211> 35

<212> DNA

＜213〉based on the HA gene order design of swine influenza virus H1N1 and the probe sequence of synthetic

<400> 1

TTTTTTTTTT TTTTTGATC[C/T] CTCACTTTGG GTCTT

<210> 2

<211> 61

<212> DNA

<400> 2

CACCCCCCTT CAATGAAACC GGCAATGGCY CCAAAHARR CCTCTDGATT GAATRGAYGG G

<210> 3

<211> 35

<212> DNA

＜213〉based on the NA gene order design of swine influenza virus H1N1 and the probe sequence of synthetic

<400> 3

TTTTTTTTTT TTTTTGGATC CCAAATCATC TCAAA

<210> 4

<211> 60

<212> DNA

<400> 4

GRTTRAAAGA CACCCAHGGT CGATTYGAVC CATGCCAGTT RTCCCTGCAH ACACAYRTGA

<210> 5

<211> 35

<212> DNA

＜213〉based on the HA gene order design of swine influenza virus H3N2 and the probe sequence of synthetic

<400> 5

TTTTTTTTTT TTTTTTTGAT GCCTGAAACC GTACC

<210> 6

<211> 60

<212> DNA

<400> 6

TTGATGCCTG AAACCGTACC AACCRTCYAT CATTCCCTCC CAACCATTTT CTATGAAACC

<210> 7

<211> 35

<212> DNA

＜213〉based on the NA gene order design of swine influenza virus H3N2 and the probe sequence of synthetic

<400> 7

TTTTTTTTTT TTTTTCTGGG TGTGTCTCCA ACAAG

<210> 8

<211> 61

<212> DNA

<400> 8

GTATCAGCTT TTCCTGAAGC ACTCCCATCA GTCATTACTA CTGTACAAG TTCCATTGAT A

<210> 9

<211> 40

<212> DNA

＜213〉based on the NPS2 gene design of porcine reproductive and respiratory syndrome virus and the probe sequence of synthetic

<400> 9

TTTTTTTTTT TTTTTTTTTT CAGTTCTACG CGGTGCAGGA

<210> 10

<211> 59

<212> DNA

<400> 10

CACCTGCTGA AACTTGCGCC GCGGTGCGGA CACAGGGATC GGCTCACTCA AGGGTGTCA

<210> 11

<211> 39

<212> DNA

＜213〉based on the gene design between PCV-II II type genome sequence 990～1500bp and the probe sequence of synthetic

<400> 11

TTTTTTTTTT TTTTTTTTTT CCGCACCTTC GGATATACT

<210> 12

<211> 60

<212> DNA

<400> 12

GGTTTCCAGT ATGTGGTTTC CGGGTCTGCA AAATTAGCAG CCCATTTGCT TTTACCACAC

<210> 13

<211> 40

<212> DNA

＜213〉based on the pK gene design of pseudoabies and the probe sequence of synthetic

<400> 13

TTTTTTTTTT TTTTTTTTTT GTCGTCGCTG ATGTCCGAGT

<210> 14

<211> 60

<212> DNA

＜213〉based on the gE gene design of pseudoabies and the probe sequence of synthetic

<400> 14

TGTGGGTCAT CACGAGCACG TACAGCCCCG ACTCGTCCGC GGTGGCGTTC GTGTGCACCT

<210> 15

<211> 19

<212> DNA

＜213〉based on the design of Y5 gene and the hybridization positive control PC Quality Control probe sequence of synthetic

<400> 15

CTCATGCCCA TGCCGATGC

<210> 16

<211> 23

<212> DNA

＜213〉based on the design of tetK gene and the hybridization negative control NC Quality Control probe sequence of synthetic

<400> 16

GTTGCTTCTG GAATGAGTTT GCT

<210> 17

<211> 50

<212> DNA

＜213〉the fixing positive control Quality Control probe sequence of chip

<400> 17

GTCACATGCG ATGGATCGAG CTCCTTTATC ATCGTTCCCA CCTTAATGCA

<210> 18

<211> 20

<212> DNA

＜213〉based on the HA gene order design of swine influenza virus H1N1 and the upstream primer sequence of synthetic

<400> 18

GGAACRTGTT ACCCAGGRSA

<210> 19

<211> 20

<212> DNA

＜213〉based on the HA gene order design of swine influenza virus H1N1 and the downstream primer sequence of synthetic

<400> 19

TCTTGATCCC TCACTTTGGG

<210> 20

<211> 19

<212> DNA

＜213〉based on the NA gene order design of swine influenza virus H1N1 and the upstream primer sequence of synthetic

<400> 20

TCGCTTGGTC AGCAAGTGC

<210> 21

<211> 20

<212> DNA

＜213〉based on the NA gene order design of swine influenza virus H1N1 and the downstream primer sequence of synthetic

<400> 21

CAGTCCATCC GTTCGGATCC

<210> 22

<211> 20

<212> DNA

＜213〉based on the HA gene order design of swine influenza virus H3N2 and the upstream primer sequence of synthetic

<400> 22

TGGACAATAG TDAADCCGGG

<210> 23

<211> 20

<212> DNA

＜213〉based on the HA gene order design of swine influenza virus H3N2 and the downstream primer sequence of synthetic

<400> 23

CCCATTGATT TGRTCGATKG

<210> 24

<211> 20

<212> DNA

＜213〉based on the NA gene order design of swine influenza virus H3N2 and the upstream primer sequence of synthetic

<400> 24

AGCAAGTGTG CATAGCATGG

<210> 25

<211> 20

<212> DNA

＜213〉based on the NA gene order design of swine influenza virus H3N2 and the downstream primer sequence of synthetic

<400> 25

TTCTGGGTGTGTCTCCAACA

<210> 26

<211> 20

<212> DNA

＜213〉based on the NPS2 gene design of porcine reproductive and respiratory syndrome virus and the upstream primer sequence of synthetic

<400> 26

CCAADAGTTC AVCCTCGAAA

<210> 27

<211> 20

<212> DNA

＜213〉based on the NPS2 gene design of porcine reproductive and respiratory syndrome virus and the downstream primer sequence of synthetic

<400> 27

AGACAAATCY AGRGGCTCGT

<210> 28

<211> 20

<212> DNA

＜213〉based on the gene design between PCV-II II type genome sequence 990～1500bp and the upstream primer sequence of synthetic

<400> 28

CTCAGGGACA ACGGAGTGAC

<210> 29

<211> 20

<212> DNA

＜213〉based on the gene design between PCV-II II type genome sequence 990～1500bp and the downstream primer sequence of synthetic

<400> 29

GTTACCGCTG GAGAAGGAAA

<210> 30

<211> 20

<212> DNA

＜213〉based on the pK gene design of pseudoabies and the upstream primer sequence of synthetic

<400> 30

ATCCCCGACG AGATCCTGTA

<210> 31

<211> 20

<212> DNA

＜213〉based on the pK gene design of pseudoabies and the downstream primer sequence of synthetic

<400> 31

CGCATGGTCA GGTAGCTGTA

Sequence table

<160> 31

<210> 1

<211> 35

<212> DNA

<400> 1

TTTTTTTTTT TTTTTGATC[C/T] CTCACTTTGG GTCTT

<210> 2

<211> 61

<212> DNA

<400> 2

CACCCCCCTT CAATGAAACC GGCAATGGCY CCAAAHARR CCTCTDGATT GAATRGAYGG G

<210> 3

<211> 35

<212> DNA

<400> 3

TTTTTTTTTT TTTTTGGATC CCAAATCATC TCAAA

<210> 4

<211> 60

<212> DNA

<400> 4

GRTTRAAAGA CACCCAHGGT CGATTYGAVC CATGCCAGTT RTCCCTGCAH ACACAYRTGA

<210> 5

<211> 35

<212> DNA

<400> 5

TTTTTTTTTT TTTTTTTGAT GCCTGAAACC GTACC

<210> 6

<211> 60

<212> DNA

<400> 6

TTGATGCCTG AAACCGTACC AACCRTCYAT CATTCCCTCC CAACCATTTT CTATGAAACC

<210> 7

<211> 35

<212> DNA

<400> 7

TTTTTTTTTT TTTTTCTGGG TGTGTCTCCA ACAAG

<210> 8

<211> 61

<212> DNA

<400> 8

GTATCAGCTT TTCCTGAAGC ACTCCCATCA GTCATTACTA CTGTACAAG TTCCATTGAT A

<210> 9

<211> 40

<212> DNA

<400> 9

TTTTTTTTTT TTTTTTTTTT CAGTTCTACG CGGTGCAGGA

<210> 10

<211> 59

<212> DNA

<400> 10

CACCTGCTGA AACTTGCGCC GCGGTGCGGA CACAGGGATC GGCTCACTCA AGGGTGTCA

<210> 11

<211> 39

<212> DNA

<400> 11

TTTTTTTTTT TTTTTTTTTT CCGCACCTTC GGATATACT

<210> 12

<211> 60

<212> DNA

<400> 12

GGTTTCCAGT ATGTGGTTTC CGGGTCTGCA AAATTAGCAG CCCATTTGCT TTTACCACAC

<210> 13

<211> 40

<212> DNA

<400> 13

TTTTTTTTTT TTTTTTTTTT GTCGTCGCTG ATGTCCGAGT

<210> 14

<211> 60

<212> DNA

<400> 14

TGTGGGTCAT CACGAGCACG TACAGCCCCG ACTCGTCCGC GGTGGCGTTC GTGTGCACCT

<210> 15

<211> 19

<212> DNA

<400> 15

CTCATGCCCA TGCCGATGC

<210> 16

<211> 23

<212> DNA

<400> 16

GTTGCTTCTG GAATGAGTTT GCT

<210> 17

<211> 50

<212> DNA

＜213〉the fixing positive control Quality Control probe sequence of chip

<400> 17

GTCACATGCG ATGGATCGAG CTCCTTTATC ATCGTTCCCA CCTTAATGCA

<210> 18

<211> 20

<212> DNA

<400> 18

GGAACRTGTT ACCCAGGRSA

<210> 19

<211> 20

<212> DNA

<400> 19

TCTTGATCCC TCACTTTGGG

<210> 20

<211> 19

<212> DNA

<400> 20

TCGCTTGGTC AGCAAGTGC

<210> 21

<211> 20

<212> DNA

<400> 21

CAGTCCATCC GTTCGGATCC

<210> 22

<211> 20

<212> DNA

<400> 22

TGGACAATAG TDAADCCGGG

<210> 23

<211> 20

<212> DNA

<400> 23

CCCATTGATT TGRTCGATKG

<210> 24

<211> 20

<212> DNA

<400> 24

AGCAAGTGTG CATAGCATGG

<210> 25

<211> 20

<212> DNA

<400> 25

TTCTGGGTGTGTCTCCAACA

<210> 26

<211> 20

<212> DNA

<400> 26

CCAADAGTTC AVCCTCGAAA

<210> 27

<211> 20

<212> DNA

<400> 27

AGACAAATCY AGRGGCTCGT

<210> 28

<211> 20

<212> DNA

<400> 28

CTCAGGGACA ACGGAGTGAC

<210> 29

<211> 20

<212> DNA

<400> 29

GTTACCGCTG GAGAAGGAAA

<210> 30

<211> 20

<212> DNA

<400> 30

ATCCCCGACG AGATCCTGTA

<210> 31

<211> 20

<212> DNA

<400> 31

CGCATGGTCA GGTAGCTGTA

Claims

1. gene chip that detects the porcine respiratory disease syndrome virus, comprise solid phase carrier and the oligonucleotide probe and the quality control oligonucleotide probe that are fixed on this solid phase carrier, it is characterized in that this above-mentioned oligonucleotide probe comprises one or more sequences of choosing from following sequence:

The NPS2 gene of porcine reproductive and respiratory syndrome virus, gene between PCV-II II type genome sequence 990～1500bp, the gE gene of pseudoabies and pK gene, the HA of swine influenza virus H1N1, NA and M gene, the dna sequence dna of choosing in the HA of swine influenza virus H3N2, the NA gene

The complementary dna sequence of the dna sequence dna of choosing in described (1);

The complementary RNA sequence of the dna sequence dna of choosing in described (1) or (2).

2. a kind of gene chip that detects the porcine respiratory disease syndrome virus according to claim 1, it is characterized in that described NPS2 gene from porcine reproductive and respiratory syndrome virus, gene between PCV-II II type genome sequence 990～1500bp, the gE gene of pseudoabies and pK gene, the HA of swine influenza virus H1N1, NA and M gene, the dna fragmentation of choosing in the HA of swine influenza virus H3N2, the NA gene have one or more dna sequence dnas in the dna sequence dna shown in SEQ ID NO:1-SEQ ID NO:14.

3. a kind of gene chip that detects the porcine respiratory disease syndrome virus according to claim 1 is characterized in that described quality control oligonucleotide probe comprises fixedly positive control probe of hybridization positive control probe, hybridization negative control probe, chip.

4. a kind of gene chip that detects the porcine respiratory disease syndrome virus according to claim 3 is characterized in that described hybridization positive control probe comprises the Y5 gene, has the dna sequence dna shown in the SEQ ID NO:15.

5. a kind of gene chip that detects the porcine respiratory disease syndrome virus according to claim 3 is characterized in that described hybridization negative control probe comprises the tetK gene, has the dna sequence dna shown in the SEQ ID NO:16.

6. a kind of gene chip that detects the porcine respiratory disease syndrome virus according to claim 3, it is characterized in that described chip fixedly the positive control probe have the dna sequence dna shown in the SEQ ID NO:17.

7. using method that detects the gene chip of porcine respiratory disease syndrome virus is characterized in that may further comprise the steps:

The genomic dna or the cDNA that prepare according to a conventional method testing sample use the middle primer of step (1) to treat survey sample gene group DNA and carry out pcr amplification and purification of target sequence;

Target sequence in the markers step (2);

With the target sequence behind the mark and gene chip hybridization claimed in claim 1;

Obtain hybridization signal and Analysis and Identification results of hybridization with biochip scanner.

8. a kind of using method that detects the gene chip of porcine respiratory disease syndrome virus according to claim 8 is characterized in that applied detection primer comprises at least one pair of in the dna sequence dna shown in SEQ ID NO:18-SEQ ID NO:31.

9. a kind of using method that detects the gene chip of porcine respiratory disease syndrome virus according to claim 8 is characterized in that using in the above-mentioned steps (5) interpretation software CapitalBio Pig Virus Detection Analysis System Analysis and Identification results of hybridization.

10. a kind of using method that detects the gene chip of porcine respiratory disease syndrome virus according to claim 10 is characterized in that using above-mentioned interpretation software CapitalBio Pig Virus Detection Analysis System to follow these steps to the Analysis and Identification results of hybridization:

Probe is described: title, symbol abbreviation and yin and yang attribute threshold value to probe arrange;

The dot matrix mode chart is set;

Chip parameter arranges;

The data analysis algorithm arranges;

The results list formatting;

Sample message arranges;

Report style arranges;

Click keys is carried out interpretation of result.