CN101020930A - Gene chip for detection and typing of bird flu virus - Google Patents

Abstract

The present invention relates to gene chip for the detection and subtyping of bird flu virus. The present invention establishes gene chip with high specificity, high sensitivity and great practicability for detection and subtyping of bird flu virus through designing and screening 50 pairs of specific primers, 2 universal primers, 52 specific probes and 3 quality control probes for the multiple RT-PCR subtyping detection of bird flu virus genes based on the gene sequences of different bird flu virus subtypes Genebank reports; and performing the effectiveness test, specificity test, sensitivity test and condition optimization of gene chip detection. The present invention establishes also the detection and subtyping process with the chip and gives the initial application.

Description

The gene chip that is used for avian flu virus detection and somatotype

Technical field:

The invention belongs to the biochip field.Particularly, the present invention relates to a kind of being used for detects gene chip with somatotype to the whole hypotypes of avian influenza virus, prepare type specificity probe, Quality Control probe that this gene chip uses, utilize this gene chip to detect method with somatotype and the multiple RT-PCR primer, multiple asymmetric RT-PCR primer and the universal primer that use in detection and the somatotype research.

Background technology

(Avian Influenza AI), is that bird that the A type influenza virus by Influenza Virus causes infects and the general name of disease in bird flu.Poultry such as chicken, turkey, duck and quail and wild bird, aquatic bird, seabird etc. all can infect, and the harm that domestic chicken and turkey are caused is the most serious, it may be the worldwide popular major cause of bird flu that migratory bird, pet bird, the fowl that looses carry that virus migrates, and pig etc. also can be used as " mixing tank " and propagate this disease.Avian influenza virus (Avian Influenza virus, AIV) the serotype hypotype is numerous, variability is strong, there are antigenic drift and antigenic shift, different subtype, different isolates virus widely different, at present in various poultry in the whole world and wild bird, be separated to thousands of strain avian influenza virus, and proved poultry or drylot feeding bird after infection, can show as inferior clinical symptom, slight respiratory system disease, egg productivity reduction or acute fatal disease.(highly pathogenic avian influenza HPAI) is defined as the category-A animal epidemic, and lists international biological weapon pact animal class transmissible disease list in, and China also classifies it as I class transmissible disease with high pathogenic avian influenza in International Office of Epizootics (OIE).

The outburst of bird flu is that a difficult problem, especially a HPAI of the global aviculture of puzzlement has constituted serious threat to world's aviculture and human health always.Since reporting for the first time that from nineteen fifty-nine the HPAI of H5 hypotype breaks out, HPAI have 24 times bigger popular, wherein 19 times is to break out in poultry, and all brings heavy economic losses at every turn.Breaking out almost of bird flu involves all over the world.H5N1 avian flu cases between fowl is successively broken out in a plurality of countries, and human world bird flu epidemic situation occurs, and total nearly 200 people in the whole world infect bird flu, and case fatality rate reaches 51.7%.The outburst of bird flu has not only caused enormous economic loss, and has brought serious public health problem.

The bird flu cause of disease is the A type influenza virus of orthomyxovirus section (Orthomyxoviridae), AIV viral nucleic acid type is the sub-thread strand RNA, gene element is 8 fragments, virus particle is spherical in shape, also can be thread, the about 80-120nm of diameter, cyst membrane is arranged, it is hundreds of fine the dashing forward of 12-14nm that there is the length of radial arrangement on the surface, can be divided into two classes, be respectively bar-shaped tripolymer hemagglutinin (HA) and be mushroom tetramer neuraminidase (NA), according to the antigenic difference of its surperficial hemagglutinin (HA), can be divided into 16 hypotypes, i.e. H ₁～H ₁₆, can be divided into 9 hypotypes, i.e. N according to the antigenic difference of neuraminidase (NA) ₁～N ₉There are numerous hypotypes in avian influenza virus, nucleic acid is for multi-segmental makes that antigenicity is drifted about easily, transgenation and constantly variation of rearrangement, can be in animal body phenotype mix and nucleocapsid is transplanted variation and is new pathogenic hypotype, and conventional detection can only be identified the only several hypotypes of China, fubaritic new anomaly that abroad have been found that and contingent, the hidden danger that has omission in causing quarantining, and gene chip can detect ten hundreds of different specific gene sequences, therefore is to carry out the strong tool that viral fast typing detects.U.S. Disease Control and Prevention Center since 2003 just the avian influenza virus somatotype detect the research work of gene chip, the research of this novel detection technique is also being set about in Hong Kong.Somatotype detects biochip technology and provides better global influenza monitoring method with help, and makes the serious strains of influenza viruses of evaluation become easier, and this also makes biochip technology become the development trend of the novel detection technique research of avian influenza virus from now on.Therefore, set up and a kind ofly both can detect 16 HA and 9 NA hypotypes special, fast, can find in time that again the high-throughput of new variation or gene rearrangement virus, quick, responsive reliable method are very necessary and timely, this is to the variation of various subtype avian influenza epidemic situations of effective monitoring and AIV and be that effectively anti-rapidly to make bird flu significant.

At present, the main diagnosis of bird flu and detection method comprise that mainly the separation of etiology, immunology, three aspect: AIV of molecular biology and HA/HI test are classical laboratory diagnostic methods, and reliable results but time are oversize; Agar diffusion (AGP) test becomes one of most widely used serological method in the AI quarantine diagnosis owing to have simple and rapid characteristics, also be one of China's bird flu diagnosis national standard appointed method, but the susceptibility of this method is lower; Neutralization test (NT) is accurate and easy to operate to be usually used in laboratory diagnosis, but needs long time and the more material of cost; Fluorescence antibody (FA) test has quick, easy, easy row, than characteristics such as sensitivities, also be usually used in clinical diagnosis; Immunoenzymatic technique (EIA) is based on enzyme linked immunosorbent assay (ELISA), although its repeatability and stability have much room for improvement, but have characteristics quick, that susceptibility is high (its susceptibility will be higher than AGP test and HI test), be usually used in gross sample and serology and detect quarantine.The research of China aspect these diagnostic techniquess is ripe, has manyly all realized commercialization, and for vital role has been brought into play in the detection diagnosis of China's avian influenza virus, but these methods once can only detect a hypotype.

Development along with Protocols in Molecular Biology, gene amplification in vitro technology PCR/RT-PCR becomes a kind of important medical diagnosis on disease technology, it can increase monomolecular target gene up to a million times in a few hours specifically, detect the existence of a trace even a virus particle (cause of disease molecule) thus, thereby greatly improved detection and analysis ability, had the characteristics of sensitivity, special, quick, accurate and easy handling the cause of disease molecule.Therefore the PCR/RT-PCR technology is being used widely aspect the detection of disease, the differential diagnosis, and becomes the emphasis direction of people's research and development.Report such as Wright is used the RT-PCR method and is distinguished A, B, the bird flu of C type, and this method key is in the design of special primer.RT-PCR reaction somatotype at N1～N9 hypotype is feasible in theory, but yet there are no successfully report.David Suarez etc. with after the fluorescent probe detection technique combines, have released the RRT-PCR technology of real-time fluorescence monitoring with the RT-PCR technology, and susceptibility is higher than common RT-PCR far away.China bird flu H5, H7, the multiple RRT-PCR detection kit of H9 hypotype were succeeded in developing in Shenzhen in 2005, this means that China's avian flu virus detection obtains important breakthrough, this achievement will allow fluorescence RT-PCR once can only detect a bird flu hypotype becomes history, realizes once detecting simultaneously three bird flu hypotypes.

A PCR reaction of conventional PCR/RT-PCR technology can only detect the substance that diagnoses a disease, and when multiple pathogenic agent polyinfection of clinical appearance or a plurality of hypotype of a kind of cause of disease, the PCR/RT-PCR reaction that just must carry out repeatedly could be made a definite diagnosis at last.Multiple PCR technique is a special P CR method, has high efficiency, systematicness, advantages such as economical and convenient only need be reacted by a PCR the discriminating detection of polyinfection pathogenic agent, just can differentiate a plurality of hypotypes of multiple pathogenic agent or a kind of cause of disease simultaneously, this is the biological reagent of conserve expensive not only, and has simplified schedule of operation, has accelerated detection diagnosis speed, have very high clinical value, a lot of scholars have carried out big quantity research.

Biochip technology is the cutting edge technology that the height that melts biology, physics, chemistry, computer science and microtronics one that grows up the nineties in 20th century intersects.Biochip technology not only has fast, the advantage of responsive, special, high-throughput and automatization, and carry out data processing and result by computer software and judge, reduce the result and judged the possibility that influenced by subjective factor, improved the reliability and stability of detected result, its original advantage is once a plurality of hypotypes of a plurality of viruses of differential diagnosis or a virus simultaneously, therefore, biochip technology has very wide application prospect in rapid differential diagnosis and context of detection.Some countries and regions such as the U.S., Hong Kong are set about research already and are detected the avian influenza virus gene chip.This technology is a kind of comparison system, perfect detection technique, the foundation of this method not only helps the effective monitoring of bird flu epidemic situation, and can be in the very first time find the new subtype virus of new variation or gene rearrangement accurately, break out the reliable technique deposit is provided for tackling new epidemic situation, the avian influenza genes chip technology will become the desirable technique of present bird flu prevention and control.

The present invention is according to the genome sequence of the different subtypes of poultry influenza virus of having delivered among the Genebank, utilize DNAStar, Bioedit, Primer5.0 and OMIGA software to carry out Analysis and Screening, and 25 pairs of Auele Specific Primers, the 1 pair of universal primer that the multiple RT-PCR somatotype that carries out the research of avian influenza virus biochip technology detects and 52 specific probes and 3 the Quality Control probes that carry out avian influenza virus chip typing technology have successfully been designed, by optimizing reaction conditions, high specificity, avian influenza virus chip typing detection method that susceptibility is high have been set up.

Summary of the invention:

The object of the invention is to provide avian flu virus detection and whole gene chips of using of subtype typings.

The 52 stripe shape specific probes that provide the avian influenza virus somatotype to use are provided another purpose of the present invention, and 3 Quality Control probes using of avian influenza virus typing gene chip.

Purpose of the present invention is achieved through the following technical solutions: the genome sequence of collecting the different subtype avian influenza viruses of delivering among the Genebank, respectively the complete genome sequence of all models AIV that delivered is analyzed with DNAStar/Bioedit software, select the conservative gene fragment as the purpose amplified fragments, utilize Primer5.0 and OMIGA software to carry out design of primers, the hypotype that can obtain standard strain is increased (in the P3 laboratory to directly carrying out RT-PCR with designed type specificity primer, according to the operation of CDC required standard), to obtaining the hypotype of standard strain, it is right that the primer of bridging PCR requirement is satisfied in design, obtains the corresponding target fragment by the bridging pcr amplification.Be connected on the T carrier in JM109 clonal expansion after the amplified production purifying reclaimed and preserve bacterial classification (for future use), be that template is divided into four groups with 25 pairs of primers and carries out multiplex PCR with the positive plasmid of preserving again, according to the nucleic acid electrophoresis result primer is screened or redesigns, up to amplifying ideal purpose band, and, be that template is carried out multiple RT-PCR to four groups of multi-primerses (in the P3 laboratory at last with the viral sample by adjusting the magnesium ion concentration in the reaction system and annealing temperature being optimized, according to CDC required standard operation) verify and do the test of susceptibility specificity.On the basis that multiple RT-PCR is set up, design 1 pair of universal primer and 25 pairs of Auele Specific Primers are modified and set up multiple asymmetric RT-PCR.Carry out multiple asymmetric PCR/RT-PCR checking with preserving bacterium liquid sample and viral sample at last, concrete process of the test is referring to embodiment 1-11.

An aspect, the invention provides a kind of gene chip that is used for avian flu virus detection and somatotype, this chip comprises: solid phase carrier and the Quality Control probe and the avian influenza virus type specificity probe that are fixed on this solid phase carrier, 5 ' end of described probe adds active group and modifies, described active group comprises amino or sulfydryl etc., there is suitable connecting arm at interval between active group and the probe, these connecting arms at interval can increase the distance between probe and the solid phase carrier (for example slide), help the formation of the crossbred of probe and detected DNA.

In a preferred embodiment of the invention, described probe 5 ' end adds NH ₂Modify NH ₂And the connecting arm between the oligonucleotide probe is T15, i.e. 15 successive thymidines.

In a preferred embodiment, shown in SEQ ID NO:1-55, its pairing separately type is as shown in table 4 respectively for the nucleotide sequence of described Quality Control probe and avian influenza virus type specificity probe.Wherein 5 ' end of the complementary probe fragment (PBA-08001-ctr1) of 3 ' of point sample Quality Control probe (PBA-08003-ctr3) end and hybridization Quality Control probe (PBA-08002-ctr2) is with fluorophor (for example, TAMRA) mark.

Solid phase carrier of the present invention can be selected from: nitrocellulose filter, nylon membrane, polystyrene and slide glass.In a specific embodiments, described solid phase carrier is a slide.Carry out activation treatment with ordinary method, make it the surface and have aldehyde radical or amino, thus can and oligonucleotide probe between form covalent linkage.

The preparation of gene chip of the present invention comprises: synthesizing of oligonucleotide probe is prepared from probe then with this area ordinary method point sample on solid phase carrier.

On the other hand, the invention provides 3 Quality Control probes and 52 specific probes that the avian influenza virus somatotype is used, shown in SEQ ID NO:1-55, its pairing separately type is as shown in table 4 respectively for its nucleotide sequence.Wherein 5 ' end of the complementary probe fragment (PBA-08001-ctr1) of 3 ' of point sample Quality Control probe (PBA-08003-ctr3) end and hybridization Quality Control probe (PBA-08002-ctr2) is with fluorophor (for example, TAMRA) mark.

Specificity and sensitivity test result prove: probe provided by the invention and the chip that uses described probe to prepare all have good type specificity and susceptibility.When utilizing the method for the invention to treat this enforcement of sample detection, can directly use gene chip of the present invention and primer, detect also result of determination according to reaction system of establishing and condition.

Therefore, on the other hand, the invention provides and use gene chip of the present invention that avian influenza virus is detected method with somatotype, in brief, this method comprises the following steps:

1) extraction of viral RNA: collected specimens, ordinary method is extracted RNA;

2) RT-PCR amplification: with the RNA that extracts in the step 1) is that template is carried out the RT-PCR amplification

3) hybridization of gene chip and washing: add step 2) amplified production and gene chip of the present invention, be suitable for reacting time enough under the condition of hybridizing with described gene chip, the washing back dries then;

4) scanning of gene chip: chip inserted carry out scanning analysis, saving result in the chip scanner;

5) chip scanning result's interpretation:, search and pairing avian influenza virus subtype when determining this micromatrix arrangement position point sample according to the micromatrix arrangement position that positive findings occurs.

Preferably, in the step 2 of described method) in the multiple asymmetric RT-PCR primer that provides in the use table 3 to carrying out multiple asymmetric RT-PCR amplification.

In a preferred embodiment of the invention, provide and used method of carrying out avian flu virus detection and somatotype of the present invention, this method comprises the following steps:

(1) extraction of collected specimens and nucleic acid: extract RNA according to a conventional method.

(2) multiple asymmetric RT-PCR: the RNA with extraction is that template is carried out multiple asymmetric RT-PCR.Reaction system: in the 0.2mL reaction tubes, add 10 μ L QIAGENE onestep RT-PCR buffer (5 *) successively, 10mM dNTP 2.0 μ L, Enzyme mix 2.0 μ L, RNase inhibitor 0.5 μ L, the upstream and downstream universal primer is respectively 0.5 μ L and 5 μ L (10 μ M), the upstream and downstream Auele Specific Primer is respectively 0.5 μ L and 1 μ L (10 μ M), and RNA template 5 μ L mend RNas-free water to 50 μ L.After sample hose put into MJ RESEARCH PTC-200 PCR instrument, following condition is set reacts: 50 ℃ of 30min; 95 ℃ of 15min; 94 ℃ of 20s, 52 ℃ of 1min, 72 ℃ of 90s, totally 20 circulations; 94 ℃ of 20s then, 70 ℃ of 90s, totally 20 circulations; Last 72 ℃ are extended 10min.

(3) cross experiment of gene chip and washing: get multiple asymmetric RT-PCR amplified production 5.2 μ L, add hybridization buffer 6 μ L, Quality Control probe 0.8 μ L mixing, 95 ℃ of sex change 5min, ice bath 3min immediately, the centrifugal 5～10s of 4000rpm puts back to ice bath with centrifuge tube; At hybridizing box (HybriCassettes ^TM, the CapitalBio Corporation) groove in add 200 μ L distilled water in case the evaporation of hybridization system is upwards put into box with chip front side.In chip point sample district, cover cover plate and hybridizing box and sealing with sample thief hybridization solution 7 μ L after the pipettor piping and druming evenly rapidly, level is put in 52 ℃ of water-baths hybridizes 2.5hr.Hybridization finishes, and the hybridizing box level is taken out, and immediately chip is taken out, put into the washings I that is preheated to 45 ℃ (2 * SSC, 0.1%SDS) in, 100rpm washs 5min.Taking out chip puts into 45 ℃ washings I and washes once.Take out chip, (0.2 * SSC, 0.1%SDS), 100rpm washs 5min, washed twice to put into the cleaning solution II that is preheated to 45 ℃.Take out chip, put into washing lotion III (0.2 * SSC), 100rpm room temperature washing 5min.Chip lixiviate in dehydrated alcohol is put into chip cartridges to chip several times then, and the centrifugal 5min of 1000r/min dries.

(4) scanning of gene chip: chip inserted in the PerkinElmer ScanArray Gx plus scanner carry out scanning analysis, saving result.

(5) chip scanning result's interpretation: interpretation is carried out in the micromatrix arrangement according to the avian influenza genes chip.

Description of drawings

Fig. 1 is illustrated to be the multiple RT-PCR agarose nucleic acid electrophoresis result who carries out with designed primer, and wherein M is DL2000 DNAMarker.Figure 1A is first group of result ( swimming lane 1,2,3,4,5,6 and 7 is respectively H1N1, H3N2, H5N1, H6, H7N1, H9N2 and negative control); Figure 1B is second group of result ( swimming lane 1,2,3,4,5,6,7,8 and 9 is respectively H2, H4, H8, H10, H11, H13, H15, H16 and negative control); Fig. 1 C is the 3rd group of result ( swimming lane 1,2,3,4,5,6 is respectively H14, N4, N6, N7, N9 and negative control); Fig. 1 D is the 4th group of result ( swimming lane 1,2,3,4,5 is respectively H12, N3, N5, N8 and negative control).

Fig. 2 is illustrated to be multiple RT-PCR specificity test nucleic acid electrophoresis result, and wherein M is DL 2000 DNAMarker.Fig. 2 A is first group of test-results ( swimming lane 1,2,3,4,5,6,7,8,9 and 10 is respectively newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, reovirus, infectious bursa of Fabricius virus, infective rhinitis, H7N1 and H5N1 mixing RNA, H3N2 and H9N2 mixing RNA, H1N1 RNA, negative control).Fig. 2 B is second group of test-results ( swimming lane 1,2,3,4,5,6,7,8 and 9 is respectively newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, reovirus, infectious bursa of Fabricius virus, infective rhinitis, H8, H15 and negative control); Fig. 2 C is the 3rd group of polyspecific test-results ( swimming lane 1,2,3,4,5,6,7,8 and 9 is respectively newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, reovirus, infectious bursa of Fabricius virus, infective rhinitis, N4, N9 and negative control); Fig. 2 D is the 4th group of test-results ( swimming lane 1,2,3,4,5,6,7,8 and 9 is respectively newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, reovirus, infectious bursa of Fabricius virus, infective rhinitis, N3, N5 and negative control).

Fig. 3 is illustrated to be the multiple asymmetric RT-PCR agarose nucleic acid electrophoresis result who carries out with designed primer, and wherein M is DL 2000 DNA Marker.Fig. 3 A is first group of test-results ( swimming lane 1,2,3,4,5,6 and 7 is respectively H1N1, H3N2, H5N1, H6, H7N1, H9N2 and negative control); Fig. 3 B is second group of test-results ( swimming lane 1,2,3,4,5,6,7,8 is respectively H2, H4, H8, H10, H11, H13, H1 5, H16 and negative control); Fig. 3 C is the 3rd group of test-results ( swimming lane 1,2,3,4,5,6 is respectively H14, N4, N6, N7, N9 and negative control); Fig. 3 D is the 4th group of test-results ( swimming lane 1,2,3,4,5 is respectively H12, N3, N5, N8 and negative control).

Fig. 4 is illustrated to be the probe micromatrix arranged distribution figure of chip surface.

Fig. 5 is illustrated to be the gene chip scanning result that related probe of application and primer carry out gene chip research: the negative contrast of Fig. 5-1; Fig. 5-2,5-3,5-4,5-5,5-6,5-7,5-8,5-9,5-10,5-11,5-12,5-13,5-14,5-15,5-16,5-17,5-18,5-19,5-20,5-21,5-22,5-23,5-24,5-25,5-26,5-27 and 5-28 are respectively H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8, N9, the hybridization scanning result of 8 common hypotype AIV and 25 hypotype AIV.

Advantage of the present invention:

1. reagent of the present invention and detection method take full advantage of the efficient amplification of round pcr, the good specificity of nucleic acid hybridization and the sensitiveness of detection technique of fluorescence, to the different shaped bird flu carry out fast, responsive, detect accurately. Be mainly manifested in the following aspects: the first, can on a chip, can identify accurately and rapidly the various hemagglutinin hypotypes (H1～16) of avian flu strain; The second, can be on same chip identify accurately and rapidly the various neuraminidase hypotypes (N1～9) of avian flu strain simultaneously; The 3rd, can also distinguish the hypotypes such as H3N2, H2N2, H1N1 and H1N2 that infect the people at same chip.

2. utilize the genetic chip that primer of the present invention and probe are set up more can satisfy high-throughout animal quarantine and epidemic monitoring purpose, thereby formulate corresponding effectively epidemic situation control measure.

Below in conjunction with specific embodiment, further set forth the present invention.It will be understood by those skilled in the art that these embodiment only to be used to the present invention is described and never scope of the present invention is constituted any restriction.Unless otherwise indicated, all scientific and technical terminologies among the application all have and the identical implication of one skilled in the art's common sense of the present invention.Arbitrary patent, patent application and the publication quoted among the application are hereby incorporated by.The experimental technique of unreceipted actual conditions in the following example, usually adopt for example people such as Sambrook of normal condition, molecular cloning: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the method for advising according to manufacturer.

Embodiment

Embodiment 1 primer design and synthetic

Genome sequence according to the different subtype avian influenza viruses of delivering among the Genebank, utilize DNAStar/Bioedit software that it is carried out sequential analysis, adopt Primer5.0, OMIGA and 25 pairs of primers of Beacon Designer2.1 software design, give birth to worker's biotechnology company limited by Shanghai and synthesize, primer sequence is as shown in table 1.

The primer of table 1. multiplex PCR/RT-PCR

Type	The primer numbering	Sequence (5 '～3 ')	The length of institute's amplified fragments
				H1	H1-F H1-R	GGAGCAATTGAGTTCAGTATC GACACTCTCCTATTGTGACTG	601bp
H3	H3-F H3-R	TGTTACCCTTATGATGTGCC CCCTGTTGCCAATTTCAGAG	669bp
				H5	H5-F H5-R	AGTGAATTGGAATATGGTAACTG AACTGAGTGTTCATTTTGTCAAT	380bp
H6	H6-F H6-R	AAGGCACTTATTGGRTCAGG GTCCTCTAGTTTCAATCTGTGG	685bp
				H7	H7-F H7-R	TCAGGWTCTTCWTTCTATGC TCYCCTTGTGCATTTTGATG	641bp
H9	H9-F H9-R	AAGAGAATGGTCCTACATCGT GGATCTTACTCGCAATGTCTG	493bp
				N1	N1-F N1-R	TCCCACTTGGAATGCAGAAC CACATGCACATTCAGACTCTTG	328bp
N2	N2-F	ATAGCATGGTCCAGCTCAAG	299bp

	N2-R	ACATGCTGAGCACTTCCTG
				H2	H2-F H2-R	CGTCATTCTTCAGGAACATGG GGCCTTGTTGCTATTTCWGG	229bp
H4	H4-F H4-R	TTGTTAYCCATTTGATGTGCC GTRACTCTTCCAGGGTTGTT	324bp
				H8	H8-F H8-R	AAGGTTGGTCATACATAGTGG GTCCTCTTACTAATGGTCTGG	444bp
H10	H10-F H10-R	CAATTCTATTGCCTACTGTTATCC TTACTYACTCTACTAGGTGCTAT	435bp
				H11	H11-F H11-R	ACTTAGAAATGTCCCAGCAA CATTTCCCTCGTCTTTGGC	437bp
H12	H12-F H12-R	AGTACAAGAACACCAGAGATT CTGGCCATCCGCCTTCTAT	537bp
				H13	H13-F H13-R	GACCCTTCTGCTCCTCATG GAAACTGATTGATTCCCCTGG	474bp
H14	H14-F H14-R	TCTCCCGACTAAACTGGCTA CTGCCGCTCTGATTCCTTAC	247bp
				H15	H15-F H15-R	GACTCCTTGACTGAGATCTGG AGTATCACATCTTTGTACCCAC	305bp
H16	H16-F H16-R	TAAACTTCTCGTGCTAATCG GTCTTCAACTTGATCCCTTC	252bp
				N3	N3-F N3-R	GGGAAAGARTGGATGCATGT GTTGTTGATTCTCATCCAAGG	366bp
N4	N4-F N4-R	GGAAGCAATCGACCATGGAT CGACACCCATCCATTAGCAT	260bp
				N5	N5-F N5-R	ACTGTTATTGGGTAATGACG TGAAAGCTCCGCTGTATCCT	459bp
N6	N6-F N6-R	TCTGCATGTCAGGACCTAAT CACTCTTCTATATGCTGTGC	246bp
				N7	N7-F N7-R	TGTGCAGAGATAAYTGGCA CCGGAATAGCCTGACCAATT	352bp
N8	N8-F N8-R	GGGCAMTGATGTATGGATGG AAGAATAGCTCCATCGTGCC	340bp
				N9	N9-F N9-R	TTCTATGCTCTCAGCCAAGG TGGCATACGCATTCAGATTC	310bp

Annotate: Y=(C, T), W=(A, T).Note：Y＝(C，T)，W＝(A，T)。

Embodiment 2 can obtain the RT-PCR amplification of the avian influenza virus subtype of standard strain

In the present embodiment,,, directly carry out conventional RT-PCR amplification as template then by extracting RNA from standard strain for 8 avian influenza virus subtypes (H1, H3, H5, H6, H7, H9, N1 and N2) that can obtain standard strain.

2.1 the extraction of template ribonucleic acid

By the method that QIAamp Viral RNA Mini Kit (available from QIAGEN company) specification sheets provides, from the avian influenza virus type strain, extract the avian influenza virus geneome RNA.

2.2RT-PCR reaction system

With reference to single stage method RT-PCR test kit (available from QIAGEN company) operation instructions, revise a little and carry out.Reaction system: in the 0.2mL reaction tubes, add QIAGENE onestep RT-PCR buffer (5 *) 10 μ L successively, 10mM dNTP 2.0 μ L, Enzyme mix 2.0 μ L, RNase inhibitot 0.5 μ L, each 0.5 μ L (10 μ M) of upstream and downstream primer, RNA template 5 μ L mend RNas-free water to 50 μ L.Wherein employed upstream and downstream primer be selected from the primer of H1, H3, H5, H6, H7, H9, N1 and one of them hypotype of N2 right.Reaction conditions is: 50 ℃ of 30min, and 95 ℃ of 15min, 94 ℃ of 30s then, 52 ℃ of 40s, 72 ℃ of 1min, totally 40 circulations, last 72 ℃ are extended 10min.After finishing, reaction gets 5 μ L products with 1.5% agarose gel electrophoresis-ethidium bromide staining analysis.

Electrophoresis result shows that amplification has obtained the target DNA fragment (having deleted 300bp) of expection size.To reclaiming product evaluations of checking order, order-checking is identified and is shown designed primer specific sensitivity, then above-mentioned amplified production is connected in the preservation of T carrier cloning for future use.

Embodiment 3 can't obtain the synthetic of the avian influenza virus gene hypotype corresponding gene fragment of standard strain

In the present embodiment, for all the other 17 hypotypes (comprising H2, H4, H8, H10, H11, H12, H13, H14, H15, H16, N3, N4, N5, N6, N7, N8 and N9) that can not obtain standard strain, then by the synthetic corresponding target dna fragmentation that obtains of bridging pcr amplification.

Above-mentioned all 17 genotypic target DNA fragments have been synthesized in the present embodiment, at this only is that example is recorded and narrated building-up process in detail with H15, the building-up process of all the other 16 genotype target DNA fragments is removed and is selected for use corresponding different primer external respectively, and other steps are all identical.

3.1 design of primers

Utilize DNAStar and Bioedit software respectively to the complete genome sequence analysis of all H15 type AIV of having delivered, adopt Primer5.0 and five upstream primer H15-F of OMIGA software design, H15 one F1, H15-F2, H15-F3, H15-F4, five downstream primer H15-R, H15-R1, H15-R2, H15-R3, H15-R4.The synthetic principle of H15-F and H15-R primer routinely designs and synthesizes.Other primer design and the synthetic requirement that then need satisfy bridging PCR, its length is 59bp, the complementary base of average 20bp is wherein respectively arranged between H15-F1/H15-R1, H15-R1/H15-F2, H15-F2/H15-R2, H15-R2/H15-F3, H15-F3/H15-R3, H15-R3/H15-F4, the H15-F4/H15-R4, and primer sequence sees Table 4.

Table 2. bridging PCR synthesizes H15 hypotype purpose fragment the primer

Numbering	Sequence (5 '～3 ')
		H15-F	GACTCCTTGACTGAGATCTGG
H15-R	AGTATCACATCTTTGTACCCAC
		H15-F1	ATAAACTGGACAAGGGACTCCTTGACTGAGATCTGGTCATACAATGCCGAACTGCTAGT
H15-R1	GAATCTGCAAGGTCAATTGTATGCTGATTCTCCATTGCTACTAGCAGTTCGGCATTGTA
		H15-F2	ACAATTGACCTTGCAGATTCTGAAATGAACAAACTCTATGAGAGAGTGAGAAGACAGCT
H15-R2	TCAAAACATCCAGTTCCATCCTCCTCGGCATTCTCCCTTAGCTGTCTTCTCACTCTCTC
		H15-F3	GATGGAACTGGATGTTTTGAGATTTTCCACCGATGTGACGATCAATGTATGGAGAGCAT
H15-R3	TCCTGTCGATATTCAGTGTGATTGTAAGTATTATTCCGTATGCTCTCCATACATTGATC
		H15-F4	CACACTGAATATCGACAGGAAGCCTTACAAAATAGGATAATGATCAATCCGGTAAAGCT
H15-R4	AAGCTAAACCATAGTATCACATCTTTGTACCCACTACTAAGCTTTACCGGATTGATCAT

3.2 dna fragmentation extends

At first carry out the DNA chain extension reaction, obtain four short-movie sections by H15-F1/H15-R1, H15-F2/H15-R2, four pairs of primers of H15-F3-H15-R3, H15-F4/H15-R4; Preceding two short-movie sections are carried out DNA chain extension, latter two short segment DNA chain extension, can obtain two moderate-length fragments; Then these two moderate-length fragments are carried out the DNA chain extension, obtain expecting that the dna fragmentation of size is desired goal gene.

3.2.1 the reaction system of short segment DNA chain extension and condition: 10 * buffer 5 μ L, dNTP 4 μ L, each 1 μ L of H15-F1/H15-R1 (H15-F2/H15-R2, H15-R2/H15-F3, H15-F3/H15-R3, H15-F4/H15-R4) upstream and downstream primer, AccuPoL ^TMDNA Polymerase 1 μ L mends sterilization deionized water to 50 μ L.Reaction conditions is: 94 ℃ of 30s, 72 ℃ of 15min.

3.2.2 the reaction system and the condition of moderate-length sheet segment DNA chain extension: 10 * buffer, 1 μ L, dNTP 4 μ L, AccuPoLTMDNA Polymerase 1 μ L, each 22 μ L of two short dna fragment products (being produced by 3.2.1) totally are 50 μ L.

Reaction conditions is: 94 ℃ of 30s, 72 ℃ of 15min.

3.3 the synthetic and evaluation of target DNA fragment

The dna fragmentation that obtains with chain extension reaction among the 3.2.2 is a template, and H15-F/H15-R is that primer carries out the PCR reaction, reaction system: 10 * buffer 5 μ L, dNTP4 μ L, each 1 μ L of upstream and downstream primer, AccuPoL ^TMDNA Polymerase 1 μ L, template 2 μ L mend sterilization deionized water to 50 μ L.Reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 52 ℃ of 40s, 72 ℃ of 1min, totally 40 circulations; 72 ℃, 10min.

Show that through electrophoresis and order-checking qualification result amplification has obtained the target DNA fragment of the 300bp of expection.Proof the present invention designs synthetic primer specific sensitivity.Target DNA fragment is connected in the T carrier cloning to be preserved for future use.

The amplification of embodiment 4 multiple RT-PCRs

25 hypotypes of avian influenza virus are divided into four groups.First group for obtaining the hypotype group of avian influenza virus standard strain.Second and third, four groups be the random packet of all the other 17 hypotypes of avian influenza virus, be specially: first group of H1, H3, H5, H6, H7, H9, N1 and N2; Second group is H2, H4, H8, H10, H11, H13, H15, H16; The 3rd group is H14, N4, N6, N7, N9; The 4th group is H12, N3, N5, N8.

4.1 multiple RT-PCR reaction system

With reference to single stage method RT-PCR test kit operation instructions, revise a little and carry out.Reaction system: in the 0.2mL reaction tubes, add 10 μ L QIAGENE onestep RT-PCR buffer (5 *) successively, 10mM dNTP 2.0 μ L, Enzyme mix 2.0 μ L, RNase inhibitor 0.5 μ L, each 0.5 μ L (10 μ M) of upstream and downstream Auele Specific Primer, RNA template 5 μ L mend RNas-free water to 50 μ L.

4.2 multiple RT-PCR reaction conditions

After sample hose put into MJ RESEARCH PTC-200 PCR instrument, following condition is set reacts: 50 ℃ of pre-sex change 30min; 95 ℃, 15min; 94 ℃, 30s, 52 ℃, 40s, 72 ℃, 1min, 40 circulations; 72 ℃, 10min.Reaction is got 5 μ l amplified productions and carry out electrophoretic analysis on 1.5% sepharose after finishing.

Embodiment 5 multiple asymmetric RT-PCR amplifications

5.1 the primer design of multiple asymmetric PCR/RT-PCR and synthetic

5 ' of primer sequence end adds that corresponding universal primer sequence (is that the specificity upstream primer adds general upstream primer in table 1, the specificity downstream primer adds general downstream primer), and carry out fluorescence at 5 ' end of downstream primer and modify, obtain asymmetric amplimer, give birth to worker's biotechnology company limited by Shanghai and synthesize, sequence sees Table 3.

The primer of the multiple asymmetric PCR/RT-PCR of table 3

Type	The primer numbering	Sequence (5`～3`)	Expanding fragment length
				General	PMA-06001-uf PMA-06002-ur	TCACTTGCTTCCGTTGAGG TAMRA-GGTTTCGGATGTTACAGCG
H1	PMA-06003-H1f PMA-06004-H1r	TCACTTGCTCCGTTGGGGGAGCAATTGAGTTCAGTATC TAMRA-GGTTTCGGATGTTACAGCGTGACACTCTCCTATTGTGACTG	601bp
				H3	PMA-06005-H3f PMA-06006-H3r	TCACTTGCTTCCGTTGAGGTGTTACCCTTATGATGTGCC TAMRA-GGTTTCGATGTTACAGCGTCCCTGTTGCCAATTTCAGAG	669bp
H5	PMA-06007-H5f PMA-06008-H5r	TCACTTGCTTCCGTTGAGGATGAATTGGAATATGGTAACTG TAMRA-GGTTTCGGATGTTACAGCGTAACTGAGTGTTCATTTTGTCAAT	380bp
				H6	PMA-06017-H6F PMA-06018-H6R	TCACTTGCTTCCGTTGAGGAAGGCACTTATTGGRTCAGG TAMRA-GGTTTCGGATGTTACAGCGTGTCCTCTAGTTTCAATCTGTGG	685bp
H7	PMA-06009-H7f PMA-06010-H7r	TCACTTGCTTCCGTTGAGTCAGGWCTTCWTTCTATGC TAMRA-GGTTTCGGATGTTACAGCGTTCYCCTTGTGCATTTTGATG	641bp

H9	PMA-06011-H9f PMA-06012-H9r	TCACTTGCTTCCGTTGAGGAAGAGAATGGTCCTACATCGT TAMRA-GGTTTCGGATGTTACAGCGTGGATCTTACTCGCAATGTCTG	493bp
				N1	PMA-06013-N1f PMA-06014-N1r	TCACTTGCTTCCGTTGAGGTCCCACTTGGAATGCAGAAC TAMRA-GGTTTCGGATGTTACAGCGTCACATGCACATTCAGACTCTTG	328bp
N2	PMA-06015-N2f PMA-06016-N2r	TCACTTGCTTCCGTTGAGGATAGCATGGTCCAGCTCAAG TAMRA-GGTTTCGGATGTTACAGCGTACATGCTGAGCACTTCCTG	299bp
				H2	PMA-06019-H2F PMA-06020-H2R	TCACTTGCTTCCGTTGAGGCGTCATTCTTCAGGAACATGG TAMRA-GGTTTCGGATGTTACAGCGTGGCCTTGTTGCTATTTCWGG	229bp
H4	PMA-06021-H4F PMA-06022-H4R	TCACTTGCTTCCGTTGAGGTTGTTAYCCATTTGATGTGCC TAMRA-GGTTTCGGATGTTACAGCGTGTRACTCTTCCAGGGTTGTT	324bp
				H8	PMA-06023-H8F PMA-06024-H8R	TCACTTGCTTCCGTTGAGGAAGGTTGGTCATACATAGTGG TAMRA-GGTTTCGGATGTTACAGCGTGTCCTCTTACTAATGGTCTGG	444bp
H10	PMA-06025-H10F PMA-06026-H10R	TCACTTGCTTCCGTTGAGGGATTGACAAGATAAGCACCGG TAMRA-GGTTTCGGATGTTACAGCGTTTACTYACTCTACTAGGTGCTAT	435bp
				H11	PMA-06027-H11F PMA-06028-H11R	TCACTTGCTTCCGTTGAGGACTTAGAAATGTCCCAGCAA TAMRA-GGTTTCGGATGTTACAGCGTCATTTCCCTCGTCTTTGGC	437bp
H12	PMA-06035-H12F PMA-06036-H12R	TCACTTGCTTCCGTTGAGGAGTACAAGAACACCAGAGATT TAMRA-GGTTTCGGATGTTACAGCGTCTGGCCATCCGCCTTCTAT	537bp
				H13	PMA-06029-H13F PMA-06030-H13R	TCACTTGCTTCCGTTGAGGGACCCTTCTGCTCCTCATG TAMRA-GGTTTCGGATGTTACAGCGTGAAACTGATTGATTCCCCTGG	474bp
H14	PMA-06037-H14F PMA-06038-H14R	TCACTTGCTTCCGTTGAGGTCTCCCGACTAAACTGGCTA TAMRA-GGTTTCGGATGTTACAGCGTCTGCCGCTCTGATTCCTTAC	247bp
				H15	PMA-06031-H15F PMA-06032-H15R	TCACTTGCTTCCGTTGAGGGACTCCTTGACTGAGATCTGG TAMRA-GGTTTCGGATGTTACAGCGTAGTATCACATCTTTGTACCCAC	305bp
H16	PMA-06033-H16F PMA-06034-H16R	TCACTTGCTTCCGTTGAGGTAAACTTCTCGTGCTAATCG TAMRA-GGTTTCGGATGTTACAGCGTGTCTTCAACTTGATCCCTTC	252bp
				N3	PMA-06049-N3F PMA-06050-N3R	TCACTTGCTTCCGTTGAGGGGGAAAGARTGGATGCATGT TAMRA-GGTTTCGGATGTTACAGCGTGTTGTTGATTCTCATCCAAGG	366bp
N4	PMA-06039-N4F PMA-06040-N4R	TCACTTGCTTCCGTTGAGGGGAAGCAATCGACCATGGAT TAMRA-GGTTTCGGATGTTACAGCGTCGACACCCATCCATTAGCAT	260bp
				N5	PMA-06051-N5F PMA-06052-N5R	TCACTTGCTTCCGTTGAGGACTGTTATTGGGTAATGACG TAMRA-GGTTTCGGATGTTACAGCGTTGCTTGTTTTGGTCCAACCG	459bp
N6	PMA-06041-N6F PMA-06042-N6R	TCACTTGCTTCCGTTGAGGACCTAATAACAATGCTTCGG TAMRA-GGTTTCGGATGTTACAGCGTCACTCTTCTATATGCTGTGC	246bp
				N7	PMA-06043-N7F PMA-06044-N7R	TCACTTGCTTCCGTTGAGGTGTGCAGAGATAAYTGGCA TAMRA-GGTTTCGGATGTTACAGCGTCCGGAATAGCCTGACCAATT	352bp
N8	PMA-06045-N8F PMA-06046-N8R	TCACTTGCTTCCGTTGAGGGGGCAMTGATGTATGGATGG TAMRA-GGTTTCGGATGTTACAGCGTAAGAATAGCTCCATCGTGCC	340bp
				N9	PMA-06047-N9F PMA-06048-N9R	TCACTTGCTTCCGTTGAGGTTCTATGCTCTCAGCCAAGG TAMRA-GGTTTCGGATGTTACAGCGTTGGCATACGCATTCAGATTC	310bp

With reference to single stage method RT-PCR test kit operation instructions, make amendment.Reaction system: in the 0.2mL reaction tubes, add 10 μ L QIAGENE onestep RT-PCR buffer (5 *) successively, 10mM dNTP 2.0 μ L, Enzyme mix 2.0 μ L, RNase inhibitor 0.5 μ L, upstream and downstream universal primer in the table 3 is respectively 0.5 μ L and 5 μ L (10 μ M), upstream and downstream Auele Specific Primer (primer in the table 3) is respectively 0.5 μ L and 1 μ L (10 μ M), and RNA template 5 μ L mend RNas-free water to 50 μ L.

After sample hose put into MJ RESEARCH PTC-200 PCR instrument, following condition is set reacts: 50 ℃ of 30min; 95 ℃ of 15min; 94 ℃ of 20s, 52 ℃ of 1min, 72 ℃ of 90s, totally 20 circulations; 94 ℃ of 20s then, 70 ℃ of 90s, totally 20 circulations; Last 72 ℃ are extended 10min.After finishing, reaction gets 5 μ L products with 1.5% agarose gel electrophoresis-ethidium bromide staining analysis.

The preparation of embodiment 6 gene chips

6.1 the design of probe is with synthetic

Genome sequence according to the different subtype avian influenza viruses of delivering among the Genebank, utilize Bioedit, Primer5.0 and OMIGA software respectively the complete genome sequence of all models AIV that delivered to be carried out Analysis and Screening, 52 specific probes and 3 Quality Control probes of carrying out avian influenza genes chip typing method have been designed, it is synthetic to be sent to Shanghai living worker's biotechnology company limited, sees Table 4.

Table 4 avian influenza genes chip probe

Type	Numbering	Probe sequence (5`～3`)
			The Quality Control probe	PBA-08001-ctr1 PBA-08002-ctr2 PBA-08003-ctr3	TAMRA-CCTCAACGGAAGCAAGTGAT NH2-T15-ATCACTTGCTTCCGTTGAGG NH2-GCTGCCTCGGCAAGGAGT-TAMRA
The H1 probe	PBA-08004-H1a PBA-08066-H1d PBA-08068-H1f	NH2-T15-TGCTTATGTCTCTGTAGTGTCTTC NH2-T15-AATAGAACCTGGAGACACAATAA NH2-T15-CGAGATATTCCCCAAGACAAGTT
			The H3 probe	PBA-08007-H3a PBA-08008-H3b	NH2-T15-CCTCGGGGTTACTTCAAAATACG NH2-T15-GGAAGCATTCCCAATGACAAACC
The H5 probe	PBA-08011-H5a PBA-08012-H5b	NH2-T15-GTCACCAATAAGGTCAACTCGATC NH2-T15-ACCATAGCAATGAGCAGGGGA
			The H6 probe	PBA-08025-H6a PBA-08026-H6b	NH2-T15-TGAGATGTTTCCCAAAAGTACATGG NH2-T15-ATGGGAACTGAAAGCATGAATTT
The H7 probe	PBA-08013-H7a PBA-08014-H7b PBA-08065-H7e	NH2-T15-CAGACCAAACTCTATGGAAGTGGA NH2-T15-GTCAAACACAGACAATGCTGCTT NH2-T15-CAAGGAAAGACCCAGCTCTGATAAT
			The H9 probe	PBA-08017-H9a PBA-08018-H9b	NH2-T15-CAAGACGCCCAATACACAAATAAT NH2-T15-AAGCATGTTCAGATTCATTCTACAG
The N1 probe	PBA-08019-N1a PBA-08020-N1b	NH2-T15-AGTTGGTTGACAATTGGAATTTCTG NH2-T15-CAAGAGTTGGAGGAACAACATACT
			The N2 probe	PBA-08022-N2a PBA-08023-N2b	NH2-T15-GCGTTTGTATCAATGGAACTTGTA NH2-T15-ATGATGGGAAAGCATGGTTACATG
The H2 probe	PBA-08027-H2a PBA-08028-H2b	NH2-T15-ACATCAACACTGAATAAGAGGTC NH2-T15-GAACAAAGGACACTGTACCAGAAT
			The H4 probe	PBA-08029-H4a PBA-08030-H4b	NH2-T15-GACAAAGGTCAACAATGGGGA NH2-T15-CTTCAACTGACGCAGAACAAA
The H8 probe	PBA-08031-H8a PBA-08032-H8b	NH2-T15-TGGAGACATCATTTTCTTATGGG NH2-T15-GCATCTTACAAGAGAATAAGGCTATT
			The H10 probe	PBA-08034-H10a PBA-08035-H10b	NH2-T15-AAAACAACTTTGTGCCTGTGGT NH2-T15-CACAAGAAAAGAATGATCTGTATGG
The H11 probe	PBA-08036-H11a PBA-08037-H11b	NH2-T15-CAGTGAAATAGAGGAGAGGATAAACC NH2-T15-AGAAGGATGCTAAAGGACAATG
			The H12 probe	PBA-08045-H12a PBA-08046-H12b	NH2-T15-TAATCACAGGGAAATCACATGGC NH2-T15-CACTAGTAAGCACTATATTGGGAA

The H13 probe	PBA-08038-H13a PBA-08039-H13b	NH2-T15-GGATGAAGATTTACTGGTATTTGATG NH2-T15-GTTCATGGAGTAGGAAATACAACC
			The H14 probe	PBA-08047-H14a PBA-08048-H14b	NH2-T15-CCATCAAGCGATAATGAGCAAAC NH2-T15-TCTTATGTCAGGCTCTATCTCTGG
The H15 probe	PBA-08040-H15a PBA-08041-H15b	NH2-T15-GCATACAATTGACCTTGCAGATTC NH2-T15-CCGATGTGACGATCAATGTATG
			The H16 probe	PBA-08043-H16b PBA-08044-H16c	NH2-T15-GACAGAACATTAGACCTGCATGAT NH2-T15-ATCATGAGGACTACAAAGAAGAG
The N3 probe	PBA-08049-N3a PBA-08050-N3b	NH2-T15-TATGTAGGGACAATTGGAAGGG NH2-T15-GATAATGATGCAAGTGCCCAGA
			The N4 probe	PBA-08051-N4a PBA-08052-N4b	NH2-T15-GGCTATGTATGTAGTGGGATATTTG NH2-T15-GATGGCACAGGCTCATGTAATAG
The N5 probe	PBA-08053-N5a PBA-08054-N5b	NH2-T15-GTTTGCCGAGATAATTGGAATGG NH2-T15-AGGGAGGTCACATTGAAGAGT
			The N6 probe	PBA-08055-N6a PBA-08056-N6b	NH2-T15-GGCAGGAAATATATTAAGGACTCA NH2-T15-CCAGCTAATAACAGAGCAGAAAC
The N7 probe	PBA-08057-N7a PBA-08058-N7b	NH2-T15-ATGTTGAAAATACCTAATGCAGG NH2-T15-AAGGGATTCGGGTTTCTAAATGG
			The N8 probe	PBA-08060-N8a PBA-08061-N8b	NH2-T15-AGCTCCATTGTGATGTGTGG NH2-T15-AACTTAAATTGGTCAGGATACAGCG
The N9 probe	PBA-08062-N9a PBA-08063-N9b	NH2-T15-TCATCACCACCCACAGTATACAA NH2-T15-AGAGCCAGGATGTCGATATGTA

6.2 the preparation of gene chip

The micromatrix of chip design is 14 * 9 array (see figure 4)s, and matrix design figure comprises that 4 part: QC are the positive reference of point sample, and 8 are repeated the site; The negative reference of NC, 10 are repeated the site; PC is the positive reference of hybridization, and 2 are repeated the site; Other site is a detection probes, 52 specific probes of 25 hypotypes, and every 2 of probe repeats the site.Entrust Huada Gene Research Center, Beijing's point sample.The solid phase carrier that chip uses as aldehyde slide (available from CELAssociates, Inc.Pearland, Texas).

Embodiment 7 utilizes the cross experiment of gene chip

(1) hybridization buffer (preparation in advance): ddH ₂O 0.84 μ L, 20 * SSC, 1.80 μ L, 50 * Denhardt ' s, 1.80 μ L, 50% T 500,3.60 μ L, 4%SDS 1.80 μ L.

(2) hybridization system: hybridization buffer 6 μ L, multiple asymmetric amplified production 5.2 μ L, Quality Control probe 0.8 μ L mixing.

(3) with 95 ℃ of sex change 5min of the hybridization system in (2), ice bath 3min immediately, ice bath 3min immediately, the centrifugal 5～10s of 4000rpm puts back to ice bath with centrifuge tube.

(4) at hybridizing box (HybriCassettes ^TM, the CapitalBio Corporation) groove in add 200 μ L distilled water in case the evaporation of hybridization system is upwards put into box with chip front side.

(5) with getting sample thief hybridization solution 7 μ L in chip point sample district after the pipettor piping and druming evenly, cover cover plate and hybridizing box and sealing rapidly, level is put in 52 ℃ of water-baths hybridizes 2.5hr.

(6) hybridization finishes, and the hybridizing box level is taken out, and immediately chip is taken out, put into the washings I that is preheated to 45 ℃ (2 * SSC, 0.1%SDS) in, 100rpm washs 5min.Taking out chip puts into 45 ℃ washings I and washes once.

(7) take out chip, (0.2 * SSC, 0.1%SDS), 100rpm washs 5min, washed twice to put into the cleaning solution II that is preheated to 45 ℃.

(8) take out chip, put into washing lotion III (0.2 * SSC), 100rpm room temperature washing 5min.

(9) chip lixiviate in dehydrated alcohol is put into chip cartridges to chip several times then, the centrifugal 5min of 1000r/min dries, and carries out chip scanning with PerkinElmer ScanArray Gx plus.

Embodiment 8 gene chips detect efficiency assay

The multiple asymmetric RT-PCR amplified production equal proportion of avian influenza virus H1N1, H3N2, H5N1, H6N2, H7N1 and H9N2 type is mixed back and chip hybridization, again the amplified production equal proportion of whole hypotypes is mixed and chip hybridization, the result shows in all corresponding detection site can both detect positive hybridization signal (shown in description of drawings 5-27,5-28).

The test of embodiment 9 gene chip detection sensitivity

Form test by plaque avian influenza virus (H1, H3, H5, H6, H7, H9, N1 and N2) allantoic fluid is carried out virus quantitatively, extract RNA with QIAamp Viral RNA Mini Kit, and it is carried out 10 times of dilutions of going forward one by one, carry out multiple asymmetric RT-PCR (seeing embodiment 5), carry out the hybridization and the washing (every type AIV makes the revision test of 3 chip micromatrixs) of gene chip then according to embodiment 6, at last the centrifugal 5min of chip 1000r/min is dried, carry out chip scanning and interpretation as a result with PerkinElmer ScanArrayGx plus.

The sensitivity test result: with H5N1 is example, and plaque adds up to: 2.47 * 10 ⁷Pfu/mL, its susceptibility can reach 247pfu/mL.

The test of embodiment 10 gene chip detection specificity

Extract each hypotype AIV, infectious bronchitis virus (IBV) AV10, Avianreovirus (ARV) AV2311 CEK3, infectious bursal disease virus (IBDV), Avian pneumo-encephalitis virus common virus nucleic acid such as (NDV) according to a conventional method respectively, carry out gene chip and detect.

Each sample repeats 3 times, and the result is consistent.Except that each hypotype AIV detected result occurs the positive hybridization signal at the corresponding positions point, other viral detected results are all negative: infectious bronchitis virus (IBV) AV10, Avianreovirus (ARV) AV2311 CEK3, infectious bursal disease virus (IBDV), Avian pneumo-encephalitis virus (NDV) gene chip detected result are all negative, except that QC and the PC positive all do not have detection signal (result is shown in description of drawings 5-1) with reference to other site the fluorescent signal; And the detected result of each hypotype AIV only positive signal (result is shown in description of drawings 5-2～5-26) occurs in corresponding site separately.

Embodiment 11 gene chips detect the Preliminary Applications test

To 395 parts of port samples (more than 20 areas of 4 provinces), adopt two kinds of detection methods of gene chip and conventional pathogen separation to carry out parallel detection respectively.Two kinds of method detected results are in full accord, show that the gene chip detecting technique of setting up has good reliability and practicality.

Sequence table

＜110〉Institute of quarantine of animals and plants, Chinese Academy of Inspection and

＜120〉be used for the gene chip of avian flu virus detection and somatotype

<130>

<160>55

<170>PatentIn version 3.1

<210>1

<211>20

<212>DNA

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<220>

＜221〉probe

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<223>

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cctcaacgga agcaagtgat 20

<210>2

<211>20

<212>DNA

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＜221〉probe

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<223>

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atcacttgct tccgttgagg 20

<210>3

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＜221〉probe

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gctgcctcgg caaggagt 18

<210>4

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tgcttatgtc tctgtagtgt cttc 24

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aatagaacct ggagacacaa taa 23

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cgagatattc cccaagacaa gtt 23

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＜221〉probe

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cctcggggtt acttcaaaat acg 23

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ggaagcattc ccaatgacaa acc 23

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gtcaccaata aggtcaac tc gatc 24

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＜221〉probe

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<400>10

accatagcaa tgagcagggg a 21

<210>11

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＜221〉probe

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tgagatgttt cccaaaagta catgg 25

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atgggaactg aaagcatgaa ttt 23

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cagaccaaac tctatggaag tgga 24

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gtcaaacaca gacaatgctg ctt 23

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caaggaaaga cccagctctg ataat 25

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＜221〉probe

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caagacgccc aatacacaaa taat 24

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aagcatgttc agattcattc tacag 25

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<223>

<400>20

gcgtttgtat caatggaact tgta 24

<210>21

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>21

atgatgggaa agcatggtta catg 24

<210>22

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>22

acatcaacac tgaataagag gtc 23

<210>23

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>23

gaacaaagga cactgtacca gaat 24

<210>24

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(21)

<223>

<400>24

gacaaaggtc aacaatgggg a 21

<210>25

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(21)

<223>

<400>25

cttcaactga cgcagaacaa a 21

<210>26

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>26

tggagacatc attttcttat ggg 23

<210>27

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(26)

<223>

<400>27

gcatcttaca agagaataag gctatt 26

<210>28

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(22)

<223>

<400>28

aaaacaactt tgtgcctgtg gt 22

<210>29

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(25)

<223>

<400>29

cacaagaaaa gaatgatctg tatgg 25

<210>30

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(26)

<223>

<400>30

cagtgaaata gaggagagga taaacc 26

<210>31

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(22)

<223>

<400>31

agaaggatgc taaaggacaa tg 22

<210>32

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>32

taatcacagg gaaatcacat ggc 23

<210>33

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>33

cactagtaag cactatattg ggaa 24

<210>34

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜221〉artificial sequence

<222>(1)..(26)

<223>

<400>34

ggatgaagat ttactggtat ttgatg 26

<210>35

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>35

gttcatggag taggaaatac aacc 24

<210>36

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>36

ccatcaagcg ataatgagca aac 23

<210>37

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>37

tcttatgtca ggctctatct ctgg 24

<210>38

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>38

gcatacaatt gaccttgcag attc 24

<210>39

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(22)

<223>

<400>39

ccgatgtgac gatcaatgta tg 22

<210>40

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>40

gacagaacat tagacctgca tgat 24

<210>41

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>41

atcatgagga ctacaaagaa gag 23

<210>42

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(22)

<223>

<400>42

tatgtaggga caattggaag gg 22

<210>43

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(22)

<223>

<400>43

gataatgatg caagtgccca ga 22

<210>44

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(25)

<223>

<400>44

ggctatgtat gtagtgggat atttg 25

<210>45

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>45

gatggcacag gctcatgtaa tag 23

<210>46

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>46

gtttgccgag ataattggaa tgg 23

<210>47

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(21)

<223>

<400>47

agggaggtca cattgaagag t 21

<210>48

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(24)

<223>

<400>48

ggcaggaaat atattaagga ctca 24

<210>49

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>49

ccagctaata acagagcaga aac 23

<210>50

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>50

atgttgaaaa tacctaatgc agg 23

<210>51

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>51

aagggattcg ggtttctaaa tgg 23

<210>52

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(20)

<223>

<400>52

agctccattg tgatgtgtgg 20

<210>53

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(25)

<223>

<400>53

aacttaaatt ggtcaggata cagcg 25

<210>54

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(23)

<223>

<400>54

tcatcaccac ccacagtata caa 23

<210>55

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜221〉probe

<222>(1)..(22)

<223>

<400>55

agagccagga tgtcgatatg ta 22

Claims (6)

1. gene chip that is used for avian flu virus detection and somatotype, comprising: solid phase carrier be fixed on Quality Control probe and avian influenza virus type specificity probe on this solid phase carrier, 5 ' end of described probe adds amino the modification.

2. the described gene chip of claim 1, wherein said solid phase carrier is selected from: nitrocellulose filter, nylon membrane, polystyrene and slide glass.

3. the described gene chip of claim 1, wherein said Quality Control probe and the pairing separately type of avian influenza virus type specificity probe and nucleotide sequence are respectively:

Type Numbering Probe sequence (5`～3`) The Quality Control probe PBA-08001-ctr1 PBA-08002-ctr2 PBA-08003-ctr3 TAMRA-CCTCAACGGAAGCAAGTGAT NH2-T15-ATCACTTGCTTCCGTTGAGG NH2-GCTGCCTCGGCAAGGAGT-TAMRA The H1 probe PBA-08004-H1a PBA-08066-H1d PBA-08068-H1f NH2-T15-TGCTTATGTCTCTGTAGTGTCTTC NH2-T15-AATAGAACCTGGAGACACAATAA NH2-T15-CGAGATATTCCCCAAGACAAGTT The H3 probe PBA-08007-H3a PBA-08008-H3b NH2-T15-CCTCGGGGTTACTTCAAAATACG NH2-T15-GGAAGCATTCCCAATGACAAACC The H5 probe PBA-08011-H5a PBA-08012-H5b NH2-T15-GTCACCAATAAGGTCAACTCGATC NH2-T15-ACCATAGCAATGAGCAGGGGA The H6 probe PBA-08025-H6a PBA-08026-H6b NH2-T15-TGAGATGTTTCCCAAAAGTACATGG NH2-T15-ATGGGAACTGAAAGCATGAATTT The H7 probe PBA-08013-H7a PBA-08014-H7b PBA-08065-H7e NH2-T15-CAGACCAAACTCTATGGAAGTGGA NH2-T15-GTCAAACACAGACAATGCTGCTT NH2-T15-CAAGGAAAGACCCAGCTCTGATAAT The H9 probe PBA-08017-H9a PBA-08018-H9b NH2-T15-CAAGACGCCCAATACACAAATAAT NH2-T15-AAGCATGTTCAGATTCATTCTACAG The N1 probe PBA-08019-N1a PBA-08020-N1b NH2-T15-AGTTGGTTGACAATTGGAATTTCTG NH2-T15-CAAGAGTTGGAGGAACAACATACT The N2 probe PBA-08022-N2a PBA-08023-N2b NH2-T15-GCGTTTGTATCAATGGAACTTGTA NH2-T15-ATGATGGGAAAGCATGGTTACATG The H2 probe PBA-08027-H2a PBA-08028-H2b NH2-T15-ACATCAACACTGAATAAGAGGTC NH2-T15-GAACAAAGGACACTGTACCAGAAT The H4 probe PBA-08029-H4a PBA-08030-H4b NH2-T15-GACAAAGGTCAACAATGGGGA NH2-T15-CTTCAACTGACGCAGAACAAA The H8 probe PBA-08031-H8a PBA-08032-H8b NH2-T15-TGGAGACATCATTTTCTTATGGG NH2-T15-GCATCTTACAAGAGAATAAGGCTATT The H10 probe PBA-08034-H10a PBA-08035-H10b NH2-T15-AAAACAACTTTGTGCCTGTGGT NH2-T15-CACAAGAAAAGAATGATCTGTATGG The H11 probe PBA-08036-H11a PBA-08037-H11b NH2-T15-CAGTGAAATAGAGGAGAGGATAAACC NH2-T15-AGAAGGATGCTAAAGGACAATG The H12 probe PBA-08045-H12a PBA-08046-H12b NH2-T15-TAATCACAGGGAAATCACATGGC NH2-T15-CACTAGTAAGCACTATATTGGGAA The H13 probe PBA-08038-H13a PBA-08039-H13b NH2-T15-GGATGAAGATTTACTGGTATTTGATG NH2-T15-GTTCATGGAGTAGGAAATACAACC

The H14 probe PBA-08047-H14a PBA-08048-H14b NH2-T15-CCATCAAGCGATAATGAGCAAAC NH2-T15-TCTTATGTCAGGCTCTATCTCTGG The H15 probe PBA-08040-H15a PBA-08041-H15b NH2-T15-GCATACAATTGACCTTGCAGATTC NH2-T15-CCGATGTGACGATCAATGTATG The H16 probe PBA-08043-H16b PBA-08044-H16c NH2-T15-GACAGAACATTAGACCTGCATGAT NH2-T15-ATCATGAGGACTACAAAGAAGAG The N3 probe PBA-08049-N3a PBA-08050-N3b NH2-T15-TATGTAGGGACAATTGGAAGGG NH2-T15-GATAATGATGCAAGTGCCCAGA The N4 probe PBA-08051-N4a PBA-08052-N4b NH2-T15-GGCTATGTATGTAGTGGGATATTTG NH2-T15-GATGGCACAGGCTCATGTAATAG The N5 probe PBA-08053-N5a PBA-08054-N5b NH2-T15-GTTTGCCGAGATAATTGGAATGG NH2-T15-AGGGAGGTCACATTGAAGAGT The N6 probe PBA-08055-N6a PBA-08056-N6b NH2-T15-GGCAGGAAATATATTAAGGACTCA NH2-T15-CCAGCTAATAACAGAGCAGAAAC The N7 probe PBA-08057-N7a PBA-08058-N7b NH2-T15-ATGTTGAAAATACCTAATGCAGG NH2-T15-AAGGGATTCGGGTTTCTAAATGG The N8 probe PBA-08060-N8a PBA-08061-N8b NH2-T15-AGCTCCATTGTGATGTGTGG NH2-T15-AACTTAAATTGGTCAGGATACAGCG The N9 probe PBA-08062-N9a PBA-08063-N9b NH2-T15-TCATCACCACCCACAGTATACAA NH2-T15-AGAGCCAGGATGTCGATATGTA

Wherein TAMRA is a fluorescence dye.

4. each described gene chip preparation method among the claim 1-3.

5. 3 Quality Control probes and the 52 stripe shape specific probes used of avian influenza virus somatotype, its nucleotide sequence and separately corresponding type be:

Type Numbering Probe sequence (5`～3`) The Quality Control probe PBA-08001-ctr1 PBA-08002-ctr2 PBA-08003-ctr3 TAMRA-CCTCAACGGAAGCAAGTGAT NH2-T15-ATCACTTGCTTCCGTTGAGG NH2-GCTGCCTCGGCAAGGAGT-TAMRA The H1 probe PBA-08004-H1a PBA-08066-H1d PBA-08068-H1f NH2-T15-TGCTTATGTCTCTGTAGTGTCTTC NH2-T15-AATAGAACCTGGAGACACAATAA NH2-T15-CGAGATATTCCCCAAGACAAGTT The H3 probe PBA-08007-H3a PBA-08008-H3b NH2-T15-CCTCGGGGTTACTTCAAAATACG NH2-T15-GGAAGCATTCCCAATGACAAACC The H5 probe PBA-08011-H5a PBA-08012-H5b NH2-T15-GTCACCAATAAGGTCAACTCGATC NH2-T15-ACCATAGCAATGAGCAGGGGA The H6 probe PBA-08025-H6a PBA-08025-H6b NH2-T15-TGAGATGTTTCCCAAAAGTACATGG NH2-T15-ATGGGAACTGAAAGCATGAATTT The H7 probe PBA-08013-H7a PBA-08014-H7b PBA-08065-H7e NH2-T15-CAGACCAAACTCTATGGAAGTGGA NH2-T15-GTCAAACACAGACAATGCTGCTT NH2-T15-CAAGGAAAGACCCAGCTCTGATAAT The H9 probe PBA-08017-H9a PBA-08018-H9b NH2-T15-CAAGACGCCCAATACACAAATAAT NH2-T15-AAGCATGTTCAGATTCATTCTACAG The N1 probe PBA-08019-N1a PBA-08020-N1b NH2-T15-AGTTGGTTGACAATTGGAATTTCTG NH2-T15-CAAGAGTTGGAGGAACAACATACT

The N2 probe PBA-08022-N2a PBA-08023-N2b NH2-T15-GCGTTTGTATCAATGGAACTTGTA NH2-T15-ATGATGGGAAAGCATGGTTACATG The H2 probe PBA-08027-H2a PBA-08028-H2b NH2-T15-ACATCAACACTGAATAAGAGGTC NH2-T15-GAACAAAGGACACTGTACCAGAAT The H4 probe PBA-08029-H4a PBA-08030-H4b NH2-T15-GACAAAGGTCAACAATGGGGA NH2-T15-CTTCAACTGACGCAGAACAAA The H8 probe PBA-08031-H8a PBA-08032-H8b NH2-T15-TGGAGACATCATTTTCTTATGGG NH2-T15-GCATCTTACAAGAGAATAAGGCTATT The H10 probe PBA-08034-H10a PBA-08035-H10b NH2-T15-AAAACAACTTTGTGCCTGTGGT NH2-T15-CACAAGAAAAGAATGATCTGTATGG The H11 probe PBA-08036-H11a PBA-08037-H11b NH2-T15-CAGTGAAATAGAGGAGAGGATAAACC NH2-T15-AGAAGGATGCTAAAGGACAATG The H12 probe PBA-08045-H12a PBA-08046-H12b NH2-T15-TAATCACAGGGAAATCACATGGC NH2-T15-CACTAGTAAGCACTATATTGGGAA The H13 probe PBA-08038-H13a PBA-08039-H13b NH2-T15-GGATGAAGATTTACTGGTATTTGATG NH2-T15-GTTCATGGAGTAGGAAATACAACC The H14 probe PBA-08047-H14a PBA-08048-H14b NH2-T15-CCATCAAGCGATAATGAGCAAAC NH2-T15-TCTTATGTCAGGCTCTATCTCTGG The H15 probe PBA-08040-H15a PBA-08041-H15b NH2-T15-GCATACAATTGACCTTGCAGATTC NH2-T15-CCGATGTGACGATCAATGTATG The H16 probe PBA-08043-H16b PBA-08044-H16c NH2-T15-GACAGAACATTAGACCTGCATGAT NH2-T15-ATCATGAGGACTACAAAGAAGAG The N3 probe PBA-08049-N3a PBA-08050-N3b NH2-T15-TATGTAGGGACAATTGGAAGGG NH2-T15-GATAATGATGCAAGTGCCCAGA The N4 probe PBA-08051-N4a PBA-08052-N4b NH2-T15-GGCTATGTATGTAGTGGGATATTTG NH2-T15-GATGGCACAGGCTCATGTAATAG The N5 probe PBA-08053-N5a PBA-08054-N5b NH2-T15-GTTTGCCGAGATAATTGGAATGG NH2-T15-AGGGAGGTCACATTGAAGAGT The N6 probe PBA-08055-N6a PBA-08056-N6b NH2-T15-GGCAGGAAATATATTAAGGACTCA NH2-T15-CCAGCTAATAACAGAGCAGAAAC The N7 probe PBA-08057-N7a PBA-08058-N7b NH2-T15-ATGTTGAAAATACCTAATGCAGG NH2-T15-AAGGGATTCGGGTTTCTAAATGG The N8 probe PBA-08060-N8a PBA-08061-N8b NH2-T15-AGCTCCATTGTGATGTGTGG NH2-T15-AACTTAAATTGGTCAGGATACAGCG The N9 probe PBA-08062-N9a PBA-08063-N9b NH2-T15-TCATCACCACCCACAGTATACAA NH2-T15-AGAGCCAGGATGTCGATATGTA

Wherein TAMRA is a fluorescence dye.

6. use each described gene chip among the claim 1-3 that avian influenza virus is detected method with somatotype, comprising:

1) extraction of viral RNA: collected specimens, ordinary method is extracted RNA;

2) RT-PCR amplification: with the RNA that extracts in the step 1) is that template is carried out the RT-PCR amplification

3) hybridization of gene chip and washing: add step 2) amplified production and gene chip of the present invention, be suitable for reacting time enough under the condition of hybridizing with described gene chip, the washing back dries then;

4) scanning of gene chip: chip inserted carry out scanning analysis, saving result in the chip scanner;

5) chip scanning result's interpretation:, search and pairing avian influenza virus subtype when determining this micromatrix arrangement position point sample according to the micromatrix arrangement position that positive findings occurs.

Images