CN101058833A - Primer system and method for detecting and analyzing avian influenza virus - Google Patents

Abstract

The invention provides a method for detecting fowl influenza virus and parting primer system and augmenting fowl influenza virus RNA asymmetrically. The invention designs and screens 25 pairs multiple asymmetrical RT-PCR primers, a pair general primer, 52 specific probes and 3 quality control probes according to the report fowl influenza virus total hypotype genome sequence in Genebank, wherein every pair in 25 pairs multiple asymmetrical RT-PCR primers is fit for a hypotype of fowl influenza virus, the primer system comprising 25 pairs multiple asymmetrical RT-PCR primers and a pair general primer can be used for detecting and parting fowl influenza virus, the primer system builds the method of asymmetrically augmenting fowl influenza virus RNA and detecting and parting fowl influenza virus. The invention provides strong specificity, high sensibility, which also proceeds the first step application.

Description

The primer system and the method that are 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 carry out Analysis and Screening, and 25 pairs of Auele Specific Primers that the multiple asymmetric RT-PCR somatotype that carries out the research of avian influenza virus biochip technology detects have successfully been designed, the 1 pair of universal primer and carry out 52 specific probes and 3 Quality Control probes of avian influenza virus chip typing technology, by optimizing reaction conditions, set up high specificity, the avian influenza virus chip typing detection method that susceptibility is high.

Summary of the invention:

The object of the invention is to provide a kind of primer system that is used for multiple asymmetric RT-PCR amplification and then is used for avian flu virus detection and somatotype.

Another object of the present invention is to provide a kind of method of utilizing described primer system to carry out avian flu virus detection and somatotype.

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 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 primer system that is used for avian flu virus detection and somatotype, comprising 25 pairs of multiple asymmetric RT-PCR primers and 1 pair of universal primer, wherein each to described multiple asymmetric RT-PCR primer each at a kind of avian influenza virus subtype, the type and the nucleotide sequence thereof of described primer correspondence are as shown in table 3.TAMRA wherein is a fluorescence dye.

Specificity and sensitivity test result prove: primer provided by the invention is to all having good type specificity and susceptibility.Can be used for treating the sample product and carry out multiple asymmetric RT-PCR amplification, and then be used for avian flu virus detection and somatotype.

On the other hand, the invention provides the method for a kind of asymmetric amplification avian influenza virus RNA, this method comprises the following steps:

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

2) multiple asymmetric RT-PCR amplification: with the RNA that extracts in the step 1) is that template is carried out multiple asymmetric RT-PCR amplification, wherein uses primer system of the present invention, and the reaction conditions of pcr amplification is: 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; The fowl that amplified production can be used for being undertaken by nucleic acid hybridization is flowed the detection and the somatotype of virus.

On the other hand, the invention provides and use primer system of the present invention that avian influenza virus is detected method with somatotype, this method comprises the following steps:

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

2) multiple asymmetric RT-PCR amplification: with the RNA that extracts in the step 1) is that template is carried out multiple asymmetric RT-PCR amplification, wherein uses the described primer system of claim 3, and the reaction conditions of amplification is: 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) hybridization and washing: adding step 2) multiple asymmetric amplified production and the gene chip that obtains, be suitable for reacting time enough under the condition of hybridizing with selected gene chip, the washing back dries then;

4) result judges: chip is inserted carry out scanning analysis in the chip scanner, and saving result, then according to the micromatrix arrangement position that positive findings occurs, pairing avian influenza virus subtype when searching this micromatrix arrangement position point sample.

In a specific embodiments of the present invention, the method for described detection and somatotype 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-200PCR 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 DL 2000DNA Marker.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 2000DNA Marker.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, H1N1RNA, 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, H15, 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.The positive probe NC of QC Quality Control probe PC feminine gender is with reference to DP (H1-16, N1-9) detection probes

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. Utilize primer system of the present invention and detection and classifying method, can identify accurately and rapidly the various neuraminidase hypotypes (N1～9) of avian flu strain simultaneously, can also distinguish the hypotypes such as H3N2, H2N2, H1N1 and H1N2 that infect the people.

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 Designer 2.1 software designs, give birth to worker's biotechnology company limited by Shanghai and synthesize, 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 N2-R	ATAGCATGGTCCAGCTCAAG ACATGCTGAGCACTTCCTG	299bp

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	H14F 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).

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 inhibitor 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 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 OMIGA software design five upstream primer H15-F, H15-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

3.2DNA fragment is extended

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, AccuPoL ^TMDNA 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, dNTP 4 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-200PCR 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 asymmetric multiplex 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-GGTTTCGGATGTTACAGCGT
H1	PMA-06003-H1f PMA-06004-H1r	TCACTTGCTTCCGTTGAGGGGAGCAATTGAGTTCAGTATC TAMRA-GGTTTCGGATGTTACAGCGTGACACTCTCCTATTGTGACTG	601bp
				H3	PMA-06005-H3f PMA-06006-H3r	TCACTTGCTTCCGTTGAGGTGTTACCCTTATGATGTGCC TAMRA-GGTTTCGGATGTTACAGCGTCCCTGTTGCCAATTTCAGAG	669bp
H5	PMA-06007-H5f PMA-06008-H5r	TCACTTGCTTCCGTTGAGGAGTGAATTGGAATATGGTAACTG TAMRA-GGTTTCGGATGTTACAGCGTAACTGAGTGTTCATTTTGTCAAT	380bp
				H6	PMA-06017-H6F PMA-06018-H6R	TCACTTGCTTCCGTTGAGGAAGGCACTTATTGGRTCAGG TAMRA-GGTTTCGGATGTTACAGCGTGTCCTCTAGTTTCAATCTGTGG	685bp
H7	PMA-06009-H7f PMA-06010-H7r	TCACTTGCTTCCGTTGAGGTCAGGWTCTTCWTTCTATGC 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

Wherein TAMRA is a fluorescence dye.

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, give birth to worker's biotechnology company limited by Shanghai and synthesize, see 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	NH2-T15-GACAGAACATTAGACCTGCATGAT

	PBA-08044-H16c	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 classical 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

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<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>21

tcacttgctt ccgttgaggt tgttayccat ttgatgtgcc 40

<210>22

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>22

ggtttcggat gttacagcgt gtractcttc cagggttgtt 40

<210>23

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>23

tcacttgctt ccgttgagga aggttggtca tacatagtgg 40

<210>24

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(41)

<223>

<400>24

ggtttcggat gttacagcgt gtcctcttac taatggtctg g 41

<210>25

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>25

tcacttgctt ccgttgaggg attgacaaga taagcaccgg 40

<210>26

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(43)

<223>

<400>26

ggtttcggat gttacagcgt ttactyactc tactaggtgc tat 43

<210>27

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>27

tcacttgctt ccgttgagga cttagaaatg tcccagcaa 39

<210>28

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>28

ggtttcggat gttacagcgt catttccctc gtctttggc 39

<210>29

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>29

tcacttgctt ccgttgagga gtacaagaac accagagatt 40

<210>30

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>30

ggtttcggat gttacagcgt ctggccatcc gccttctat 39

<210>31

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(38)

<223>

<400>31

tcacttgctt ccgttgaggg acccttctgc tcctcatg 38

<210>32

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(41)

<223>

<400>32

ggtttcggat gttacagcgt gaaactgatt gattcccctg g 41

<210>33

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>33

tcacttgctt ccgttgaggt ctcccgacta aactggcta 39

<210>34

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>34

ggtttcggat gttacagcgt ctgccgctct gattccttac 40

<210>35

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>35

tcacttgctt ccgttgaggg actccttgac tgagatctgg 40

<210>36

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(42)

<223>

<400>36

ggtttcggat gttacagcgt agtatcacat ctttgtaccc ac 42

<210>37

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>37

tcacttgctt ccgttgaggt aaacttctcg tgctaatcg 39

<210>38

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>38

ggtttcggat gttacagcgt gtcttcaact tgatcccttc 40

<210>39

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>39

tcacttgctt ccgttgaggg ggaaagartg gatgcatgt 39

<210>40

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(41)

<223>

<400>40

ggtttcggat gttacagcgt gttgttgatt ctcatccaag g 41

<210>41

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>41

tcacttgctt ccgttgaggg gaagcaatcg accatggat 39

<210>42

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>42

ggtttcggat gttacagcgt cgacacccat ccattagcat 40

<210>43

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>43

tcacttgctt ccgttgagga ctgttattgg gtaatgacg 39

<210>44

<211>40

<212>DNA

＜213〉primer

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>44

ggtttcggat gttacagcgt tgcttgtttt ggtccaaccg 40

<210>45

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>45

tcacttgctt ccgttgagga cctaataaca atgcttcgg 39

<210>46

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>46

ggtttcggat gttacagcgt cactcttcta tatgctgtgc 40

<210>47

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(38)

<223>

<400>47

tcacttgctt ccgttgaggt gtgcagagat aaytggca 38

<210>48

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>48

ggtttcggat gttacagcgt ccggaatagc ctgaccaatt 40

<210>49

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>49

tcacttgctt ccgttgaggg ggcamtgatg tatggatgg 39

<210>50

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>50

ggtttcggat gttacagcgt aagaatagct ccatcgtgcc 40

<210>51

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(39)

<223>

<400>51

tcacttgctt ccgttgaggt tctatgctct cagccaagg 39

<210>52

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜221〉primer

<222>(1)..(40)

<223>

<400>52

ggtttcggat gttacagcgt tggcatacgc attcagattc 40

Claims (3)

1. primer system that is used for avian flu virus detection and somatotype, comprising 25 pairs of multiple asymmetric RT-PCR primers and 1 pair of universal primer, wherein each is to each a kind of hypotype at avian influenza virus for wherein said multiple asymmetric RT-PCR primer, and pairing type of described primer and nucleotide sequence thereof are respectively: Type The primer numbering Sequence (5`～3`) General PMA-06001-uf PMA-06002-ur TCACTTGCTTCCGTTGAGG TAMRA-GGTTTCGGATGTTACAGCGT H1 PMA-06003-H1f PMA-06004-H1r TCACTTGCTTCCGTTGAGGGGAGCAATTGAGTTCAGTATC TAMRA-GGTTTCGGATGTTACAGCGTGACACTCTCCTATTGTGACTG H3 PMA-06005-H3f PMA-06006-H3r TCACTTGCTTCCGTTGAGGTGTTACCCTTATGATGTGCC TAMRA-GGTTTCGGATGTTACAGCGTCCCTGTTGCCAATTTCAGAG H5 PMA-06007-H5f PMA-06008-H5r TCACTTGCTTCCGTTGAGGAGTGAATTGGAATATGGTAACTG TAMRA-GGTTTCGGATGTTACAGCGTAACTGAGTGTTCATTTTGTCAAT H6 PMA-06017-H6F PMA-06018-H6R TCACTTGCTTCCGTTGAGGAAGGCACTTATTGGRTCAGG TAMRA-GGTTTCGGATGTTACAGCGTGTCCTCTAGTTTCAATCTGTGG H7 PMA-06009-H7f PMA-06010-H7r TCACTTGCTTCCGTTGAGGTCAGGWTCTTCWTTCTATGC TAMRA-GGTTTCGGATGTTACAGCGTTCYCCTTGTGCATTTTGATG H9 PMA-06011-H9f PMA-06012-H9r TCACTTGCTTCCGTTGAGGAAGAGAATGGTCCTACATCGT TAMRA-GGTTTCGGATGTTACAGCGTGGATCTTACTCGCAATGTCTG N1 PMA-06013-N1f PMA-06014-N1r TCACTTGCTTCCGTTGAGGTCCCACTTGGAATGCAGAAC TAMRA-GGTTTCGGATGTTACAGCGTCACATGCACATTCAGACTCTTG N2 PMA-06015-N2f PMA-06016-N2r TCACTTGCTTCCGTTGAGGATAGCATGGTCCAGCTCAAG TAMRA-GGTTTCGGATGTTACAGCGTACATGCTGAGCACTTCCTG H2 PMA-06019-H2F PMA-06020-H2R TCACTTGCTTCCGTTGAGGCGTCATTCTTCAGGAACATGG TAMRA-GGTTTCGGATGTTACAGCGTGGCCTTGTTGCTATTTCWGG H4 PMA-06021-H4F PMA-06022-H4R TCACTTGCTTCCGTTGAGGTTGTTAYCCATTTGATGTGCC TAMRA-GGTTTCGGATGTTACAGCGTGTRACTCTTCCAGGGTTGTT H8 PMA-06023-H8F PMA-06024-H8R TCACTTGCTTCCGTTGAGGAAGGTTGGTCATACATAGTGG TAMRA-GGTTTCGGATGTTACAGCGTGTCCTCTTACTAATGGTCTGG H10 PMA-06025-H10F PMA-06026-H10R TCACTTGCTTCCGTTGAGGGATTGACAAGATAAGCACCGG TAMRA-GGTTTCGGATGTTACAGCGTTTACTYACTCTACTAGGTGCTAT H11 PMA-06027-H11F PMA-06028-H11R TCACTTGCTTCCGTTGAGGACTTAGAAATGTCCCAGCAA TAMRA-GGTTTCGGATGTTACAGCGTCATTTCCCTCGTCTTTGGC H12 PMA-06035-H12F PMA-06036-H12R TCACTTGCTTCCGTTGAGGAGTACAAGAACACCAGAGATT TAMRA-GGTTTCGGATGTTACAGCGTCTGGCCATCCGCCTTCTAT H13 PMA-06029-H13F PMA-06030-H13R TCACTTGCTTCCGTTGAGGGACCCTTCTGCTCCTCATG TAMRA-GGTTTCGGATGTTACAGCGTGAAACTGATTGATTCCCCTGG H14 PMA-06037-H14F PMA-06038-H14R TCACTTGCTTCCGTTGAGGTCTCCCGACTAAACTGGCTA TAMRA-GGTTTCGGATGTTACAGCGTCTGCCGCTCTGATTCCTTAC H15 PMA-06031-H15F PMA-06032-H15R TCACTTGCTTCCGTTGAGGGACTCCTTGACTGAGATCTGG TAMRA-GGTTTCGGATGTTACAGCGTAGTATCACATCTTTGTACCCAC H16 PMA-06033-H16F PMA-06034-H16R TCACTTGCTTCCGTTGAGGTAAACTTCTCGTGCTAATCG TAMRA-GGTTTCGGATGTTACAGCGTGTCTTCAACTTGATCCCTTC N3 PMA-06049-N3F PMA-06050-N3R TCACTTGCTTCCGTTGAGGGGGAAAGARTGGATGCATGT TAMRA-GGTTTCGGATGTTACAGCGTGTTGTTGATTCTCATCCAAGG N4 PMA-06039-N4F PMA-06040-N4R TCACTTGCTTCCGTTGAGGGGAAGCAATCGACCATGGAT TAMRA-GGTTTCGGATGTTACAGCGTCGACACCCATCCATTAGCAT N5 PMA-06051-N5F PMA-06052-N5R TCACTTGCTTCCGTTGAGGACTGTTATTGGGTAATGACG TAMRA-GGTTTCGGATGTTACAGCGTTGCTTGTTTTGGTCCAACCG N6 PMA-06041-N6F PMA-06042-N6R TCACTTGCTTCCGTTGAGGACCTAATAACAATGCTTCGG TAMRA-GGTTTCGGATGTTACAGCGTCACTCTTCTATATGCTGTGC N7 PMA-06043-N7F PMA-06044-N7R TCACTTGCTTCCGTTGAGGTGTGCAGAGATAAYTGGCA TAMRA-GGTTTCGGATGTTACAGCGTCCGGAATAGCCTGACCAATT N8 PMA-06045-N8F PMA-06046-N8R TCACTTGCTTCCGTTGAGGGGGCAMTGATGTATGGATGG TAMRA-GGTTTCGGATGTTACAGCGTAAGAATAGCTCCATCGTGCC N9 PMA-06047-N9F PMA-06048-N9R TCACTTGCTTCCGTTGAGGTTCTATGCTCTCAGCCAAGG TAMRA--GGTTTCGGATGTTACAGCGTTGGCATACGCATTCAGATTC

Wherein TAMRA is a fluorescence dye.

2. the method for an asymmetric amplification avian influenza virus RNA, this method comprises the following steps:

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

2) multiple asymmetric RT-PCR amplification: with the RNA that extracts in the step 1) is that template is carried out multiple asymmetric RT-PCR amplification, wherein uses the described primer system of claim 3, and the reaction conditions of amplification is: 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; The fowl that amplified production can be used for being undertaken by nucleic acid hybridization is flowed the detection and the somatotype of virus.

3. method that is used for avian flu virus detection and somatotype, this method comprises the following steps:

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

2) multiple asymmetric RT-PCR amplification: with the RNA that extracts in the step 1) is that template is carried out multiple asymmetric RT-PCR amplification, wherein uses the described primer system of claim 3, and the reaction conditions of amplification is: 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) hybridization and washing: adding step 2) multiple asymmetric amplified production and the gene chip that obtains, be suitable for reacting time enough under the condition of hybridizing with selected gene chip, the washing back dries then;

4) result judges: chip is inserted carry out scanning analysis in the chip scanner, and saving result, then according to the micromatrix arrangement position that positive findings occurs, pairing avian influenza virus subtype when searching this micromatrix arrangement position point sample.

Images