CN107312872A - Differentiate multiple PCR primer group, kit and the detection method of H5 subtype avian influenza virus and its NA hypotypes simultaneously - Google Patents
Differentiate multiple PCR primer group, kit and the detection method of H5 subtype avian influenza virus and its NA hypotypes simultaneously Download PDFInfo
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Abstract
The invention discloses multiple PCR primer group, kit and the detection method for differentiating H5 subtype avian influenza virus and its NA hypotypes simultaneously.Parting and detection primer composition such as SEQ ID NO that the present invention is set up for 6 genes of M, H5, N1, N2, N6 and N8 gene of avian influenza virus:Shown in 1~12.Primer composition of the present invention and kit can differentiate H5 subtype avian influenza virus and its NA hypotypes simultaneously, and high specificity, sensitivity is high, cross reaction is small, reproducible, is detected by multiplexed PCR amplification, can be by the judgements of intuitively characteristic bands, to distinguish the H5 avian influenza virus of different NA hypotypes.The present invention can greatly reduce testing cost, and simple to operate, and the used time is brief, the investigation of prevalence virus and prevention and control to H5 subtype avian influenza virus, ensure that the healthy and sustainable development of aviculture is significant.
Description
Technical field
The invention belongs to field of molecular detection, H5 subtype avian influenza virus is differentiated simultaneously more particularly, to a kind of
And its multiple PCR primer group, kit and the detection method of NA hypotypes.
Background technology
Bird flu (Avian Influenza, AI) is a kind of birds acute infection as caused by avian influenza virus (AIV)
Disease, meanwhile, the avian influenza virus of some hypotypes can infect the mankind.Avian influenza virus is that minus strand single-stranded RNA virus mesh just glues disease
Malicious section's Influenzavirus A influenza virus, its genome is made up of 8 genetic fragments, respectively PB2, PB1, PA, HA, NP,
NA, M and NS, it is antigenic different from neuraminidase (NA) according to its glycoprotein hemagglutinin (HA), can be 16 kinds by AIV points
HA hypotypes (H1-H16), 9 kinds of NA hypotypes (N1-N9).Bird flu is caused a disease according to the pathogenic highly pathogenic bird flu that is divided into low
Property bird flu, part H5 and H7 are highly pathogenic bird flu, can result in the mortality of chicken group, particularly again and again quick-fried in recent years
The H5 epidemic situations of hair, the aquaculture to multiple countries and regions brings huge economic loss.In China, H5N1 subtype avian influenzas disease
Poison is isolated in Guangdong Province first in 1996, and H5N1 epidemic situations are broken out in subsequent 2003 Qinghai lake, cause substantial amounts of wild bird
Death, H5N1 propagate rapidly progressively spread to from Asia Europe and Africa including 56 countries and regions, recent years,
H5N2, H5N6, H5N8 subtype avian influenza virus also at home and abroad successive Outbreak in different regions, cause substantial amounts of poultry dead
Die and slaughtered.In addition, part H5 subtype avian influenza virus there occurs antigenic variation, the ability propagated across inter-species is obtained direct
Infection people simultaneously causes the case of death also to point out H5 hypotypes not only to bring huge injury to aquaculture, while still human world
Sanitarian huge potential safety hazard.
The quick detection and diagnostic method for setting up avian influenza virus not only facilitate early detection and symptom management, while
Be conducive to the monitoring of bird flu and the deposit of detection technique, the appearance for reply flu outbreak provides the Monitoring Data of early stage simultaneously
The effective precautionary measures are taken in time.The traditional detection method of bird flu includes the separation and identification of virus, and separation AIV's is conventional
Method has chick embryo culture partition method, HA the and NA hypotypes of virus can be further discriminated between additionally by Serologic detection, due to fowl
The numerous and new variant of influenza subtype continuously emerges, although virus purification and the goldstandard that identification is detection virus, but behaviour
Make cumbersome, detection cycle is long, and easily influenceed to make testing result inaccurate and timely by factors such as antibody, be unfavorable for examining for disease
Break and for the control of epidemic situation；With biotechnology increasingly renewal and develop rapidly, modern molecular biology means gradually by
For detecting AIV, such as real-time fluorescence quantitative PCR, ring mediated isothermal amplification method, gene chips and high-flux sequence, this
Although class method specificity and sensitiveness are preferably, the requirement for instrument and equipment is higher, popularizes not enough at present.Polymerization
PCR (PCR) is simple to operate, and instrument requirements are not high, time-consuming short, while the degree of accuracy is high, also there is good specificity
With sensitiveness, as a result more directly perceived, it is easy to judge, Sample storage is convenient, and inspection again is also convenient for, and the same day can complete, and set up multiple
PCR can amplify multiple target gene fragments simultaneously and then realize the quick diagnosis of multiple pathogens and reduce inspection cost.
At present, these four NA hypotypes are relatively conventional with N1, N2, N6 and N8 for the popular H5 subtype avian influenza virus of China,
And the outburst of the H5 avian influenza of difference NA hypotypes all has the characteristics of morbidity is anxious, propagation is fast, symptom is similar, thus it is only right with clinic
Specific NA hypotypes are not can determine that in the observation of the state of an illness, and the inspection in terms of more detection method at present lays particular emphasis on HA
Survey, for that can detect H5 HA with the multiple PCR method of its four kinds common NA hypotypes then there is not yet report simultaneously.
The content of the invention
It is an object of the invention to provide differentiate H5 subtype avian influenza virus and its multiple PCR primer of NA hypotypes simultaneously
Group, kit and detection method.
The technical solution used in the present invention is：
Differentiate H5 subtype avian influenza virus and its multiple PCR primer composition of NA hypotypes, its nucleotide sequence point simultaneously
It is not as follows：
For the primer of avian influenza virus M genes：
MU：5’-TTCTAACCGACGTCGAAAC-3’；
ML：5’-TTAGTCAGAGTTGACARGAT-3’；
For the primer of avian influenza virus H gene：
H5U：5’-ATGAACACTCARTATGAGG-3’；
H5L：5’-GCATTATCCYTAATCTGTAG-3’；
For the primer of avian influenza virus N1 genes：
N1U：5’-TTCGAGTCTGTTGCTTGGT-3’；
N1L：5’-GGAGTATTCCTCATAGTGAT-3’；
For the primer of avian influenza virus N2 genes：
N2U：5’-CAATTGGCTCTGYTTGTCT-3’；
N2L：5’-TGGTYCCCTGYCCAATTGC-3’；
For the primer of avian influenza virus N6 genes：
N6U：5’-TGCAATCAGAATAGGTGAGG-3’；
N6L：5’-TGCTTGTGTGCGTCATCRT-3’；
For the primer of avian influenza virus N8 genes：
N8U：5’-TTGATGCTGTRGCATGGTC-3’；
N8L：5’-CATGCGACTAGTGCATGAAC-3’；
Above-mentioned base R=A/G, Y=C/T.
Differentiate H5 subtype avian influenza virus and its kit of NA hypotypes simultaneously, it is characterised in that：Containing respectively for fowl
The primer of M, H5, N1, N2, N6 and N8 gene order design of influenza virus.
As the further preferred of mentioned reagent box, the nucleotide sequence of the primer is as follows：
For the primer of avian influenza virus M genes：
MU：5’-TTCTAACCGACGTCGAAAC-3’；
ML：5’-TTAGTCAGAGTTGACARGAT-3’；
For the primer of avian influenza virus H gene：
H5U：5’-ATGAACACTCARTATGAGG-3’；
H5L：5’-GCATTATCCYTAATCTGTAG-3’；
For the primer of avian influenza virus N1 genes：
N1U：5’-TTCGAGTCTGTTGCTTGGT-3’；
N1L：5’-GGAGTATTCCTCATAGTGAT-3’；
For the primer of avian influenza virus N2 genes：
N2U：5’-CAATTGGCTCTGYTTGTCT-3’；
N2L：5’-TGGTYCCCTGYCCAATTGC-3’；
For the primer of avian influenza virus N6 genes：
N6U：5’-TGCAATCAGAATAGGTGAGG-3’；
N6L：5’-TGCTTGTGTGCGTCATCRT-3’；
For the primer of avian influenza virus N8 genes：
N8U：5’-TTGATGCTGTRGCATGGTC-3’；
N8L：5’-CATGCGACTAGTGCATGAAC-3’；
Above-mentioned base R=A/G, Y=C/T.
As the further preferred of mentioned reagent box, primer pair MU and ML, primer pair H5U and H5L, primer pair N1U and
N1L, primer pair N2U and N2L, primer pair N6U and N6L, primer pair N8U and N8L mol ratio are (2~4)：(2~4)：(1~
2)：(1~2)：(2~4)：(1~2).
As the further preferred of mentioned reagent box, also containing reaction solution, archaeal dna polymerase, negative control.
As the further preferred of mentioned reagent box, reaction solution contains 10 × PCR buffer, 2.5mM MgCl2、2.5mM
DNTP, three's volume ratio is：2：2：2.5.
As the further preferred of mentioned reagent box, archaeal dna polymerase is Ex Taq polymerases.
Differentiate the detection method of H5 subtype avian influenza virus and its NA hypotypes, including following step while non-diseases is diagnosed
Suddenly：
(1) measuring samples nucleic acid is extracted；
(2) measuring samples nucleic acid is subjected to reverse transcription, obtains measuring samples cDNA；
(3) measuring samples cDNA is carried out as template using the primer in foregoing Primer composition or aforementioned agents box
Multiplexed PCR amplification；
(4) after gel electrophoresis, according to amplified fragments band and size, judged result：
If there is band in, at least band of 158bp, 215bp two then contains H5 subtype avian influenzas disease in sample
Poison；
If the band of 158bp, 215bp two, also characteristic bands of 324bp sizes then contain H5N1 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 396bp sizes then contain H5N2 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 635bp sizes then contain H5N6 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 503bp sizes then contain H5N8 in sample；
In addition to case above, it is judged as not containing H5 subtype avian influenza virus.
As the further preferred of above-mentioned detection method, 25 μ L system components of multiplexed PCR amplification are：Testing sample cDNA
1 μ L, 10 × PCR buffer 2 μ L, 2.5mM MgCl2The μ of 2 μ L, 2.5mM dNTP, 2 μ L, Ex Taq polymerase 0.25
L, the final concentration of 20pmol/uL of each primer, 25 μ L are complemented to RNA-free water.
As the further preferred of above-mentioned detection method, the condition of multiplexed PCR amplification is：94 DEG C of 3min, then 94 DEG C
30s, 54~56 DEG C of 30s, 72 DEG C of 1min, 30~35 circulations, 72 DEG C of 5~10min of extension.
The beneficial effects of the invention are as follows：
The parting and detection that the present invention is set up for 6 genes of M, H5, N1, N2, N6 and N8 gene of avian influenza virus are drawn
Compositions such as SEQ ID NO:Shown in 1~12.Primer composition of the present invention and kit can differentiate H5 subtype avian influenzas simultaneously
Virus and its NA hypotypes, high specificity, sensitivity are high, and cross reaction is small, reproducible, is detected, can led to by multiplexed PCR amplification
The judgement of intuitively characteristic bands is crossed, to distinguish the H5 avian influenza virus of different NA hypotypes.The present invention can greatly reduce detection
Cost, and it is simple to operate, and the used time is brief, the investigation of prevalence virus and prevention and control to H5 subtype avian influenza virus, ensures aviculture
Healthy and sustainable development it is significant.
Brief description of the drawings
Fig. 1：In the agarose gel electrophoresis figure of the multiplex PCR of clinical Simple infection H5 subtype avian influenza virus, figure, swimming lane
1 is H5N1, and swimming lane 2 is H5N2, and swimming lane 3 is H5N6, and swimming lane 4 is H5N8, and swimming lane 5 is Newcastle Disease Virus template, and swimming lane 6 is
DEPC water, swimming lane 7 is 100bp Ladder Marker；
Fig. 2：The agarose gel electrophoresis figure of the multiplex PCR of multi-template；In figure, swimming lane 1 and 2 is H5N1 and H5N2 mixing
Template reaction band, swimming lane 3 and 4 is H5N1 and H5N6 hybrid template reaction band, and swimming lane 5 and 6 is the mixed of H5N1 and H5N8
Shuttering reacts band, and swimming lane 7 and 8 is H5N2 and H5N6 hybrid template reaction band, and swimming lane 9 and 10 is H5N2's and H5N6
Hybrid template reacts band, and swimming lane 11 and 12 is H5N6 and H5N8 hybrid template reaction band, and swimming lane 13 is 100bp
Ladder Marker；
Fig. 3：Single primer amplification 105The sensitivity test result of~1pg/ μ L cDNA templates；The M that is identified in figure, H5,
N1, N2, N6, N8 are respectively corresponding single primer amplification result, and each single primer amplification corresponds to 10 respectively from left to right5~1pg/ μ L
CDNA templates, Marker be 100bp Ladder Marker；
Fig. 4：Primer composition amplification concentration is 105The sensitivity test result of~1pg/ μ L cDNA templates；In figure, swimming
Road M is 100bp Ladder Marker, and swimming lane is respectively 10 from left to right5~1pg/ μ L cDNA templates.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail.Reality used in following embodiments
Proved recipe method is conventional method unless otherwise specified.Material, reagent used in following embodiments etc., unless otherwise specified,
Commercially obtain.
Embodiment 1, while differentiating the design of the multiple PCR primer composition of H5 subtype avian influenza virus and its NA hypotypes
A large amount of corresponding sequences are downloaded in GenBank according to M, H5, N1, N2, N6 and N8 gene of avian influenza virus, to each
The sequence of genetic fragment is analyzed and compared, and finds out each genetic fragment with respect to conservative region, designs specific primer, then right
Designed primer is analyzed and screened, and multiple PCR primer composition (being shown in Table 1) of the present invention is obtained, by Shanghai
Invitrogen is synthesized, PAGE purifying.
Table 1, primer information
Primer information as shown in table 1, annexs base code R=A/G, Y=C/T, according to the poison of actually detected pathogen
The different actual amplified production length obtained to detection of strain can be expected on the length of amplified production fluctuation 3bp up and down.
Embodiment 2, while differentiating the kit of H5 subtype avian influenza virus and its NA hypotypes
Differentiate H5 subtype avian influenza virus and its kit of NA hypotypes simultaneously, including such as SEQ ID NO:Shown in 1~12
While differentiate the multiple PCR primer composition of H5 subtype avian influenza virus and its NA hypotypes, reaction solution, Ex Taq polymerases.
Primer pair MU and ML, primer pair H5U and H5L, primer pair N1U and N1L, primer pair in multiple PCR primer composition
N2U and N2L, primer pair N6U and N6L, primer pair N8U and N8L molar concentration rate are 2：2：1：1：2：1；Reaction solution contains 10
×PCR buffer、2.5mM MgCl2, 2.5mM dNTP, three's volume ratio is：2：2：2.5.
Embodiment 3, while differentiating the detection method of H5 subtype avian influenza virus and its NA hypotypes
(1) nucleic acid extraction：Viral nucleic acid is extracted using TRIzol viral nucleic acids extracting method, concretely comprised the following steps：750 μ L's
TRIzol and 250 μ L viral sample allantoic fluid are fully mixed, and stand 5min；Add 200 μ L chloroforms and fully mix standing 5min；
4 DEG C of 12000rpm centrifuge 10min, and upper honest and upright and thrifty 450 μ L are sucked into new clean EP manages, and adds 500 μ L isopropanol, stands
Abandoned after 20min, 4 DEG C of 12000rpm centrifugations 10min after supernatant, adherent addition 1ML 70% ice ethanol, 12000rpm centrifugations 5min
Supernatant is abandoned, 70% ice ethanol of addition is then repeated and washed once again, EP pipes are inverted to the RNA-free water that 25 μ L are added after drying
Elution, obtains viral sample nucleic acid.
(2) reverse transcription：Reverse transcription reaction system is carried out with reference to Promega company reverse transcriptase (article No. M1705) specification,
The viral sample nucleic acid of acquisition is subjected to reverse transcription according to following reaction system and reaction condition, viral sample cDNA is obtained；Instead
Responsive transcription system：μ L, 50mmol/L the Random Primer (9mer) of 5 × Reverse Transcriptase Buffer 8
μ L, the 5U/ μ L MLV of 4 μ L, 40U Ribonuclease Inhibitor of 2mL, dNTP Mixture (10mM/L) 0.5
The μ L of Reverse Transcriptase 1, the μ L of viral nucleic acid 25；42 DEG C of reverse transcription temperature, reacts 1.5h.
(3) multiplexed PCR amplification：It is 25 μ L by multi-PRC reaction cumulative volume by viral sample cDNA and the article of kit
Configure, system component is：1 μ L, 10 × PCR buffer of viral sample cDNA 2 μ L, 2.5mM MgCl22 μ L, 2.5mM
μ L, the SEQ ID NO of 2 μ L, Ex Taq polymerase of dNTP 0.25:Each primer of primer shown in 1~12 is final concentration of
20pmol/uL, 25 μ L are complemented to RNA-free water；Reaction condition is：94 DEG C of 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C
1min, 30 circulations, 72 DEG C of extension 5min are placed in 4 DEG C.
(4) Ago-Gel of preparation 1.5%, treats that gelling is solid, takes each μ L of amplified production 5 and 16 × loading of μ L
Buffer, which is mixed, adds loading hole, 100bp Ladder Marker, 110V, 40min is added at end, in gel imager
Observe each amplified fragments；According to amplified production clip size judged result：
If there is band in, at least band of 158bp, 215bp two then contains H5 subtype avian influenzas disease in sample
Poison；
If the band of 158bp, 215bp two, also characteristic bands of 324bp sizes then contain H5N1 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 396bp sizes then contain H5N2 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 635bp sizes then contain H5N6 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 503bp sizes then contain H5N8 in sample；
In addition to case above, it is judged as not containing H5 subtype avian influenza virus.
Embodiment 4, specific test
Respectively using the single positive sample of many plants of known viruse nucleic acid as template, including H5N1, H5N2, H5N6, H5N8,
Set control be：Newcastle Disease Virus template；Negative control：DEPC water, is carried out multiple using the Primer composition of embodiment 1
PCR is expanded.
When multiple PCR primer composition is detected to single template, as shown in figure 1, template be H5N1, H5N2, H5N6,
H5N8 has amplified M, H5 and respective NA three band, wherein M correspondence 158bp, H5 correspondence 215bp, N1 correspondences respectively
324bp, N2 correspondence 396bp, N6 correspondence 635bp, N8 correspondence 503bp band, Newcastle Disease Virus template and DEPC water are equal
Without any band.
Respectively using the mixing cDNA samples of a variety of cause of diseases as template, including H5N1 and H5N2 hybrid template, H5N1 with
H5N6 hybrid template, H5N1 and H5N8 hybrid template, H5N2 and H5N6 hybrid template, H5N2 and H5N6 hybrid guided mode
The hybrid template of plate, H5N6 and H5N8, multiplexed PCR amplification is carried out using the Primer composition of embodiment 1..
When multiple PCR primer composition is detected to hybrid template, as shown in Fig. 2 H5N1, H5N2, H5N6, H5N8 two
The hybrid template amplification of two combinations produces 4 bands, and hybrid template is not in interference effect in amplification.
Above result of the test shows：The multiple PCR primer combination single template of analyte detection and hybrid template of the present invention is special
Different in nature strong, cross reaction is small between primer, can be flowed the H5 fowl of distinguishing different NA hypotypes by the judgements of intuitively characteristic bands
Influenza Virus.
Embodiment 5, sensitivity and replica test
After reverse transcription, cDNA template concentrations are determined using nucleic acid-protein content meter, then cDNA templates are carried out
The serial dilution of 10 times of multiple proportions, it is 10 to obtain concentration5~1pg/ μ L cDNA templates, are entered with single primer and Primer composition respectively
Performing PCR is expanded, and is tested and be not repeated 3 times the nucleic acid concentration that determines each DNA profiling on the same day, is expanded with template highest extension rate and is in
The positive is its PCR susceptibility.
Single primer amplification result is as shown in figure 3, integrate each primer sensitivity, and the minimum nucleic acid concentration that can be detected is about
100pg/uL；
Primer composition amplification as shown in figure 4, for M, H5, N1, N2, N6, N8 gene it is minimum can detect to
100pg/ μ L sample of nucleic acid.
Above result of the test shows：The multiplex PCR list primer and Primer composition of the present invention has 100pg/ μ L detection
Sensitivity, and primer combination after interfere it is small, repeatability it is excellent.
Embodiment 6, clinical sample are examined
The detection of avian influenza virus multiplex PCR is carried out to clinical 50 parts of samples, including 36 parts of pathological material of diseases and 14 parts of cotton swabs.
The testing result of table 2, clinical sample
The testing result of clinical sample is as shown in table 2, in 50 clinical samples, 18 parts of negative sample, 32 parts of positive, its
In 1 part of H5N1,10 parts of H5N2,20 parts of H5N6,1 part of H5N8.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou City Huanan Nonda Biology Medicine Co., Ltd
<120>Differentiate multiple PCR primer group, kit and the detection side of H5 subtype avian influenza virus and its NA hypotypes simultaneously
Method
<130>
<160> 12
<170> PatentIn version 3.5
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Claims (10)
1. differentiate H5 subtype avian influenza virus and its multiple PCR primer composition of NA hypotypes simultaneously, its nucleotide sequence difference
It is as follows：
For the primer of avian influenza virus M genes：
MU：5’-TTCTAACCGACGTCGAAAC-3’；
ML：5’-TTAGTCAGAGTTGACARGAT-3’；
For the primer of avian influenza virus H5 genes：
H5U：5’-ATGAACACTCARTATGAGG-3’；
H5L：5’-GCATTATCCYTAATCTGTAG-3’；
For the primer of avian influenza virus N1 genes：
N1U：5’-TTCGAGTCTGTTGCTTGGT-3’；
N1L：5’-GGAGTATTCCTCATAGTGAT-3’；
For the primer of avian influenza virus N2 genes：
N2U：5’-CAATTGGCTCTGYTTGTCT-3’；
N2L：5’-TGGTYCCCTGYCCAATTGC-3’；
For the primer of avian influenza virus N6 genes：
N6U：5’-TGCAATCAGAATAGGTGAGG-3’；
N6L：5’-TGCTTGTGTGCGTCATCRT-3’；
For the primer of avian influenza virus N8 genes：
N8U：5’-TTGATGCTGTRGCATGGTC-3’；
N8L：5’-CATGCGACTAGTGCATGAAC-3’；
Above-mentioned base R=A/G, Y=C/T.
2. differentiate H5 subtype avian influenza virus and its kit of NA hypotypes simultaneously, it is characterised in that：Flowed containing fowl is directed to respectively
The primer of M, H5, N1, N2, N6 and N8 gene order design of Influenza Virus.
3. kit according to claim 2, it is characterised in that：The nucleotide sequence of the primer is as follows：
For the primer of avian influenza virus M genes：
MU：5’-TTCTAACCGACGTCGAAAC-3’；
ML：5’-TTAGTCAGAGTTGACARGAT-3’；
For the primer of avian influenza virus H gene：
H5U：5’-ATGAACACTCARTATGAGG-3’；
H5L：5’-GCATTATCCYTAATCTGTAG-3’；
For the primer of avian influenza virus N1 genes：
N1U：5’-TTCGAGTCTGTTGCTTGGT-3’；
N1L：5’-GGAGTATTCCTCATAGTGAT-3’；
For the primer of avian influenza virus N2 genes：
N2U：5’-CAATTGGCTCTGYTTGTCT-3’；
N2L：5’-TGGTYCCCTGYCCAATTGC-3’；
For the primer of avian influenza virus N6 genes：
N6U：5’-TGCAATCAGAATAGGTGAGG-3’；
N6L：5’-TGCTTGTGTGCGTCATCRT-3’；
For the primer of avian influenza virus N8 genes：
N8U：5’-TTGATGCTGTRGCATGGTC-3’；
N8L：5’-CATGCGACTAGTGCATGAAC-3’；
Above-mentioned base R=A/G, Y=C/T.
4. kit according to claim 3, it is characterised in that：Primer pair MU and ML, primer pair H5U and H5L, primer pair
N1U and N1L, primer pair N2U and N2L, primer pair N6U and N6L, primer pair N8U and N8L mol ratio are (2~4)：(2~4)：
(1~2)：(1~2)：(2~4)：(1~2).
5. kit according to claim 2, it is characterised in that：Also contain reaction solution, archaeal dna polymerase, negative control.
6. kit according to claim 5, it is characterised in that：Reaction solution contains 10 × PCR buffer, 2.5mM
MgCl2, 2.5mM dNTP, three's volume ratio is：2：2：2.5.
7. kit according to claim 5, it is characterised in that：Archaeal dna polymerase is Ex Taq polymerases.
8. differentiating the detection method of H5 subtype avian influenza virus and its NA hypotypes while non-diseases is diagnosed, comprise the following steps：
(1) measuring samples nucleic acid is extracted；
(2) measuring samples nucleic acid is subjected to reverse transcription, obtains measuring samples cDNA；
(3) measuring samples cDNA is utilized into any one of the Primer composition or claim 2~7 of claim 1 as template
Primer in kit carries out multiplexed PCR amplification；
(4) after gel electrophoresis, according to amplified fragments band and size, judged result：
If there is band in, at least band of 158bp, 215bp two then contains H5 subtype avian influenza virus in sample；
If the band of 158bp, 215bp two, also characteristic bands of 324bp sizes then contain H5N1 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 396bp sizes then contain H5N2 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 635bp sizes then contain H5N6 in sample；
If the band of 158bp, 215bp two, also characteristic bands of 503bp sizes then contain H5N8 in sample；
In addition to case above, it is judged as not containing H5 subtype avian influenza virus.
9. detection method according to claim 8, it is characterised in that：25 μ L system components of multiplexed PCR amplification are：It is to be measured
1 μ L, 10 × PCR buffer of sample cDNA 2 μ L, 2.5mM MgCl22 μ L, 2.5mM dNTP 2 μ L, Ex Taq
Polymerase 0.25 μ L, the final concentration of 20pmol/uL of each primer, 25 μ L are complemented to RNA-free water.
10. detection method according to claim 8, it is characterised in that：The condition of multiplexed PCR amplification is：94 DEG C of 3min, so
94 DEG C of 30s, 54~56 DEG C of 30s, 72 DEG C of 1min, 30~35 circulations afterwards, 72 DEG C extend 5~10min.
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