CN107529560A - Avian influenza virus H7N9 hypotype fluorescence RT PCR primers group, probe groups, kit and method - Google Patents
Avian influenza virus H7N9 hypotype fluorescence RT PCR primers group, probe groups, kit and method Download PDFInfo
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Abstract
The invention provides a kind of avian influenza virus H7N9 hypotypes fluorescence RT PCR primers group and the probe groups being used cooperatively with the primer sets, universal primer and probe including being directed to H7 antigens, the specific primer of N9 ANTIGEN DESIGNThes and probe and avian influenza virus respectively, the a large amount of, fragment of high special can be amplified, dual RT PCR amplifications are realized by once-through operation, the detection to avian influenza virus and H7N9 hypotypes is realized according to the collection change of different fluorescence signals.Present invention also offers a kind of avian influenza virus H7N9 hypotypes fluorescence RT PCR kits and corresponding application method, the cyst membrane and nucleocapsid destroyed by the agent of nucleic acid targeted release outside virus, nucleoid RNA is discharged, and suitable liquid environment is provided for RT PCR reactions by PCR mixed liquors, in order to follow-up detection and analysis.Product provided by the invention and method have the advantages that it is sensitive, special, stably, easy, favorable repeatability, carry out extensive Pathogen test suitable for a line quarantine functionary.
Description
Technical field
The invention belongs to virus detection techniques field, more particularly to a kind of avian influenza virus H7N9 hypotype fluorescence RT-PCRs
Primer sets, probe groups, kit and method.
Background technology
Avian influenza virus, belong to influenza A virus, the pathogenic difference according to avian influenza virus to chicken and turkey,
It is divided into high, medium and low/non-pathogenic three-level.Due to avian influenza virus hemagglutinin structure the features such as, nonspecific infection birds, work as disease
Poison in a replication process match somebody with somebody again by producer, causes structure to change, and obtains the ability of infection people, is only possible to cause people to feel
Contaminate the generation of bird flu disease.So far find that the avian influenza virus subtype of energy direct infection people has：H5N1、H7N1、 H7N2、
H7N3, H7N7, H9N2 and H7N9 hypotype.Wherein, highly pathogenic H5N1 hypotypes and in March, 2013 find first on human body
New bird flu H7N9 hypotypes are particularly noticeable, not only cause the injures and deaths of the mankind, while inflicted heavy losses on poultry farming.
The method of diagnosis H7N9 subtype avian influenza virus is mainly virus purification, serological test and general both at home and abroad at present
Logical RT-PCR, substance fluorescence RT-PCR etc..Virus purification, the serological diagnostic method of classics are wasted time and energy, and are unsuitable for clinical inspection
Survey, do not meet the requirement of bio-safety regulations, the requirement of quick detection can not be met.Regular-PCR is not simply failed to pathological material of disease sample
In virus carry out quantitative detection, and electrophoresis detection is needed after amplified reaction, is not only unsuitable for the screening of high-volume sample, and
And the easy false positive caused by operational pollution.Substance fluorescence RT-PCR, it is only capable of examining bird flu H7 hypotypes or N9 hypotypes
Survey.Double PCR, which is once tested, has completed double fluorescent RT-PCR that avian influenza virus H7 hypotypes and N9 hypotypes tentatively confirm also
Progressively develop, but its difficult point is the control to the specificity, sensitiveness of dual amplification.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of avian influenza virus H7N9 hypotype fluorescence RT-PCRs to draw
Thing group, probe groups, kit and method.
Concrete technical scheme of the present invention is as follows：
The invention provides a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR primer sets, including following primer：
H7-F：5'-TCACAGCAAATACAGGGAAGAG-3'(is as shown in SEQ ID NO.1)；
H7-R：5'-CCCGAAGCTAAACCAGAGTATC-3'(is as shown in SEQ ID NO.2)；
N9-F：5'-TAGCAATGACACACACTAGTCAAT-3'(is as shown in SEQ ID NO.3)；
N9-R：5'-ATTACCTGGATAAGGGTCATTACACT-3'(is as shown in SEQ ID NO.4)；
HA-F：5'-CTTCTAACCGAGGTCGAAACGTA-3'(is as shown in SEQ ID NO.5)；
HA-R：5'-GGTGACAGGATTGGTCTTGTCTTTA-3'(is as shown in SEQ ID NO.6).
H7-F, H7-R, N9-F and N9-R primer are to be directed to H7 antigens and N9 ANTIGEN DESIGNThes respectively, are respectively provided with good
Specificity, the fragment of high special can be amplified, and impurity is few, is also provided convenience for follow-up identification operation；
HA-F, HA-R primer are the universal primers of avian influenza virus, all avian influenza virus are respectively provided with good expanding effect, together
When to non-avian influenza virus also have height specificity.
Present invention also offers a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR probe groups, it is characterised in that including
Following probe：
H7-P5：5'-ROX-TGACCCAGTCAAACTAAGCAGCGG-MGB-3'(is as shown in SEQ ID NO.7)；
N9-p7：5'-JOE-AGACAATCCCCGACCGAATGACCC-MGB-3'(is as shown in SEQ ID NO.8).
HA-P2：5'-CY5-TCAGGCCCCCTCAAAGCCGAG-MGB-3'(is as shown in SEQ ID NO.9).
Above-mentioned probe is universal for H7 antigentic specificities primer, N9 antigentic specificities primer and bird flu respectively
Design of primers, different fluorescence labelings is connected to, is used in combination to foregoing corresponding primer, passes through different fluorescence signals
Collection change realize detection to avian influenza virus and H7N9 hypotypes.
Invention additionally provides a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including above-mentioned primer
Group and probe groups.
Further, the kit also includes PCR mixed liquors, and the PCR mixed liquors include following composition：
PH is 8.8 250~300mM of bicine/KOH, 0.8~1M of KCL, 25~30mM of MnCl2 and volume fraction
For 40% glycerine.
Mn2+ is added in mixed liquor as confactor, suitable working environment can be provided for reverse transcriptase, be easy to
It plays reverse transcriptase activity
Further, following composition is also included in the PCR mixed liquors：
1~1.2mM of dNTP and reverse transcriptase 0.1~0.12U/ μ L.
DNTP, reverse transcriptase are all added in PCR mixed liquors, can directly use, avoid as an entirety
Various ingredients need to add respectively cumbersome during experiment every time, and can avoid causing reagent dirt because repeatedly opening, repeatedly drawing
Dye or failure.
Further, the primer sets and the probe groups are added in the PCR mixed liquors, in the primer sets
Concentration of each primer in the PCR mixed liquors is 2.5~3 μM, and each probe is in the PCR mixed liquors in the probe groups
In concentration be 1.25~1.5 μM.
Primer and probe is all added in PCR mixed liquors, can also directly use, avoid more as an entirety
It is secondary to take the substantial amounts of time and efforts of consumption, and the pollution of primer and probe can be avoided to greatest extent.
Further, following composition is also included in the PCR mixed liquors：
10~15mM of 20~30mM of phytic acid and methionine.
Phytic acid and methionine are respectively provided with good anti-oxidation function, and PCR mixed liquor characteristicses stabilization, extension can be kept to make
With the time limit, and property is gentle, nonhazardous effect.
Further, the kit also includes nucleic acid targeted release agent, and nucleic acid targeted release agent described in per mL is included such as
Lower composition：
40mg bovine serum albumin(BSA)s, 8 μ L Tween-20s, 17.5mg KCL, 0.5mg Proteinase Ks and 20mg NaOH.
The effect of nucleic acid targeted release agent is by the cyst membrane outside virus and nucleocapsid destruction, and nucleoid RNA is discharged
Come, in order to subsequent operation.
Invention further provides a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR method, comprise the following steps：
S1：Isometric nucleic acid targeted release agent is added into testing sample, is gently mixed；The sample mixed is existed
10min is heated at 99 DEG C；After heating terminates, sample is placed in 2~3min of balance at room temperature, it is standby as template after centrifugation；
S2：RT-PCR amplifications are carried out to obtained RNA sample, respectively to universal Flu-A, H7 hypotypes and N9 hypotypes
Carry out fluorescence signal detection.
Further, in the step S2, the reaction system of the RT-PCR mainly includes following composition：
Template ribonucleic acid 0.2ng/ μ L, sense primer are each 0.5 μM, anti-sense primer is each 0.5 μM, probe is each 0.25 μM, dNTP
0.2mM and reverse transcriptase 0.02U/ μ L；
The reaction condition of the RT-PCR is as follows：
50℃10min；95℃2min；95 DEG C of 15s, 60 DEG C of 35s, totally 6 circulations；95 DEG C of 15s, 60 DEG C of 35s, totally 40
Circulation.
When being detected using quantitative real time PCR Instrument, the universal Flu-A fluorescence signal of CY5 Air conduct measurements is selected
(5'-CY5,3'-BHQ)；Select ROX Air conduct measurement bird flu H7 hypotypes fluorescence signals (5'-ROX, 3'-BHQ)；Selection JOE leads to
Road detection bird flu N9 gene by fluorescence signal (5'-JOE, 3'-BHQ).Determination methods are as follows：
(1) sample fluorescence passage (CY5) CT value≤35 are detected, report Flu-A is positive；Detect sample fluorescence passage
(ROX) CT value≤35, detection sample fluorescence passage (JOE) CT value≤35, report bird flu H7N9 hypotypes are positive；
(2) sample fluorescence passage (CY5) CT value≤35 are detected, report Flu-A is positive；Detect sample fluorescence passage
(ROX) CT value≤35, sample fluorescence passage (JOE) CT values are detected>40, report bird flu H7 hypotype are positive；
(3) sample fluorescence passage (CY5/JOE/ROX) 35 ＜ CT value≤40 are detected to be suspected to be first stream/H7N9 bird flus, are needed
Again detect, if 35 ＜ CT value≤40 are detected again, and amplification curve has obvious Exponential growth stage, while negative control CT
It is worth for No CT, the positive can be reported as, be otherwise feminine gender；
(4) it is No CT to detect sample fluorescence passage (CY5/JOY/ROX) CT values, is reported as feminine gender.
Beneficial effects of the present invention are as follows：The invention provides a kind of avian influenza virus H7N9 hypotype fluorescence RT-PCRs to draw
Thing group and the probe groups being used cooperatively with the primer sets, primer sets include respectively for H7 antigens, N9 ANTIGEN DESIGNThes it is special
Property primer and avian influenza virus universal primer, a large amount of, fragment of high special can be amplified, and impurity is few,
Provided convenience for follow-up identification operation；Universal probe, H7 probes and N9 probes are connected to different fluorescence
Mark, is expanded by dual RT-PCR, is realized according to the collection of different fluorescence signals change to avian influenza virus and H7N9 hypotypes
Detection.
Present invention also offers a kind of avian influenza virus H7N9 hypotype fluorescence RT-PCR kits and corresponding user
Method, the cyst membrane and nucleocapsid destroyed by the agent of nucleic acid targeted release outside virus, nucleoid RNA is discharged, and pass through PCR
Mixed liquor provides suitable liquid environment for RT-PCR reactions, in order to follow-up detection and analysis.Drawn by provided by the invention
Thing group, probe groups, kit and method to avian influenza virus H7N9 hypotypes carry out RT-PCR detections, have it is sensitive, special,
The advantages that stable, simplicity, favorable repeatability, extensive Pathogen test is carried out suitable for a line quarantine functionary.
Brief description of the drawings
Fig. 1 is the testing result of HA gradient dilutions in experimental example 1；
Fig. 2 is the standard curve of HA gradient dilutions in experimental example 1；
Fig. 3 is the testing result of H7 gradient dilutions in experimental example 1；
Fig. 4 is the standard curve of H7 gradient dilutions in experimental example 1；
Fig. 5 is the testing result of N9 gradient dilutions in experimental example 1；
Fig. 6 is the standard curve of N9 gradient dilutions in experimental example 1；
Fig. 7 is 10 repetition testing results of HA in experimental example 2；
Fig. 8 is 10 repetition testing results of H7 in experimental example 2；
Fig. 9 is 10 repetition testing results of N9 in experimental example 2.
Embodiment
The present invention is described in further detail with following examples below in conjunction with the accompanying drawings.
Embodiment 1
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR primer sets, including following primer：
H7-F：5'-TCACAGCAAATACAGGGAAGAG-3'；
H7-R：5'-CCCGAAGCTAAACCAGAGTATC-3'；
N9-F：5'-TAGCAATGACACACACTAGTCAAT-3'；
N9-R：5'-ATTACCTGGATAAGGGTCATTACACT-3'；
HA-F：5'-CTTCTAACCGAGGTCGAAACGTA-3'；
HA-R：5'-GGTGACAGGATTGGTCTTGTCTTTA-3'.
Embodiment 2
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR probe groups, including following probe：
H7-P5：5'-ROX-TGACCCAGTCAAACTAAGCAGCGG-MGB-3'；
N9-p7：5'-JOE-AGACAATCCCCGACCGAATGACCC-MGB-3'；
HA-P2：5'-CY5-TCAGGCCCCCTCAAAGCCGAG-MGB-3'.
Embodiment 3
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2.
Embodiment 4
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2, in addition to PCR mixed liquors, the PCR mixed liquors include following composition：
The bicine/KOH 250mM, KCL 1M, MnCl that PH is 8.8225mM and the glycerine that volume fraction is 40%.
Embodiment 5
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2, in addition to PCR mixed liquors, the PCR mixed liquors include following composition：
The bicine/KOH 300mM, KCL 0.8M, MnCl that PH is 8.8230mM and volume fraction be 40% it is sweet
Oil.
Embodiment 6
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2, in addition to the PCR mixed liquors described in embodiment 4, also include following composition in the PCR mixed liquors：
DNTP 1mM and reverse transcriptase 0.1U/ μ L.
Embodiment 7
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2, in addition to the PCR mixed liquors described in embodiment 5, also include following composition in the PCR mixed liquors：
DNTP 1.2mM and reverse transcriptase 0.12U/ μ L.
Embodiment 8
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2, in addition to the PCR mixed liquors described in embodiment 6, primer sets and probe groups are added to PCR mixed liquors
In, concentration of each primer in PCR mixed liquors is 2.5 μM in primer sets, and each probe is in PCR mixed liquors in probe groups
Concentration is 1.25 μM.
Embodiment 9
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the primer sets described in embodiment 1 and implementation
Probe groups described in example 2, in addition to the PCR mixed liquors described in embodiment 7, primer sets and probe groups are added to PCR mixed liquors
In, concentration of each primer in PCR mixed liquors is 3 μM in primer sets, and each probe is dense in PCR mixed liquors in probe groups
Degree is 1.5 μM.
Embodiment 10
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the PCR mixed liquors described in embodiment 8, institute
Stating also includes following composition in PCR mixed liquors：
Phytic acid 30mM and methionine 10mM.
Embodiment 11
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the PCR mixed liquors described in embodiment 9, institute
Stating also includes following composition in PCR mixed liquors：
Phytic acid 20mM and methionine 15mM.
Embodiment 12
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the PCR mixed liquors described in embodiment 6, also
Including nucleic acid targeted release agent, nucleic acid targeted release agent described in per mL includes following composition：
40mg bovine serum albumin(BSA)s, 8 μ L Tween-20s, 17.5mg KCL, 0.5mg Proteinase Ks and 20mg NaOH.
Embodiment 13
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, including the PCR mixed liquors described in embodiment 9, also
Including nucleic acid targeted release agent, nucleic acid targeted release agent described in per mL includes following composition：
40mg bovine serum albumin(BSA)s, 8 μ L Tween-20s, 17.5mg KCL, 0.5mg Proteinase Ks and 20mg NaOH.
Embodiment 14
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR method, primer sets, the embodiment 2 of the offer of Application Example 1
The kit that the probe groups and embodiment 12 of offer provide, specifically comprises the following steps：
S1.1：Isometric nucleic acid targeted release agent is added into testing sample, is gently mixed；The sample that will be mixed
10min is heated at 99 DEG C；After heating terminates, sample is placed in 2~3min of balance at room temperature, it is standby as template after centrifugation；
S2：RT-PCR amplifications are carried out to obtained RNA sample, respectively to universal Flu-A, H7 hypotypes and N9 hypotypes
Fluorescence signal detection is carried out, wherein the reaction system of the RT-PCR includes following composition：
Template ribonucleic acid 0.2ng/ μ L, sense primer (HA-F, H7-F, N9-F) are each 0.5 μM, anti-sense primer (HA-R, H7-R,
N9-R) each 0.5 μM, probe (HA-P2, H7-P5, N9-P7) each 0.25 μM and volume fraction be 20% PCR mixed liquors, use
Deionized water complements to 25 μ L；
The reaction condition of the RT-PCR is as follows：
50℃10min；95℃2min；95 DEG C of 15s, 60 DEG C of 35s, totally 6 circulations；95 DEG C of 15s, 60 DEG C of 35s, totally 40
Circulation.
Embodiment 15
A kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR method, the kit that Application Example 13 provides, specific bag
Include following steps：
S1.1：Isometric nucleic acid targeted release agent is added into testing sample, is gently mixed；The sample that will be mixed
10min is heated at 99 DEG C；After heating terminates, sample is placed in 2~3min of balance at room temperature, it is standby as template after centrifugation；
S2：RT-PCR amplifications are carried out to obtained RNA sample, respectively to universal Flu-A, H7 hypotypes and N9 hypotypes
Fluorescence signal detection is carried out, wherein the reaction system of the RT-PCR includes following composition：
Template ribonucleic acid 0.2ng/ μ L, the μ L of PCR mixed liquors 5,25 μ L are complemented to deionized water；
The reaction condition of the RT-PCR is as follows：
50℃10min；95℃2min；95 DEG C of 15s, 60 DEG C of 35s, totally 6 circulations；95 DEG C of 15s, 60 DEG C of 35s, totally 40
Circulation.
Reference examples 1
Using bird flue virus H 5 N 1 subtype RNA as template, fluorescence RT-PCR inspection is carried out using the method described in embodiment 14
Survey.
Reference examples 2
Using avian influenza virus H7N7 hypotypes RNA as template, fluorescence RT-PCR inspection is carried out using the method described in embodiment 15
Survey.
Reference examples 3
Using avian influenza virus H5N9 hypotypes RNA as template, fluorescence RT-PCR inspection is carried out using the method described in embodiment 14
Survey.
Reference examples 4
Using the geneome RNA of I types duck hepatitis virus (DHV) as template, fluorescence is carried out using the method described in embodiment 15
RT-PCR is detected.
Reference examples 5
Using the geneome RNA of NDV (NDV) as template, fluorescence RT- is carried out using the method described in embodiment 14
PCR is detected.
Reference examples 6
Using the geneome RNA of duck tembusu virus (DTMUV) as template, carried out using the method described in embodiment 15 glimmering
Light RT-PCR is detected.
Experimental example 1
The making of standard curve
Choose avian influenza virus H7N9 positives and carry out gradient dilution, respectively obtain 106、105、 104、103And 102
The sample of the order of magnitude, and the avian influenza virus H7N9 hypotype fluorescence RT-PCR methods that Application Example 14 provides are to above-mentioned sample
Detected respectively, while negative control is set, make universal amplification, H7 amplifications and N9 amplifications respectively of testing result
Standard curve.
As shown in figs. 1 to 6, the standard curve of universal amplification, H7 amplifications and N9 amplifications is linear good for experimental result
Good, coefficient correlation reaches more than 0.99.This shows that detection method stability provided by the invention is good, reliability is high.
Experimental example 2
Repeated experiment
Choose avian influenza virus H7N9 positives, the avian influenza virus H7N9 hypotype fluorescence that Application Example 15 provides
RT-PCR method, detection 10 times is repeated, and compare the difference of 10 testing results.
As shown in figs. 7-9, ten secondary response result curves of universal amplification, H7 amplifications and N9 amplifications are equal for experimental result
Highly overlap.Show that primer provided by the invention has good specificity, experimental method repeatability is preferable, suitable for reply
Extensive sample detection.
Experimental example 3
Specific test
Respectively Application Example 14 and 15 provide avian influenza virus H7N9 hypotype fluorescence RT-PCR methods, to H5N1,
6 kinds of viruses of H7N7, H5N9, DHV, NDV, DTMUV synchronize detection, while set up H7N9 positive controls and blank control,
Examine the specificity of this method.
Experimental result shows that only the testing result of H7N9 positive controls is the positive, other 6 kinds of viruses and negative control
Testing result be feminine gender.Illustrate that this method specificity is good, be not likely to produce false positive results.
Experimental example 4
Sensitivity tests
By the H7N9 of purifying with PBS dilute to obtain 1000copies/mL, 800copies/mL, 500copies/mL,
300copies/mL, 200copies/mL and 100copies/mL, the avian influenza virus H7N9 provided using embodiment 14 are sub-
Type fluorescence RT-PCR method is detected respectively, analyzes the sensitiveness of this method.
Test result indicates that result is still positive when H7N9 concentration is 300copies/mL, and when concentration is
Result is then feminine gender during 200copies/mL, and 300copies/mL, sensitiveness can be reached by illustrating the limit of identification of this method
Preferably.
Experimental example 5
Coincidence rate is tested
102 parts of clinical anus swab samples are taken, the avian flu that the traditional RT-PCR method of application and embodiment 15 provide respectively
Above-mentioned anus swab samples are detected by malicious H7N9 hypotypes fluorescence RT-PCR method, are compared testing result and are determined for compliance with rate.
Experimental result shows that the coincidence rate of negative sample and positive is 100%, batch in and batch between CT values variation
Coefficient≤5%.Show this method repeatability preferably, be adapted to analyze batch samples.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the present invention's
Protection domain.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
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Claims (10)
1. a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR primer sets, it is characterised in that including following primer：
H7-F：5'-TCACAGCAAATACAGGGAAGAG-3'；
H7-R：5'-CCCGAAGCTAAACCAGAGTATC-3'；
N9-F：5'-TAGCAATGACACACACTAGTCAAT-3'；
N9-R：5'-ATTACCTGGATAAGGGTCATTACACT-3'；
HA-F：5'-CTTCTAACCGAGGTCGAAACGTA-3'；
HA-R：5'-GGTGACAGGATTGGTCTTGTCTTTA-3'.
2. a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR probe groups, it is characterised in that including following probe：
H7-P5：5'-ROX-TGACCCAGTCAAACTAAGCAGCGG-MGB-3'；
N9-P7：5'-JOE-AGACAATCCCCGACCGAATGACCC-MGB-3'；
HA-P2：5'-CY5-TCAGGCCCCCTCAAAGCCGAG-MGB-3'.
3. a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit, it is characterised in that including drawing described in claim 1
Probe groups described in thing group and claim 2.
4. a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit as claimed in claim 3, it is characterised in that also wrap
PCR mixed liquors are included, the PCR mixed liquors include following composition：PH is 8.8 250~300mM of bicine/KOH, KCL 0.8
~1M, MnCl225~30mM and the glycerine that volume fraction is 40%.
5. avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit as claimed in claim 4, it is characterised in that the PCR
Also include following composition in mixed liquor：
1~1.2mM of dNTP and reverse transcriptase 0.1~0.12U/ μ L.
6. a kind of avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit as claimed in claim 5, it is characterised in that described
Primer sets and the probe groups are added in the PCR mixed liquors, and each primer is in the PCR mixed liquors in the primer sets
Concentration be 2.5~3 μM, concentration of each probe in the PCR mixed liquors is 1.25~1.5 μM in the probe groups.
7. avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit as claimed in claim 6, it is characterised in that the PCR
Also include following composition in mixed liquor：
10~15mM of 20~30mM of phytic acid and methionine.
8. avian influenza virus H7N9 hypotypes fluorescence RT-PCR kit as claimed in claim 4, it is characterised in that also including core
For acid cut to releasing agent, nucleic acid targeted release agent described in per mL includes following composition：
40mg bovine serum albumin(BSA)s, 8 μ L Tween-20s, 17.5mg KCL, 0.5mg Proteinase Ks and 20mg NaOH.
A kind of 9. avian influenza virus H7N9 hypotypes fluorescence RT-PCR method, it is characterised in that comprise the following steps：
S1：Isometric nucleic acid targeted release agent is added into testing sample, is gently mixed；By the sample mixed at 99 DEG C
Heat 10min；After heating terminates, sample is placed in 2~3min of balance at room temperature, it is standby as template after centrifugation；
S2：RT-PCR amplifications are carried out to obtained RNA sample, universal Flu-A, H7 hypotypes and N9 hypotypes carried out respectively
Fluorescence signal detects.
10. avian influenza virus H7N9 hypotypes fluorescence RT-PCR method as claimed in claim 9, it is characterised in that the step
In S2, the reaction system of the RT-PCR mainly includes following composition：
Template ribonucleic acid 0.2ng/ μ L, sense primer are each 0.5 μM, anti-sense primer is each 0.5 μM, probe is each 0.25 μM, dNTP 0.2mM
And reverse transcriptase 0.02U/ μ L；
The reaction condition of the RT-PCR is as follows：
50℃10min；95℃2min；95 DEG C of 15s, 60 DEG C of 35s, totally 6 circulations；95 DEG C of 15s, 60 DEG C of 35s, totally 40 circulations.
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