CN106636475A - Primer set for detecting American canine influenza virus subtype H3N8 and application thereof - Google Patents
Primer set for detecting American canine influenza virus subtype H3N8 and application thereof Download PDFInfo
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Abstract
The invention discloses a primer set for detecting American canine influenza virus subtype H3N8 and application thereof. The primer set provided by the invention comprises a primer pair A and a primer pair B; the primer pair A is composed of a primer F1 and a primer R1; the primer pair B is composed of a primer F2 and a primer R2; the primer F1 is as shown in a sequence 1; the primer R1 is as shown in a sequence 2; the primer F2 is as shown in a sequence 3; and the primer R2 is as shown in a sequence 4. The application of the primer set comprises (c1) assistant identification of the American canine influenza virus subtype H3N8; and (c2) assistant identification of that whether a to-be-detected sample contains the American canine influenza virus subtype H3N8. The primer set has good specificity and high sensitivity when used for detection and can detect a clinical sample within 4 to 5 h; and the primer set is simple to operate, easy to promote and convenient for basic-level operation and application, and is of great application value to diagnosis and epidemiological investigation of diseases caused by the American canine influenza virus subtype H3N8.
Description
Technical field
The present invention relates to a kind of primer sets of detection North America H3N8 hypotype canine influenza virus and its application.
Background technology
In recent years, the report of influenza a virus infection dog continuously emerges, including H3N8 subtype influenza virus, H3N2 hypotypes
There is H3N8 in the country such as influenza virus, H5N1 subtype influenza virus and H1N1 subtype influenza virus, the U.S., Britain, Australia
The report of hypotype canine influenza virus epidemic situation.H3N8 hypotypes canine influenza virus can have effect spread in dog, cause heating, cough,
The influenza-like symptoms such as rhinorrhea, serious causes dog hemorrhagic pneumonia and causes death.
At present, China not yet has dog to infect the report of North America H3N8 hypotype canine influenza virus.With pet market in recent years
Extension, and various countries police dog, army dog introduce a fine variety work increasingly frequently, which increase North America H3N8 hypotype canine influenza virus it is incoming in
The possibility of state, accordingly, it would be desirable to set up a kind of method for quick for North America H3N8 hypotype canine influenza virus.
The authentication method of existing North America H3N8 hypotype canine influenza virus is hemagglutination-inhibition test.Hemagglutination-inhibition test is related to
The separation of cause of disease, operation is relatively cumbersome, and easily influences each other between each hypotype, and sensitiveness is relatively low, takes time and effort.
The content of the invention
It is an object of the invention to provide a kind of primer sets of detection North America H3N8 hypotype canine influenza virus and its application.
Present invention firstly provides a kind of primer sets, including primer pair first and primer pair B；The primer pair first is by primer
F1 and primer R1 is constituted；The primer pair B is made up of primers F 2 and primer R2.
The primers F 1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) by sequence 1 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 1
The DNA molecular of identical function；
The primer R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) by sequence 2 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 2
The DNA molecular of identical function.
The primers F 2 is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 3 of sequence table；
(b2) by sequence 3 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 3
The DNA molecular of identical function；
The primer R2 is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 4 of sequence table；
(b4) by sequence 4 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 4
The DNA molecular of identical function.
The present invention also protects a species-specific primers pair, is the primer pair first or the primer pair B.
The purposes of the primer sets or the special primer pair is following (c1) auxiliary identification North America H3N8 hypotype dog influenzas
Virus；(c2) whether North America H3N8 hypotype canine influenza virus are contained in auxiliary identification sample to be tested.
The present invention also protects the primer sets or the special primer to the application in reagent preparation box；The kit
Purposes be following (c1) or (c2)：(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) identification is aided in treat test sample
Whether contain North America H3N8 hypotype canine influenza virus in this.
The present invention also protects a kind of kit, including the primer sets or the special primer pair；The use of the kit
Way is following (c1) or (c2)：(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) in auxiliary identification sample to be tested
Whether North America H3N8 hypotype canine influenza virus are contained.
The kit also includes standard items plasmid.Specifically, the kit includes standard items plasmid first and/or mark
Quasi- quality grain second.The standard items plasmid first is the plasmid of DNA molecular shown in the sequence 5 with sequence table.The standard quality
Grain first is the plasmid of DNA molecular shown in the 376-465 positions nucleotides of sequence 5 with sequence table.The standard items plasmid first is
The recombinant plasmid that the MCS that DNA molecular shown in the sequence 5 of sequence table inserts pUC57 carriers is obtained.The standard items
Plasmid first is the MCS that DNA molecular shown in the 376-465 positions nucleotides of sequence 5 by sequence table inserts pUC57 carriers
The recombinant plasmid for obtaining.The standard items plasmid second is the plasmid of DNA molecular shown in the sequence 6 with sequence table.The standard
Quality grain second is the plasmid of DNA molecular shown in the 868-1012 positions nucleotides of sequence 6 with sequence table.The standard items plasmid
Second is the recombinant plasmid that the MCS of the pUC57 carriers of DNA molecular insertion shown in sequence 6 by sequence table is obtained.The mark
Quasi- quality grain second is the polyclonal of the pUC57 carriers of DNA molecular insertion shown in the 868-1012 positions nucleotides of sequence 6 by sequence table
The recombinant plasmid that site obtains.
The present invention also protects the material for detecting DNA molecular first and the material for detecting DNA molecular second preparing examination
Application in agent box；The DNA molecular first is the target sequence of primer pair first described in the H3N8 hypotype canine influenza virus cDNA of North America；
The DNA molecular second is the target sequence of primer pair B described in the H3N8 hypotype canine influenza virus cDNA of North America；The kit
Purposes is following (c1) or (c2)：(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) auxiliary identification sample to be tested
In whether contain North America H3N8 hypotype canine influenza virus.
The present invention also protects the material for detecting DNA molecular first or the material for detecting DNA molecular second preparing examination
Application in agent box；The DNA molecular first is the target sequence of primer pair first described in the H3N8 hypotype canine influenza virus cDNA of North America；
The DNA molecular second is the target sequence of primer pair B described in the H3N8 hypotype canine influenza virus cDNA of North America；The kit
Purposes is following (c1) or (c2)：(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) auxiliary identification sample to be tested
In whether contain North America H3N8 hypotype canine influenza virus.
The DNA molecular first is concrete as shown in the 376-465 positions nucleotides of sequence 5 of sequence table.The DNA molecular second tool
Body is as shown in the 868-1012 positions nucleotides of sequence 6.
The present invention also protects a kind of method of auxiliary identification North America H3N8 hypotype canine influenza virus, comprises the steps：With
The cDNA of virus to be measured is template, and the primer pair first and the primer pair B is respectively adopted carries out quantitative fluorescent PCR, if
The North America H3N8 hypotype canine influenza virus of positive amplification curve, virus to be measured for candidate are shown, if being unsatisfactory for above-mentioned condition, treating
Survey non-north american H3N8 hypotype canine influenza virus of the virus for candidate.
The present invention also protects in a kind of auxiliary identification sample to be tested the whether side containing North America H3N8 hypotype canine influenza virus
Method, comprises the steps：As template, the primer pair first and the primer pair B is respectively adopted to be carried out cDNA with sample to be tested
Quantitative fluorescent PCR, if showing that positive amplification curve, sample to be tested are doubtful containing North America H3N8 hypotype canine influenza virus, such as
Fruit is unsatisfactory for that above-mentioned condition, sample to be tested are doubtful not to contain North America H3N8 hypotype canine influenza virus.
The present invention also protects a kind of method of auxiliary identification North America H3N8 hypotype canine influenza virus, comprises the steps：With
The cDNA of virus to be measured is template, and using the primer pair first or the primer pair B quantitative fluorescent PCR is carried out, if showing sun
Property amplification curve, virus to be measured for candidate North America H3N8 hypotype canine influenza virus, if not showing positive amplification curve, to be measured
Virus is the non-north american H3N8 hypotype canine influenza virus of candidate.
The present invention also protects in a kind of auxiliary identification sample to be tested the whether side containing North America H3N8 hypotype canine influenza virus
Method, comprises the steps：CDNA with sample to be tested carries out fluorescence as template using the primer pair first or the primer pair B
Quantitative PCR, if showing that positive amplification curve, sample to be tested are doubtful containing North America H3N8 hypotype canine influenza virus, if do not shown
Show that positive amplification curve, sample to be tested are doubtful and do not contain North America H3N8 hypotype canine influenza virus.
In any of the above methods described, during using the primer pair first, the response procedures of quantitative fluorescent PCR are：95℃
10min；95 DEG C of 15s, 59 DEG C of 1min, 40 circulations.
In any of the above methods described, during using the primer pair B, the response procedures of quantitative fluorescent PCR are：95℃
10min；95 DEG C of 15s, 58 DEG C of 1min, 40 circulations.
In any of the above methods described, during using the primer pair first, the reaction system of quantitative fluorescent PCR is：SYBR
Premix Ex Taq (2X) 10 μ L, the μ L of primers F 1 0.5, the μ L of primer R1 0.5, the μ L of template 2, plus distilled water is to 20 μ l.System
In, the concentration of primers F 1 and primer R1 in reaction system is 0.5 μM.
In any of the above methods described, during using the primer pair B, the reaction system of quantitative fluorescent PCR is：SYBR
Premix Ex Taq (2X) 10 μ L, the μ L of primers F 2 0.5, the μ L of primer R2 0.5, cDNA2 μ L, plus distilled water is to 20 μ l.System
In, the concentration of primers F 2 and primer R2 in reaction system is 0.5 μM.
In any of the above methods described, quantitative fluorescent PCR adopts the dyestuffs of SYBR Green I.SYBR Green I are a kind of
It is incorporated into the dyestuff with green excitation wavelength in all double-stranded DNA minor groove regions, under free state, SYBR
Green I sends faint fluorescence, once but combined with double-stranded DNA, fluorescence intensity is greatly enhanced.Therefore, it can according to fluorescence
Signal detection goes out double-stranded DNA quantity present in PCR system.Although quantitative fluorescent PCR (dye method) is non-specific detection,
As long as design of primers is special, the factors such as non-specific amplification, primer dimer are excluded, and carry out reaction condition optimization, be not required to
Want to be detected under the conditions of probe, it is time saving and energy saving to save money.
Detected using the primer sets of present invention offer, kit, specific good, sensitiveness is high, it is only necessary to 4-5 hours
Time clinical sample can be detected, simple to operate, easy popularization, be easy to basic unit operate and apply, for North America
H3N8 hypotype canine influenza virus disease diagnosis and epidemiology survey have great using value.
Description of the drawings
Fig. 1 is the result of embodiment 3.
Fig. 2 is the result of embodiment 4.
Fig. 3 is the result of embodiment 5.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, and as a result makes even
Average.
SYBR Premix Ex Taq(2X)：Life Technologies, Inc. of the U.S. (Life Technologies), article No.
4461693。
The design of embodiment 1, primer sets
Multiple primer pairs are obtained through a large amount of sequence analyses, the specificity to each primer pair and sensitivity by preliminary experiment
Detected, finally filtered out two primer pairs for identifying H3N8 hypotype canine influenza virus.
Primer pair first (target sequence is located at HA genes, and annealing temperature is 59 DEG C) is made up of primers F 1 and primer R1.
Primer pair B (target sequence is located at NA genes, and annealing temperature is 58 DEG C) is made up of primers F 2 and primer R2.
Primers F 1 (sequence 1 of sequence table)：5'-TTGCTACCCATATGACATCCCTGAC-3'；
Primer R1 (sequence 2 of sequence table)：5'-GTGAATCCCTCTGCTGTGAATTCCA-3'.
Primers F 2 (sequence 3 of sequence table)：5'-CCCCAATGAAGGGAAGGTGGAATGC-3；
Primer R2 (sequence 4 of sequence table)：5'-ATCCTCTCCCCTAGGGGTGTCAGTG-3'.
The foundation of embodiment 2, method
1st, the total serum IgE and reverse transcription for extracting sample to be tested is cDNA.
Sample to be tested can be test serum or virus to be measured.Test serum can be hepatic tissue, the brain tissue of dog, the dog of dog
Lung tissue, Nasal swabs of dog etc..
2nd, as template, the primer pair first designed using embodiment 1 carries out quantitative fluorescent PCR to the cDNA for being obtained with step 1, real
When monitor fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 1 0.5,
The μ L of primer R1 0.5, cDNA 2 μ L, plus distilled water is to 20 μ l.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 59 DEG C of 1min, 40 circulations.
3rd, as template, the primer pair B designed using embodiment 1 carries out quantitative fluorescent PCR to the cDNA for being obtained with step 1, real
When monitor fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 2 0.5,
The μ L of primer R2 0.5, cDNA2 μ L, plus distilled water is to 20 μ l.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 58 DEG C of 1min, 40 circulations.
4th, result judgement is carried out
When sample to be tested is test serum, if step 2 and step 3 show positive amplification curve, sample to be tested being
The tissue with North America H3N8 hypotype canine influenza virus of candidate.
When sample to be tested is to be measured viral, if step 2 and step 3 show positive amplification curve, virus to be measured being
The North America H3N8 hypotype canine influenza virus of candidate.
The foundation of embodiment 3, calibration curve
Gene template copy number=DNA mass concentrations/DNA molecular amount.
First, the preparation of standard items plasmid first
DNA molecular shown in the sequence 5 of sequence table is North America H3N8 hypotype canine influenza virus HA genetic fragments.By sequence table
Sequence 5 shown in DNA molecular insert pUC57 carriers MCS, obtain standard items plasmid first.
2nd, the preparation of standard items plasmid second
DNA molecular shown in the sequence 6 of sequence table is North America H3N8 hypotype canine influenza virus NA genetic fragments.By sequence table
Sequence 6 shown in DNA molecular insert pUC57 carriers MCS, obtain standard items plasmid second.
3rd, the foundation of calibration curve first
1st, with 10 times of gradient dilution standard items plasmid first of distilled water, concentration is obtained for 1.0 × 108-1.0×103Copy number/μ
Each dilution of L.
2nd, as template, the primer pair first designed using embodiment 1 carries out quantitative fluorescent PCR to the dilution for being obtained with step 1,
Real-time monitoring fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 1 0.5,
The μ L of primer R1 0.5, the μ L of template 2, plus distilled water is to 20 μ l.In system, the concentration of primers F 1 and primer R1 in reaction system is equal
For 0.5 μM.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 59 DEG C of 1min, 40 circulations.
With ct values as ordinate, built as abscissa with the concentration denary logarithm value of dilution Plays quality grain first
Day-mark directrix curve, is shown in Figure 1A.Calibration curve equation is：CT=-3.051logC0+36.583 (coefficient correlations：R2=0.988).
4th, the foundation of calibration curve second
1st, with 10 times of gradient dilution standard items plasmid second of distilled water, concentration is obtained for 1.0 × 109-1.0×103Copy number/μ
Each dilution of L.
2nd, as template, the primer pair B designed using embodiment 1 carries out quantitative fluorescent PCR to the dilution for being obtained with step 1,
Real-time monitoring fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 2 0.5,
The μ L of primer R2 0.5, cDNA2 μ L, plus distilled water is to 20 μ l.In system, the concentration of primers F 2 and primer R2 in reaction system is equal
For 0.5 μM.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 58 DEG C of 1min, 40 circulations.
With ct values as ordinate, built as abscissa with the concentration denary logarithm value of dilution Plays quality grain second
Day-mark directrix curve, is shown in Figure 1B.Calibration curve equation is：CT=-3.166logC0+38.797 (coefficient correlations：R2=0.99).
In conjunction with the embodiments the calibration curve of 2 method and embodiment 3, can calculate H3N8 hypotypes dog stream in sample to be tested
The copy number of Influenza Virus.
Embodiment 4, specificity
H3N2 hypotypes canine influenza virus used in the present embodiment are strain A/canine/Beijing/364/2009
(H3N2), it is recorded in following document：A Multiplex RT-PCR Assay for Detection
andDifferentiation of Avian-Origin Canine H3N2,Equine-Origin H3N8,Human-
Origin H3N2,and H1N1/2009 Canine Influenza Viruses；Chenxi Wang,Qian Wang,
Junyi Hu,Honglei Sun,Juan Pu,Jinhua Liu,Yipeng Sun；PLoSONE 12(1):e0170374.
H3N2 hypotypes swine influenza virus used in the present embodiment is strain A/swine/Guangdong/7/06 (H3N2),
It is recorded in following document：Identification of swine influenza A virus andStenotrophomonas
maltophilia co-infection inChinese pigs；Dongjun Hou,Yuhai Bi,Honglei Sun,Jun
Yang,Guanghua Fu,Yipeng Sun,Jinhua Liu,and Juan Pu；Virology Journal 2012,9:
169。
H3N2 subtype avian influenza virus used in the present embodiment are strain A/duck/Anhui/D293/2014 (H3N2),
It is recorded in following document：The foundation of North America H7 subtype avian influenza virus RT-PCR detection methods；Wang Chenxi, Acquaintance clouds, Sun Honglei,
Pu Juan；Chinese Veterinary Journal, (volume 52) the 6th phase in 2016,28-32.
First, the specificity of primer pair first
1st, the total serum IgE and reverse transcription for extracting sample to be tested is cDNA.
Sample to be tested is respectively：H3N2 hypotype canine influenza virus, H3N2 hypotype swine influenza viruses, H3N2 subtype avian influenzas disease
Poison.
2nd, as template, the primer pair first designed using embodiment 1 is carried out the cDNA for being obtained with standard items plasmid first or step 1
Quantitative fluorescent PCR, real-time monitoring fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 1 0.5,
The μ L of primer R1 0.5, the μ L of template 2, plus distilled water is to 20 μ l.In system, the concentration of primers F 1 and primer R1 in reaction system is equal
For 0.5 μM.
Replace cDNA with 2 μ L distilled waters, as negative control.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 59 DEG C of 1min, 40 circulations.
As a result Fig. 2A is seen.In Fig. 2A, 1,2,3,4,5 represent successively standard items plasmid first, H3N2 hypotype canine influenza virus,
H3N2 hypotype swine influenza viruses, H3N2 subtype avian influenza virus and negative control.Only there is specific amplification in standard items plasmid first
Curve, and H3N2 hypotype canine influenza virus, H3N2 hypotype swine influenza viruses, H3N2 subtype avian influenza virus and negative sample are not
There is amplification curve, show that set up fluorescence quantifying PCR method is respectively provided with preferable specificity.
2nd, the specificity of primer pair B
1st, the total serum IgE and reverse transcription for extracting sample to be tested is cDNA.
Sample to be tested is respectively：H3N2 hypotype canine influenza virus, H3N2 hypotype swine influenza viruses, H3N2 subtype avian influenzas disease
Poison.
2nd, as template, the primer pair B designed using embodiment 1 is carried out the cDNA for being obtained with standard items plasmid second or step 1
Quantitative fluorescent PCR, real-time monitoring fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 2 0.5,
The μ L of primer R2 0.5, cDNA2 μ L, plus distilled water is to 20 μ l.In system, the concentration of primers F 2 and primer R2 in reaction system is equal
For 0.5 μM.
Replace cDNA with 2 μ L distilled waters, as negative control.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 58 DEG C of 1min, 40 circulations.
As a result Fig. 2 B are seen.In Fig. 2 B, 1,2,3,4,5 represent successively standard items plasmid first, H3N2 hypotype canine influenza virus,
H3N2 hypotype swine influenza viruses, H3N2 subtype avian influenza virus and negative control.Only there is specific amplification in standard items plasmid second
Curve, and H3N2 hypotype canine influenza virus, H3N2 hypotype swine influenza viruses, H3N2 subtype avian influenza virus and negative sample are not
There is amplification curve, show that set up fluorescence quantifying PCR method is respectively provided with preferable specificity.
Embodiment 5, sensitivity
First, the sensitivity of primer pair first
1st, with 10 times of gradient dilution standard items plasmid first of distilled water, 2.5 × 10 are obtained6copies/μL-25copies/μL
Each dilution.
2nd, as template, the primer pair first designed using embodiment 1 carries out quantitative fluorescent PCR to the dilution for being obtained with step 1,
Real-time monitoring fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 1 0.5,
The μ L of primer R1 0.5, the μ L of template 2, plus distilled water is to 20 μ l.In system, the concentration of primers F 1 and primer R1 in reaction system is equal
For 0.5 μM.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 59 DEG C of 1min, 40 circulations.
As a result Fig. 3 A are seen.In Fig. 3 A, 1 to 6 is followed successively by DNA concentration each dilution from high to low.As a result show, adopt
Said method detects that the lowest detection of template Plays quality grain first is limited to 25copies/ μ L.
2nd, the sensitivity of primer pair B
1st, with 10 times of gradient dilution standard items plasmid second of distilled water, 3.7 × 10 are obtained7copies/μL-37copies/μL
Each dilution.
2nd, as template, the primer pair B designed using embodiment 1 carries out quantitative fluorescent PCR to the dilution for being obtained with step 1,
Real-time monitoring fluorescence intensity.
The reaction system (20 μ L) of quantitative fluorescent PCR：SYBR Premix Ex Taq (2X) 10 μ L, the μ L of primers F 2 0.5,
The μ L of primer R2 0.5, cDNA2 μ L, plus distilled water is to 20 μ l.In system, the concentration of primers F 2 and primer R2 in reaction system is equal
For 0.5 μM.
The response procedures of quantitative fluorescent PCR：95℃10min；95 DEG C of 15s, 58 DEG C of 1min, 40 circulations.
As a result Fig. 3 B are seen.In Fig. 3 B, 1 to 7 is followed successively by DNA concentration each dilution from high to low.As a result show, adopt
Said method detects that the lowest detection of template Plays quality grain second is limited to 37copies/ μ L.
SEQUENCE LISTING
<110>China Agricultural University
<120>A kind of primer sets of detection North America H3N8 hypotype canine influenza virus and its application
<130> GNCYX170234
<160> 6
<170> PatentIn version 3.5
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ccccaatgaa gggaaggtgg aatgc 25
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tattcgtctc agggagcaaa agcaggggat atttctgtca atcatgaaga caaccattat 60
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cacactgtgt ctgggacacc atgcagtagc aaatggaaca ttggtaaaaa caatgagtga 180
tgatcaaatt gaggtgacaa atgctacaga attagttcag agcatttcaa tggggaaaat 240
atgcaacaaa tcatatagaa ttctagatgg aagaaattgc acattaatag atgcaatgct 300
aggagacccc cactgtgacg cctttcagta tgagagttgg gacctcttta tagaaagaag 360
cagcgctttc agcaattgct acccatatga catccctgac tatgcaccgc tccgatccat 420
tgtagcatcc tcagggacag tggaattcac agcagaggga ttcacatgga caggtgtaac 480
tcaaaacgga agaagtggag cctgcaaaag gggatcagcc gatagtttct ttagccgact 540
gaattggcta acaaaatctg gaagctctta ccccacattg aatgtgacaa tgcctaacaa 600
taaaaatttc gacaagctat acatctgggg gattcatcac ccgagctcaa atcaagagca 660
gacaaaattg tacatccaag aatcaggacg agtaacagtc tcaacaaaaa gaagtcaaca 720
aacaataatc cctaacatcg gatctagacc gttggtcaga ggtcaatcag gcaggataag 780
catatactgg accattgtaa aacctggaga tatcctaatg ataaacagta atggcaactt 840
agttgcaccg cggggatatt ttaaattgaa cacagggaaa agctctgtaa tgagatccga 900
tgtacccata gacatttgtg tgtctgaatg tattacacca aatggaagca tctccaacga 960
caagccattc caaaatgtga acaaagttac atatggaaaa tgccccaagt atatcaggca 1020
aaacacttta aagctggcca ctgggatgag gaatgtacca gaaaagcaaa ccagaggaat 1080
ctttggagca atagcgggat tcatcgaaaa cggctgggaa ggaatggttg atgggtggta 1140
tgggttccga tatcaaaact ctgaaggaac agggcaagct gcagatctaa agagcactca 1200
agcagccatc gaccagatta atggaaagtt aaacagagtg attgaaagaa ccaatgagaa 1260
attccatcaa atagagaagg aattctcaga agtagaagga agaattcagg acttggagaa 1320
atatgtagaa gacaccaaaa tagacctatg gtcctacaat gcagaattgc tggtggctct 1380
agaaaatcaa catacaattg acttaacaga tgcagaaatg aataaattat ttgagaagac 1440
tagacgccag ttaagagaaa acgcagaaga catgggaggt ggatgtttca agatttacca 1500
caaatgtgat aatgcatgca ttgaatcaat aagaactggg acatatgacc attacatata 1560
caaagatgaa gcattaaaca atcgatttca gatcaaaggt gtagagttga aatcaggcta 1620
caaagattgg atactgtgga tttcattcgc catatcatgc ttcttaattt gcgttgttct 1680
attgggtttc attatgtggg cttgccaaaa aggcaacatc agatgcaaca tttgcatttg 1740
agtaaactga tagttaaaaa cacccttgtt tctactaata cgagacgata ta 1792
<210> 6
<211> 1478
<212> DNA
<213>Artificial sequence
<400> 6
gaattcagga gcaaagcagg agtttaaaat gaatccaaat caaaagataa tagcaattgg 60
atctgcatca ttggggatat taatcattaa tgtcattctc catgtagtca gcattatagt 120
aacagtactg gtcctcaata acaatagaac agatctgaac tgcaaaggga cgatcataag 180
agagtacaat gaaacagtaa gagtagaaaa acttactcaa tggtataata tcagtacaat 240
taagtacata gagagacctt caaatgaata ttacatgaac aacactgaac cactttgtga 300
ggcccaaggc tttgcaccat tttccaaaga taatggaata cgaattgggt cgagaggcca 360
tgtttttgtg ataagagaac cttttgtatc atgttcaccc tcagaatgta gaaccttttt 420
cctcacacag ggctcattac tcaatgacaa acattctaac ggcacaataa aggatcgaag 480
tccgtatagg actctgatga gtgtcaaaat agggcaatca cctaatgtat atcaagctaa 540
atttgaatcg gtggcatggt cagcaacagc atgccatgat ggaaaaaaat ggatgacagt 600
tggagtcaca gggcccgaca atcaagcaat tgcagtagtg aactatggag gtgttccggt 660
tgatattatc aattcatggg caggggatat tttaagaacc caagaatcat catgcacctg 720
cattaaagga gactgttatt gggtaatgac tgatggaccg gcaaataggc aagctaatta 780
taggatattc aaagcaaaag atggaagagt aattggacaa actgatataa gtttcaatgg 840
gggacacata gaggagtgtt cttgttaccc caatgaaggg aaggtggaat gcatatgcag 900
agacaattgg actggaacaa atagaccaat tctggtaata tcttctgatc tatcgtacac 960
agttggatat ttgtgtgctg gcattcccac tgacacccct aggggagagg atagtcaatt 1020
cacaggctca tgtacaagtc ctttgggaaa taaaggatac ggtgtcaaag gtttcgggtt 1080
tcgacaagga actgacgtat gggccggaag gacaattagt aggacttcaa gatcaggatt 1140
cgaaataata aaaatcagga atggttggac acagaatagt aaggaccaaa tcaggaggca 1200
agtgattatc gatgacccaa attggtcagg atatagcggt tctttcacat tgccggttga 1260
attaacaaaa aaaggatgtt tggtcccctg tttctgggtt gaaatgatta gaggtaaacc 1320
tgaagaaaca acaatatgga cctctagcag ctccattgtg atgtgtggag tagatcataa 1380
aattgccagt tggtcatggc acgatggagc aattcttccc tttgacatcg ataagatgta 1440
atttacgaaa aaaactcctt gtttctactc ctgaattc 1478
Claims (10)
1. primer sets, including primer pair first and primer pair B；The primer pair first is made up of primers F 1 and primer R1；The primer
Second is made up of primers F 2 and primer R2；
The primers F 1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) by sequence 1 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 1 identical
The DNA molecular of function；
The primer R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) by sequence 2 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 2 identical
The DNA molecular of function；
The primers F 2 is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 3 of sequence table；
(b2) by sequence 3 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 3 identical
The DNA molecular of function；
The primer R2 is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 4 of sequence table；
(b4) by sequence 4 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 4 identical
The DNA molecular of function.
2. a species-specific primers pair, are the primer pair B in the primer pair first or the claim 1 in claim 1.
3. primer sets described in claim 1 or special primer described in claim 2 are to the application in reagent preparation box；The examination
The purposes of agent box is following (c1) or (c2)：(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) identification is aided in treat
Whether contain North America H3N8 hypotype canine influenza virus in test sample sheet.
4. a kind of kit, including special primer pair described in primer sets described in claim 1 or claim 2；The kit
Purposes be following (c1) or (c2)：(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) identification is aided in treat test sample
Whether contain North America H3N8 hypotype canine influenza virus in this.
5. it is used to detect the material of DNA molecular first and for detecting application of the material of DNA molecular second in reagent preparation box；Institute
State the target sequence that DNA molecular first is primer pair first in the H3N8 hypotype canine influenza virus cDNA of North America；The DNA molecular second is North America
The target sequence of primer pair B in H3N8 hypotype canine influenza virus cDNA；The purposes of the kit is following (c1) or (c2)：
(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) whether North America H3N8 hypotypes are contained in auxiliary identification sample to be tested
Canine influenza virus；
The primer pair first is made up of primers F 1 and primer R1；
The primers F 1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) by sequence 1 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 1 identical
The DNA molecular of function；
The primer R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) by sequence 2 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 2 identical
The DNA molecular of function；
The primer pair B is made up of primers F 2 and primer R2；
The primers F 2 is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 3 of sequence table；
(b2) by sequence 3 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 3 identical
The DNA molecular of function；
The primer R2 is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 4 of sequence table；
(b4) by sequence 4 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 4 identical
The DNA molecular of function.
6. it is used to detect the material of DNA molecular first or for detecting application of the material of DNA molecular second in reagent preparation box；Institute
State the target sequence that DNA molecular first is primer pair first in the H3N8 hypotype canine influenza virus cDNA of North America；The DNA molecular second is North America
The target sequence of primer pair B in H3N8 hypotype canine influenza virus cDNA；The purposes of the kit is following (c1) or (c2)：
(c1) auxiliary identification North America H3N8 hypotype canine influenza virus；(c2) whether North America H3N8 hypotypes are contained in auxiliary identification sample to be tested
Canine influenza virus；
The primer pair first is made up of primers F 1 and primer R1；
The primers F 1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) by sequence 1 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 1 identical
The DNA molecular of function；
The primer R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) by sequence 2 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 2 identical
The DNA molecular of function；
The primer pair B is made up of primers F 2 and primer R2；
The primers F 2 is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 3 of sequence table；
(b2) by sequence 3 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 3 identical
The DNA molecular of function；
The primer R2 is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 4 of sequence table；
(b4) by sequence 4 is through the replacement of one or several nucleotides and/or disappearance and/or adds and has with sequence 4 identical
The DNA molecular of function.
7. a kind of method of auxiliary identification North America H3N8 hypotype canine influenza virus, comprises the steps：With the cDNA of virus to be measured
For template, the primer pair first and the primer pair B being respectively adopted in claim 1 carries out quantitative fluorescent PCR, if
The North America H3N8 hypotype canine influenza virus of positive amplification curve, virus to be measured for candidate are shown, if being unsatisfactory for above-mentioned condition, treating
Survey non-north american H3N8 hypotype canine influenza virus of the virus for candidate.
8. a kind of whether method containing North America H3N8 hypotype canine influenza virus in auxiliary identification sample to be tested, including following step
Suddenly：As template, the primer pair first and the primer pair B being respectively adopted in claim 1 is carried out cDNA with sample to be tested
Quantitative fluorescent PCR, if showing that positive amplification curve, sample to be tested are doubtful containing North America H3N8 hypotype canine influenza virus, such as
Fruit is unsatisfactory for that above-mentioned condition, sample to be tested are doubtful not to contain North America H3N8 hypotype canine influenza virus.
9. a kind of method of auxiliary identification North America H3N8 hypotype canine influenza virus, comprises the steps：With the cDNA of virus to be measured
For template, quantitative fluorescent PCR is carried out using the primer pair first or the primer pair B in claim 1, if showing sun
Property amplification curve, virus to be measured for candidate North America H3N8 hypotype canine influenza virus, if not showing positive amplification curve, to be measured
Virus is the non-north american H3N8 hypotype canine influenza virus of candidate.
10. a kind of whether method containing North America H3N8 hypotype canine influenza virus in auxiliary identification sample to be tested, including following step
Suddenly：CDNA with sample to be tested carries out fluorescence as template using the primer pair first or the primer pair B in claim 1
Quantitative PCR, if showing that positive amplification curve, sample to be tested are doubtful containing North America H3N8 hypotype canine influenza virus, if do not shown
Show that positive amplification curve, sample to be tested are doubtful and do not contain North America H3N8 hypotype canine influenza virus.
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| CN201710119861.5A CN106636475B (en) | 2017-03-01 | 2017-03-01 | Primer group for detecting North American H3N8 subtype canine influenza virus and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110592276A (en) * | 2019-07-08 | 2019-12-20 | 北京世纪元亨动物防疫技术有限公司 | Specific primer and kit for detecting canine influenza virus |
| CN114908196A (en) * | 2022-06-14 | 2022-08-16 | 中国疾病预防控制中心病毒病预防控制所 | Reagent for specifically detecting virus and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592276A (en) * | 2019-07-08 | 2019-12-20 | 北京世纪元亨动物防疫技术有限公司 | Specific primer and kit for detecting canine influenza virus |
| CN114908196A (en) * | 2022-06-14 | 2022-08-16 | 中国疾病预防控制中心病毒病预防控制所 | Reagent for specifically detecting virus and application thereof |
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| Publication number | Publication date |
|---|---|
| CN106636475B (en) | 2020-05-22 |
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