CN108384891A - Triple RT-PCR Detection Kit for H9 Subtype Avian Influenza Virus, Duck Tembusu Virus and Duck Circovirus - Google Patents
Triple RT-PCR Detection Kit for H9 Subtype Avian Influenza Virus, Duck Tembusu Virus and Duck Circovirus Download PDFInfo
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Abstract
The invention discloses a triple RT-PCR detection kit for H9 subtype avian influenza virus, duck tembusu virus and duck circovirus, which contains three pairs of specific primers. Experiments prove that the kit can simultaneously detect three pathogens of H9 subtype avian influenza virus, duck tembusu virus and duck circovirus only by one RT-PCR reaction, and has the advantages of strong specificity, high sensitivity, low cost, high efficiency and the like; in addition, the invention can directly judge the amplification result by utilizing the difference of the lengths of the amplified fragments in the design of the primers, and is more convenient, intuitive and practical. The method can simultaneously detect 1pg H9 subtype avian influenza virus RNA, 50pg duck tembusu virus RNA and 10ng duck circovirus DNA at the lowest, provides an accurate detection method for the early onset of the three viruses, has an important significance for timely cutting off the spread of the viruses, has a wide application prospect, and has an important practical significance for the sustainable development of the duck breeding industry.
Description
Technical field
The present invention relates to RT-PCR detection technique fields, specifically a kind of H9 subtype avian influenza virus, duck tembusu virus
With the triple RT-PCR detection kits of duck circovirus.
Background technology
Bird flu is birds infection and disease caused by different subtype virus in being belonged to by orthomyxovirus section influenza A
General name.H9 subtype avian influenza virus（H9AIV）It is in generally down to medium virulence, but it is widely distributed, can cause the immune suppression of duck
The mixed infection with other ducks disease is made and then caused, can also cause being decreased obviously for laying rate, extreme influence after laying ducks infection
The economic benefit of laying duck, therefore it is very important to the harm of duck culturing industry.
Duck tembusu virus（DTMUV）It is found in the coastal regions in east China such as Fujian China, Zhejiang, Jiangsu in 2010 first,
And gradually spread to national most area.It can lead to laying duck egg production degradation after duck tembusu virus infected duck, lay eggs
Rate can be down to from 90% within 10%, or even total crop failure；Duckling can be caused the nerves such as astasia occur, collapse in the ground after infection duckling
Symptom, and then duckling mortality is caused to increase, seriously up to 80%, bring great economic loss to duck culturing industry.
Duck circovirus（DuCV）It is common in duck, duck circovirus can cause that duck growth retardation, feather be in disorder, weight loss
Etc. symptoms, can infected poultry immune system, cause immunosupress.
Mixed infection makes the diagnosis difficulty of duck disease larger, only cannot make diagnosis by clinical manifestation, need be by
In molecular diagnostic techniques.The methods of separation, agar gel diffusion test and enzyme-linked immunosorbent assay of virus are unfavorable than relatively time-consuming
In the prevention of duck disease.
The conventional method that zoonosis is made a definite diagnosis mainly by the separation of cause of disease, Electronic Speculum observation, the methods of serological Identification,
But this method is unfavorable for the early diagnosis and quickly control of Animal diseases.
The rise of molecular biology, RT-PCR technology is because with easy to operate, sensibility is high, high specificity and reproducible
The advantages that be widely used to the detection of animal pathogenic microorganism, especially in the antidiastole of clinical several cause of disease mixed infections
With unique advantage and very high practical value.
Since there are several templates and primers in same RT-PCR systems, therefore, it is necessary to prevent between primer and template
Non-specific binding, in order to avoid the appearance of non-characteristic band.Meanwhile the size of target fragment has suitable gradient and each primer to move back
Fiery condition is as identical as possible, and to ensure respective amplified production amount relative equilibrium, condition is groped particularly significant.
Invention content
Present invention aims at the problems mentioned above in the background art are solved, a kind of high efficiency, low cost, sensitivity are provided
Property good, specific high, quickly and easily H9 subtype avian influenza virus, duck tembusu virus and triple RT-PCR inspections of duck circovirus
Test agent box, and identification or auxiliary identify primer pair groups of these three viruses.
The present invention in order to achieve the above objectives, using following technical scheme：H9 subtype avian influenza virus, duck tembusu virus and
The triple RT-PCR primer groups of duck circovirus, including three pairs of specific primers are primer 1 and primer 2, primer 3 and primer respectively
4, primer 5 and primer 6, they are respectively provided with the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6.
Further, the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6
Molar ratio is（2-1）:（2-1）:（2-1）:（2-1）:（1-0.5）:（1-0.5）.
Further, the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6
Molar ratio be 2:2:1:1:1:1.
Above-mentioned H9 subtype avian influenza virus, duck tembusu virus and the triple RT-PCR primer groups of duck circovirus expand in PCR
The annealing temperature of application in terms of increasing, the PCR amplification is 50.2-60 DEG C.
Further, the annealing temperature of the PCR amplification is 53.4 DEG C.
H9 subtype avian influenza virus, duck tembusu virus and the triple RT-PCR detection kits of duck circovirus, including inspection
Survey H9 subtype avian influenza virus, duck tembusu virus and the PCR reagent of duck circovirus, One-Step RT-PCR kits, sun
Property comparison liquid and negative controls.
Further, the PCR reagent includes being drawn by H9AIV upstream and downstream primers, DTMUV upstream and downstream primers and DuCV upstream and downstream
The PCR primer group of object composition；
The H9AIV upstream and downstream primers are the primer 1 and the primer 2 respectively；
Final concentration of the primer 1 in the PCR reagent in the PCR primer group is specially 2 μm of ol/L；
Final concentration of the primer 2 in the PCR reagent in the PCR primer group is specially 2 μm of ol/L；
The DTMUV upstream and downstream primers are the primer 3 and the primer 4 respectively；
Final concentration of the primer 3 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
Final concentration of the primer 4 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
The DuCV upstream and downstream primers are the primer 5 and the primer 6 respectively；
Final concentration of the primer 5 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
Final concentration of the primer 6 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
The One-StepRT-PCR kits include 2 × one-stepreactionmix, TransScript one-step
Emzyme mix and ddH2O；
The positive control solution is H9 subtype avian influenza virus cDNA, duck tembusu virus cDNA and duck circovirus DNA；
The negative controls are ddH2O。
Above-mentioned H9 subtype avian influenza virus, duck tembusu virus and the triple RT-PCR detection kits of duck circovirus exist
The annealing temperature of application in terms of PCR amplification, the PCR amplification is 50.2-60 DEG C.
Further, the annealing temperature of the PCR amplification is 53.4 DEG C.
Compared with prior art, advantages of the present invention and generation good effect are：
The present invention is carried out at the same time detection for shortage at present to H9 subtype avian influenza virus, duck tembusu virus and duck circovirus
With effective reliable technology of diagnosis, three pairs of specific primers of inventor's research and design establish H9 subtype avian influenzas accordingly
Virus, triple RT-PCR detection methods of duck tembusu virus and duck circovirus, and it is prepared for corresponding detection kit；This
Invention has many advantages, such as that high efficiency, low cost, sensibility are good, specific high, quick and convenient, and the present invention utilizes in design of primers
The different of expanding fragment length directly judge amplification so that this method is easier, intuitive and practical in result judgement；It answers
The pathogen that can detect and differentiate H9 subtype avian influenza virus duck tembusu virus and duck circovirus simultaneously with the present invention, is used for
The duck circovirus mixed infection of duck tembusu virus sum caused by hypoimmunity caused by H9 subtype avian influenza virus infects；
The present invention most low energy detects 1pgH9 subtype avian influenza virus RNA, 50pg duck tembusu virus RNA and 10ng ducks annulus disease simultaneously
Malicious DNA provides accurate detection method for three kinds of viral morbidity early stages, is of great significance to cutting off its propagation in time,
Duck aquaculture sustainable development is had practical significance, is had broad application prospects, it, can be simultaneously in clinical application
Cost and the pollution for reducing the time, have highly important practical value and practice significance.
Description of the drawings
Fig. 1 is triple successful Test Drawings of RT-PCR reaction condition optimizations, in figure：M is molecular weight standard 100bp DNA
Ladder, 1 is H9AIV, DTMUV and DuCV；2 be H9 AIV；3 be DTMUV；4 be DuCV.
Fig. 2 is triple RT-PCR sensitivity tests result electrophoretograms, in figure：M is molecular weight standard 100bpDNAladder,
1 is 50ng H9AIV, 50ng DTMUV and 50ng DuCV；2 be 10ng H9AIV, 10ng DTMUV and 10ng DuCV；3 are
100pg H9AIV, 100pg DTMUV and 100pg DuCV；4 be 50pg H9AIV, 50pg DTMUV and 50pg DuCV；5 are
10pg H9AIV, 10pg DTMUV and 10pg DuCV；6 be 1pg H9AIV, 1pg DTMUV and 1pg DuCV；7 be negative control
（ddH2O）.
Fig. 3 is triple RT-PCR specific tests result electrophoretograms, in figure：M is molecular weight standard 100bpDNAladder,
1 is H9AIV RNA, and 2 be DTMUV RNA, and 3 be DuCV DNA, and 4 be the mixing sample of H9AIV RNA and DTMUV RNA（Concentration
Than 2:1）, 5 be the mixing sample of DTMUV RNA and DuCV RNA（Concentration ratio 1:1）, 6 be the mixed of H9AIV RNA and DuCV RNA
Close sample（Concentration ratio 2:1）, 7 be the mixing sample of H9AIV RNA, DTMUV RNA and DuCV RNA（Concentration ratio 2:1:1）, 8 are
H5 subtype avian influenza virus RNA；9 be H7 subtype avian influenza virus RNA, and 10 be Muscovy duck parvovirus RNA, and 11 be duck plague virus
DNA, 12 be Duck Paramyxovirus disease RNA, and 13 be duck hepatitis virus RNA, and 14 be Egg Drop syndrome virus DNA, and 15 be negative control
（ddH2O）.
Fig. 4 is triple RT-PCR partial clinicals sample detection result electrophoretograms, in figure：M is molecular weight standard
100bpDNAladder, 1 is positive control（H9 subtype avian influenza virus cDNA, duck tembusu virus cDNA and duck circovirus
DNA）, 2-15 is respectively the clinical sample testing result of the purposeful band in part.
Specific implementation mode
Experimental method used in following embodiment is all made of conventional method as not having specified otherwise；Material used,
Reagent etc. does not have specified otherwise such as, is commercially available.Specific material therefor and reagent are as follows：
H9 subtype avian influenza virus（H9AIV）, duck tembusu virus（DTMUV）And duck circovirus（DuCV）, H7 hypotype fowl stream
Influenza Virus（H7AIV）, Muscovy duck parvovirus（MPV）, duck plague virus（DPV）, Duck Paramyxovirus disease（NDV）, duck hepatitis virus（DHV）
And Egg Drop syndrome virus（EDS）It is provided by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute poultry diease research department.
Viral RNA/DNA rapidly purifies kit and One-Step RT-PCR kits are purchased from the full formula gold biology in Beijing
Science and Technology Ltd., PMD-18T are purchased from Dalian treasured bioengineering Co., Ltd, and plastic recovery kit is purchased from Guangzhou Dongsheng biology section
Skill Co., Ltd；The C1000PCR instruments of BIO-RAD companies of U.S. production, the low-temperature and high-speed centrifugation of HERMLE companies of Germany production
Machine etc..
Embodiment 1, the design of primer and synthesis
According to existing disclosed H9 subtype avian influenza virus（H9AIV）, duck tembusu virus（DTMUV）And duck circovirus
（DuCV）Gene conserved sequence, verified by Blast, designed and synthesized three pairs of specific primers（Table 1）.
Table 1 identifies the primer sequence of H9AIV, DTMUV and DuCV
Embodiment 2, triple RT-PCR identify H9 subtype avian influenza virus（H9AIV）, duck tembusu virus（DTMUV）With duck annulus
Virus（DuCV）
Step 1: the foundation of triple RT-PCR systems
（1）The preparation of sample to be tested
Kit specification, extraction H9 subtype avian influenza virus RNA, duck tembusu virus are rapidly purified according to viral RNA/DNA
RNA and duck circovirus DNA.With reference to Sambrook methods measure nucleic acid concentration and purity, be stored in -70 DEG C it is spare.
（2）The optimization of triple RT-PCR reaction conditions
RT-PCR is carried out using one-step method using One Step RT-PCR kits.To each loop parameters of RT-PCR and each primer
Concentration etc. optimizes, with the RT-PCR patterns that determination is best.
It is optimized by primer concentration, each reaction temperature, time and the cycle-index etc. to RT-PCR, is finally determined
The best effort final concentration of H9 AIV-F and H9 AIV-R primers is that 2 μm of ol/L, DTMUV-F and DTMUV-R draw in RT-PCR
The best effort final concentration of object is 1 μm of ol/L, and the best effort final concentration of DuCV-F and DuCV-R primers is 1 μm of ol/L.
Reaction system（50μl）：1 Step Enzyme Mix of Prime Script 2 μ l, 2 × 1 Step Buffer 25
μ l, final concentration are H9AIV-F the and H9AIV-R primers of 2 μm of ol/L, and final concentration is the DTMUV-F and DTMUV- of 1 μm of ol/L
R primers and final concentration are DuCV-F the and DuCV-R primers of 1 μm of ol/L, H9AIV, DTMUV and DuCV totally 4 μ L（Volume is
2:1:1）As hybrid template, 50 μ l are supplied with the dH2O of no RNase.
The optimum response pattern of RT-PCR is 50 DEG C of reverse transcriptions 30min, 95 DEG C of denaturation 3min, subsequently into 94 DEG C of denaturation
50s, 53.4 DEG C of annealing 50s, the cycle of 70 DEG C of extension 50s carries out 30 cycles altogether, finally again after 70 DEG C extend 10min, in
4 DEG C reaction was completed.
Step 2: the sensitivity tests of triple RT-PCR
By step 1（1）In the sample that is prepared as H9 subtype avian influenza virus, duck tembusu virus and duck circovirus
The RNA of template, add keeps its final concentration consistent in same test tube and with ddH2O adjustment in right amount, then carries out 10 multiple proportions gradient dilutions,
According to step 1（2）PCR reaction conditions after middle optimization are expanded, its sensibility is detected；Setting simultaneously is with ddH2O replacements etc.
Measure the negative control of template.
After reaction, 50 μ l RT-PCR products is taken to be mixed with 5 μ l bromjophenol blues, the electrophoresis in 10g/L Ago-Gels,
After ethidium bromide staining, observation under ultraviolet light is taken pictures, and is made comparisons with DNA standard molecular weights, is analyzed and record result.In purple
With the segment needed for blade cutting under outer lamp, then purify recycling with DNA segment plastic recovery kit.Take appropriate purifying recycling
PCR product, with PMD-18T（From PME-18T kits）It is connected 4 hours in 16 DEG C, converts DH5 α escherichia coli.Picking
37 DEG C of cultures of the white colony grown on the Selective agar medium containing ampicillin, Rapid identification is carried out with PCR method, positive
Clone bacterium send Dalian Bao Sheng Bioisystech Co., Ltd to be sequenced, and sequencing result carries out Blast and compares analysis.
The results are shown in Figure 2 for agarose gel electrophoresis, which detects 1pg H9 subtype avian influenza diseases
Malicious RNA, 50pg duck tembusu virus RNA and 10ng duck circovirus RNA.It is more than lowest detection line that H9 is sub- in the template of each concentration
Type avian influenza virus, which expands, obtains the band that size is about 732bp, and it is about 419bp that duck tembusu virus, which expands and obtains size,
Band, duck circovirus, which expands, obtains the band that size is about 245bp, and sequencing result further demonstrates H9 hypotype fowl
Influenza virus, duck tembusu virus and duck circovirus RT-PCR amplified productions, size are respectively 732bp, 419bp and 245bp,
It is consistent with experimental design size, and the homology of the nucleic acid sequence of PCR product and the gene homologous segment of design of primers template
Unanimously.Justify the above result shows that detecting H9 subtype avian influenza virus, duck tembusu virus and duck simultaneously using triple RT-PCR
Circovirus virus has higher sensitivity.
Step 3: the specific test of triple RT-PCR
（1）The preparation of sample to be tested
Rapidly purify kit specification according to viral RNA/DNA, extraction H9AIV RNA, DTMUV RNA, DuCV RNA,
H5AIV RNA、H7AIV RNA、MPV RNA、DPV DNA、NDV RNA、DHV RNA、EDS DNA.With reference to Sambrook methods
Measure nucleic acid concentration and purity, be stored in -70 DEG C it is spare.
（2）Triple RT-PCR amplifications
According to step 1（2）PCR reaction conditions after middle optimization are expanded, the difference is that template difference is as follows：H9AIV
The mixing sample of RNA, DTMUV RNA, DuCV DNA, H9AIV RNA and DTMUV RNA（Concentration ratio 2:1）, DTMUV RNA and
The mixing sample of DuCV RNA（Concentration ratio 1:1）, the mixing sample of H9AIV RNA and DuCV RNA（Concentration ratio 2:1）, H9AIV
The mixing sample of RNA, DTMUV RNA and DuCV RNA（Concentration ratio 2:1:1）, H5AIV RNA；H7AIV RNA, MPV RNA,
DPV DNA, NDV RNA, DHV RNA, EDS DNA, negative control（ddH2O）.
After reaction, 50 μ lRT-PCR products is taken to be mixed with 5 μ l bromjophenol blues, the electrophoresis in 10g/L Ago-Gels, warp
After ethidium bromide staining, observation under ultraviolet light is taken pictures, and is made comparisons with DNA standard molecular weights, is analyzed and record result.Ultraviolet
With the segment needed for blade cutting under lamp, then purify recycling with DNA segment plastic recovery kit.Take appropriate purifying recycling
PCR product, with PMD-18T（From PME-18T kits）It is connected 4 hours in 16 DEG C, converts DH5 α escherichia coli.Picking
37 DEG C of cultures of the white colony grown on the Selective agar medium containing ampicillin, Rapid identification is carried out with PCR method, positive
Clone bacterium send Dalian Bao Sheng Bioisystech Co., Ltd to be sequenced, and sequencing result carries out Blast and compares analysis.
The results are shown in Figure 3 for agarose gel electrophoresis, and all samples nucleic acid-templated containing H9AIV, DTMUV and DuCV are equal
The amplified band being consistent with experimental design size can be amplified, i.e. H9AIV expands to obtain the purpose band that size is about 732bp,
DTMUV expands to obtain the purpose band that size is about 419bp, and DuCV expands to obtain the purpose band that size is about 245bp；And its
It compares pathogen in same position but without any amplified band.Sequencing result further demonstrates H9AIV, DTMUV and DuCV
RT-PCR amplified productions, size are respectively 732bp, 419bp and 245bp, are consistent with experimental design size, and the core of PCR product
Acid sequence is consistent with the homology of gene homologous segment of design of primers template.This using triple RT-PCR the result shows that examined
Surveying H9AIV, DTMUV and DuCV has stronger specificity.
The assembling of embodiment 3, detection kit
According to the result of study of Examples 1 and 2, assembling detection kit is with easy to use.
H9 subtype avian influenza virus, duck tembusu virus and the triple RT-PCR detection kits of duck circovirus, including inspection
Survey H9 subtype avian influenza virus, duck tembusu virus and the PCR reagent of duck circovirus, One-StepRT-PCR kits, sun
Property comparison liquid and negative controls.
The PCR reagent includes being made of H9AIV upstream and downstream primers, DTMUV upstream and downstream primers and DuCV upstream and downstream primers
PCR primer group；
The H9AIV upstream and downstream primers are the primer 1 and the primer 2 respectively；
The DTMUV upstream and downstream primers are the primer 3 and the primer 4 respectively；
The DuCV upstream and downstream primers are the primer 5 and the primer 6 respectively；
The One-Step RT-PCR kits include 2 × one-step reaction mix（It is slow containing 6 kinds of dNTP, RT reactions
Fliud flushing forms and PCR reaction buffers composition）、Trans Script one-step emzyme mix（Containing reverse transcriptase and
PCRTaq enzymes）And ddH2O；
The positive control solution is H9 subtype avian influenza virus cDNA, duck tembusu virus cDNA and duck circovirus DNA；It is described
Negative controls are ddH2O。
Embodiment 4, triple RT-PCR detect clinical sample
Using triple RT-PCR methods of foundation, 250 that 2016~2017 Jiangxi, Fujian, Henan, Anhui province duck group are collected
Part clinical sample is detected, and detection H9AIV viruses infect 13 parts（Infection rate is 6.28%）, 7 parts of duck tembusu virus infection
（Infection rate is 2.80%）, 12 parts of duck circovirus infection（Infection rate is 5.04%）, wherein H9AIV viruses and duck circovirus is mixed
Infection 2 is closed, part amplification is as shown in Figure 4.
Using conventional PCR method, 250 parts that 2016~2017 Jiangxi, Fujian, Henan, Anhui province duck group collect are faced
Bed sample is detected, and is as a result compared with triple RT-PCR testing results, the coincidence rate 100% of the two.
Therefore, primer sets of the invention, kit and the detection method thus established can be used for whether identifying sample to be tested
Infect H9 subtype avian influenza virus, duck tembusu virus and duck circovirus：If obtaining the segment of 732bp, in sample to be tested
It is on the contrary then do not have containing H9 subtype avian influenza virus；If obtaining the segment of 419bp, duck Tan Busu diseases are contained in sample to be tested
Poison, it is on the contrary then do not have；If obtaining the segment of 245bp, contain duck circovirus in sample to be tested, it is on the contrary then do not have.
Above-described embodiment is only the more excellent embodiment of the present invention, every according to the technical essence of the invention to implementing above
Any simple modification, modification and the alternate variation that example is made, belong in the range of technical solution of the present invention.
Sequence table
<110>Animal and veterinary research institute of Jiangxi Academy of Agricultural Sciences, Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>H9 subtype avian influenzas, duck Tan Busu and the triple RT-PCR detection kits of duck circovirus
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Claims (9)
1.H9 subtype avian influenza virus, duck tembusu virus and the triple RT-PCR primer groups of duck circovirus, which is characterized in that packet
Three pairs of specific primers are included, are primer 1 and primer 2, primer 3 and primer 4, primer 5 and primer 6 respectively, they are respectively provided with sequence
The base sequence of list SEQ.ID.No.1 to SEQ.ID.No.6.
2. H9 subtype avian influenza virus according to claim 1, duck tembusu virus and the triple RT-PCR of duck circovirus
Primer sets, which is characterized in that the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer
6 molar ratio is（2-1）:（2-1）:（2-1）:（2-1）:（1-0.5）:（1-0.5）.
3. H9 subtype avian influenza virus according to claim 2, duck tembusu virus and the triple RT-PCR of duck circovirus
Primer sets, which is characterized in that the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer
6 molar ratio is 2:2:1:1:1:1.
4. H9 subtype avian influenza virus as claimed in claim 3, duck tembusu virus and the triple RT-PCR of duck circovirus draw
Application of the object group in terms of PCR amplification, which is characterized in that the annealing temperature of the PCR amplification is 50.2-60 DEG C.
5. H9 subtype avian influenza virus according to claim 4, duck tembusu virus and the triple RT-PCR of duck circovirus
Application of the primer sets in terms of PCR amplification, which is characterized in that the annealing temperature of the PCR amplification is 53.4 DEG C.
6.H9 subtype avian influenza virus, duck tembusu virus and the triple RT-PCR detection kits of duck circovirus, feature exist
In including PCR reagent, the One-Step RT-PCR of detection H9 subtype avian influenza virus, duck tembusu virus and duck circovirus
Kit, positive control solution and negative controls.
7. H9 subtype avian influenza virus according to claim 6, duck tembusu virus and the triple RT-PCR of duck circovirus
Detection kit, which is characterized in that the PCR reagent includes by H9 AIV upstream and downstream primers, DTMUV upstream and downstream primers and DuCV
The PCR primer group of upstream and downstream primer composition；
The H9AIV upstream and downstream primers are the primer 1 and the primer 2 respectively；
Final concentration of the primer 1 in the PCR reagent in the PCR primer group is specially 2 μm of ol/L；
Final concentration of the primer 2 in the PCR reagent in the PCR primer group is specially 2 μm of ol/L；
The DTMUV upstream and downstream primers are the primer 3 and the primer 4 respectively；
Final concentration of the primer 3 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
Final concentration of the primer 4 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
The DuCV upstream and downstream primers are the primer 5 and the primer 6 respectively；
Final concentration of the primer 5 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
Final concentration of the primer 6 in the PCR reagent in the PCR primer group is specially 1 μm of ol/L；
The One-Step RT-PCR kits include 2 × one-step reaction mix, Trans Script one-
Step emzyme mix and ddH2O；
The positive control solution is H9 subtype avian influenza virus cDNA, duck tembusu virus cDNA and duck circovirus DNA；
The negative controls are ddH2O。
8. H9 subtype avian influenza virus as claimed in claim 7, duck tembusu virus and the triple RT-PCR inspections of duck circovirus
Application of the test agent box in terms of PCR amplification, which is characterized in that the annealing temperature of the PCR amplification is 50.2-60 DEG C.
9. H9 subtype avian influenza virus according to claim 8, duck tembusu virus and the triple RT-PCR of duck circovirus
Application of the detection kit in terms of PCR amplification, which is characterized in that the annealing temperature of the PCR amplification is 53.4 DEG C.
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Application publication date: 20180810 |