CN106811551A - The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method - Google Patents
The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method Download PDFInfo
- Publication number
- CN106811551A CN106811551A CN201710173030.6A CN201710173030A CN106811551A CN 106811551 A CN106811551 A CN 106811551A CN 201710173030 A CN201710173030 A CN 201710173030A CN 106811551 A CN106811551 A CN 106811551A
- Authority
- CN
- China
- Prior art keywords
- fadv
- quantitative pcr
- probe
- seq
- pcr detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Primer pair, probe, kit and method the invention discloses a kind of type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, the detection method is the SEQ ID No with sample DNA as template:1 and SEQ ID No:2 is primer, SEQ ID No:3 is probe, carries out fluorescent quantitative PCR, gathers fluorescence signal.Fluorescent quantitative PCR detection method of the invention can realize it is quick, accurate, convenient and swift, specifically detected for the type aviadenovirus nucleic acid of FAdV 4, sensitivity is high, high specificity, reproducible, has broad application prospects.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly to a kind of fluorescence quantitative PCR detection FAdV-4 types fowl adenopathy
The primer pair of poison, probe, kit and method.
Background technology
Aviadenovirus (Fowl adenovirus, FAdV) belongs to Adenoviridae, and A~E groups can be divided into according to antigenicity.From chicken
It is one of common infectious disease pathogens of the birds such as chicken, duck, goose that I crowds of FAdV of separation have 12 serotypes (FAdV 1-12).From
Since 2013, the country has broken out a kind of highly pathogenic FAdV for causing IBH and HPS and has infected, and mainly encroaches on 3~5 week old young
Chicken, especially encroaches on 4 week old.To in June, 2015, the disease is popular on a large scale in the whole nation, more in acute onset, and it is multiple be born in 20~
30 Day-old Broiler Chickens, 4~8d is peak mortality after morbidity, and 8~15d of the course of disease, the death rate is 20%~100%, is caused to poultry husbandry
Serious economic loss.Ox steps on cloud et al. and gathered 240 parts of samples in east and North China region in 2015, wherein being separated to 30 plants
I group I fowl adenovirus strain, the Phylogenetic tree through hexon genes shows that separation strains are respectively I crowds of C and the genotype of E two, FAdV-
4 and FAdV-8b type serotypes, and 4 type strains than 8b type strains popular, more commonly 9~December is the sick high-incidence season, China
The popularity in middle area is even more serious.Recent years, FAdV infection has been in Global prevalence, and atypia lesion and Asia can be caused to face
Bed symptom, shows as hepatitis, alpastic anemia, bleeding, slight breathing problem and egg production and declines, and serious causes chicken
Inclusion body hepatitis (IBH), pericarditis syndrome (HPS), gizzard erosion and ulcer (GEU) etc..
Since FAdV-4 types aviadenovirus breaks out, also without a kind of method of effective quick diagnosis.Detection at present
FAdV-4 type aviadenovirus, mainly still SPF chicken embryos expand poison and regular-PCR conventional method, but, the SPF chicken embryos expansion poison cycle is long,
Operation is more complicated, cumbersome, and regular-PCR it is very low for viral level in the case of be difficult to detect, therefore be badly in need of setting up a kind of
Easy to operate, quick, high sensibility detection method.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of primer of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus
To, probe, kit and method, the kit and method can it is easy, rapidly, sensitively, specifically to FAdV-4 type aviadenovirus
Detected.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, there is provided a kind of primer pair of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus,
The primer pair has such as SEQ ID No:1 and SEQ ID No:Base sequence shown in 2.
In another aspect of this invention, a kind of probe of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus is additionally provided,
The probe has such as SEQ ID No:Base sequence shown in 3.
Preferably, the 5 ' of the probe are combined with FAM, and the 3 ' of the probe is combined with BHQ1.
In another aspect of this invention, a kind of reagent of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus is additionally provided
Box, the kit includes such as SEQ ID No:1 and SEQ ID No:Primer pair and SEQ ID No shown in 2:Shown in 3
Probe.
Preferably, the kit also includes：Containing FAdV-4 type aviadenovirus ON1 strain hexon genes the 852nd~
1518 positive recombinant plasmid standard items of nucleotide sequence.
In another aspect of this invention, a kind of method of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus is additionally provided,
Comprise the following steps：With sample DNA as template, SEQ ID No:1 and SEQ ID No:2 is primer, SEQ ID No:3 is spy
Pin, carries out fluorescent quantitative PCR, gathers fluorescence signal.
Wherein in some embodiments, the reaction system of the fluorescent quantitative PCR is：Premix Ex Taq 10μ
The μ L of L, ROX Reference Dye II 0.2, the μ L of DNA profiling 2, SEQ ID No:The μ L of 1 primer 0.4, SEQ ID No:2 primers
0.4μL、SEQ ID No:The μ L of 3 probe 0.8, plus sterile purified water to 20 μ L.
Wherein in some embodiments, the reaction condition of the fluorescent quantitative PCR is：95 DEG C of predegeneration 30sec；95
DEG C denaturation 5sec, 60 DEG C annealing extend 34sec, 40 circulation.
Compared with prior art, the invention has the advantages that：
1st, fluorescent quantitative PCR detection method of the invention need to only be collected can extract the tissue of minim DNA, collection port
Chamber and cloacal swabs just can be detected, be adapted to quick epidemiology disease and learn investigation；
2nd, fluorescent quantitative PCR detection method of the invention only needs synthetic primer pair, probe to be made kit, just can be square
Just thousands of samples are efficiently detected, it is convenient cheap；
3rd, the minimum detectable template concentrations of fluorescence quantitative RT-PCR detecting method of the invention are 22.8 copy/μ L, than
Conventional PCR method is higher by about 100 times, and other common poultry diease viruses do not find cross reaction, in batch without specific amplification
1% is respectively less than with interassay coefficient of variation, detection method of the invention is illustrated and kit sensitivity is high, high specificity, repeatability
It is good, have broad application prospects.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is the PCR amplification figures of the embodiment of the present invention 1；
Fig. 2 be the embodiment of the present invention 1 FAdV-4 qPCR melt curve analysis and canonical plotting；
Fig. 3 is the FAdV-4TaqMan probe qPCR dynamic curve diagrams of the embodiment of the present invention 2；
Fig. 4 is the specific outcome figure of the FAdV-4Taqman fluorescent quantitative PCR detection methods of the embodiment of the present invention 3；
Fig. 5 is that the embodiment of the present invention 4 attacks toxin expelling scatter diagram after poison；
Fig. 6 is that the HN viruses of the embodiment of the present invention 4 respectively organize organ distribution result figure in chicken body.
Specific embodiment
In the following example, the experimental technique of unreceipted actual conditions, generally routinely condition, such as《Molecular Cloning: A Laboratory
Guide》(Pehanorm Brooker J, Russell D W, are write, Huang Peitang, and Wang Jiaxi, Zhu Houchu are waited and translated Molecular Cloning:A Laboratory guides, the 3rd
Version, Beijing：Science Press, 2002) described in method carry out.
The foundation of embodiment 1FAdV-4 type aviadenovirus TaqMan real-time fluorescence quantitative PCR detection methods
1. material
1.1 main agents and instrument
Fluorescent quantitation TaqMan Universal PCR Master Mi × be purchased from Applied Biosystems companies；
The type fluorescent quantitation instruments of Applied Biosystems 7500；RNA reverse transcription reagent box Prime Script RT kit are purchased from
Takara companies；TIANamp viral RNAs extracts kit is purchased from QIAGEN companies.
1.2 virus stains
HN strains (FAdV) and DAdV-8b from being clinically separated gained 2016.6 months, MD-CIV988, CLV-J, NDV,
IBV, MG, H9N2, H5N6 are provided by Shanghai veterinary institute.
2. method
2.1 standard plasmids build and design of primers
With reference to FAdV-4 types aviadenovirus ON1 strain hexon gene (accession number in GenBank：GU188428 852)~
Specific primer, FAdV-4-F1 are designed at 1518bp：5'-CAACTACATCGGGTTCAGGGATAACTTC-3'(SEQ ID
NO：4)；FAdV-4-667R：5'CCAGTTTCTGTGGTGGTTGAAGGGGTT-3'(SEQ ID NO：5) purpose fragment, is expanded
It is 667bp.Isolated viral DNA, amplification, recovery purifying PCR primer are extracted using genome DNA extracting reagent kit, and is connected to
PGEM-Teasy carriers, build pGEM-Teasy-hexon plasmids and using it as standard items.Meanwhile, by BLAST in NCBI
Compare analysis and look for conservative specific regions, specific primer and Taqman probes, sequence such as table 1 below are designed in this place.
The specific primer of table 1 and Taqman probe sequences
The extraction of 2.2 sample nucleics
DNA nucleic acid extractions are carried out to chicken sample lapping liquid and DNA virus, extracting method is taken out with reference to Ezup pillar viral DNAs
Extraction reagent kit specification.For RNA virus, with reference to viral RNA extracts kit specification, extract RNA is carried out anti-extracting method
Transcription synthesis cDNA：Template is the viral RNA for extracting, and influenza virus primer is Unit12A, remaining RNA virus reverse transcription primer
It is Olig (dT)18。
Reverse transcription synthesis cDNA is carried out, reverse transcription system (50 μ L) is RNA templates 10 μ L, primer 2 μ L, 5 × reverse transcription
10 μ L, d NTP (10mmol/L) of Buffer 2 μ L, Ribonuclease Inhibitor (40U/ μ L) 1 μ L, reverse transcriptase M-
The 24 μ L of μ L, DEPCH2O of MLV (200U) 1.42 DEG C of 1h, 70 DEG C of water-baths 4min, 10 DEG C of 4min.- 20 DEG C save backup.
2.3qPCR reaction systems and condition
FAdV-4 TaqMan fluorescent quantitative PCR reaction systems：2×Premi×E×Taq(Probe qPCR)10μ
L, 50 × RO × μ L of Reference Dye II 0.2, μ L, Hexon-1293F, Hexon-1387R primers (10 μ of DNA profiling 2
Mol/L) 0.8 μ L of each 0.4 μ L, FAM-probe (10 μm of ol/L), are mended to 20 μ L with sterilizing ddH2O.Reaction condition after optimization
For：95℃30sec；95 DEG C of 5sec, 60 DEG C of 35sec, and gather 40 circulations of fluorescence.
3. result
The structure and PCR results of 3.1pGEM-Teasy-hexon
Extract FAdV DNA PCR and expand 667bp fragments (see Figure 1A), recovery product connects pGEM-Teasy carriers, uses
Blue hickie screening, chooses positive bacteria sequencing, correct plasmid is sequenced and is named as pGEM-Teasy-hexon plasmids.Plasmid is built with 10
Dilute again, take 2.28 × 109~2.28copies/ μ L concentration enters performing PCR with Hexon-1293F, Hexon-1387R as primer
Amplification.Result is shown in that Figure 1B shows, PCR most low energy detects 2.28 × 103copies/μL.In Figure 1A, M:DNA molecular amount standard
(DL2000)；1：Positive control；2：Hexon genes.In Figure 1B, 1~12 is respectively the dilution of pGEM-Teasy-Hexon plasmids
2.28×109~2.28copies/ μ L；M:DNA molecular amount standard (DL2000).
3.2qPCR standard curves and melt curve analysis
PGEM-Teasy-hexon plasmids will be built with 10 times of doubling dilutions, 3 × 10 will be taken7~3 × 100Copies/ μ L, press
Above-mentioned 2.3 method carries out FAdV-4 dye method SYBR Green II qPCR, while melt curve analysis analysis is carried out, simple spike,
Without non-specific fluorescence (see Fig. 2A), illustrate that primer used is special, it is quantitative accurate without primer dimer.
Next, carrying out FAdV-4TaqManqPCR, the logarithm value and phase of initial template concentration according to above-mentioned reaction condition
The Ct values answered draw out standard curve (Fig. 2 B), as a result show：(the R of y=-3.5237 ×+35.9882=0.9976), qPCR expands
Increasing Efficiency reaches 92% in 90%-120% is interval, and this shows qPCR response availabilities and different log of gradient values and Ct values
Between good linear relationship is presented, with good linearity.
The sensitivity of embodiment 2 is detected with repeatability
With pGEM-Teasy-hexon plasmids as template, by 2.28 × 107~2.28 × 10110 times of dilutions of copies/ μ L,
Fluorescent quantitative PCR is carried out respectively, and Ct values are considered as feminine gender more than 35.Draw the standard curve of various concentrations.Result shows：Should
Method has sensitivity very high, and minimum detectable template concentrations are 22.8copies/ μ L (Fig. 3), higher than regular-PCR sensitivity
100 times.In Fig. 3,1~7 correspondence standard plasmid is with 10 times of diluted concentrations 2.28 × 107~2.28 × 101copies/μL。
With pGEM-Teasy-hexon plasmids 2.28 × 104With 2.28 × 105The DNA conducts of two dilution factors of copies/ μ L
The two dilution factors are carried out 3 replications by template in different time sections, carry out 3 repetitions simultaneously to same template every time
3 repeating holes are determined and set, real-time fluorescence quantitative PCR is carried out according to the amplification system and condition of embodiment 1.Result shows：
Within-run and between-run analysis coefficient is respectively less than 1%, illustrates that the FAdV-4 fluorescence quantifying PCR methods set up have preferably repeatability,
Result is reliable and stable (table 2).
The repeatability of table 2FAdV-4Taqman fluorescent quantitative PCR detection methods
The specificity experiments of embodiment 3FAdV-4 type aviadenovirus real time fluorescence quantifying PCR methods
Collect laboratory cause the ill common virus MD-CVI988 of poultry, CLV-J, MG, NDV, IBV, H9N2, H5N6,
FAdV-8b virus, the specificity for detecting FAdV-4 types aviadenovirus real time fluorescence quantifying PCR method of the present invention, system and
Amplification reaction condition is expanded with reference to above example 1.Result as shown in figure 4, MD-CVI988, CLV-J, MG, NDV, IBV,
H9N2, H5N6, FAdV-8b, aseptic double-distilled water, without amplification curve, are negative, the positive curve of only FAdV-4 amplifications.Show this
Other viruses of fluorescence quantifying PCR method detection do not have cross reaction with FAdV-4, further illustrate the method with very high
Specificity.
The TaqMan real time fluorescence quantifying PCR methods of the FAdV-4 type aviadenovirus that embodiment 4 is set up using the present invention enter
Row clinical sample is detected
Laboratory clinical separates adenovirus HN strains (KY379035) and takes viral dilution into 1 × 106The μ L of TCID 50/100,
53 week old SPF chickens are injected through chest muscle.The oral cavity of 3 days and cloacal swabs after poison are attacked in collection, plus the aseptic PBS concussions of 1mL are mixed
Even 12000rpm 2min, take its 200uL supernatant dirty with reference to DNA kits extraction DNA, and bloodletting 3 chicken tissues of collection
Device, weighs plus PBS makes its concentration for 0.33g/mL, takes 100uL lapping liquids and extracts DNA.Using the FAdV- for setting up
4TaqManqPCR methods, detection HN strains are bred and toxin expelling situation in chicken body.
As a result：HN strains attack poison infection SPF chickens, and toxin expelling (see Fig. 5) have occurred in 2d after infection, oral cavity and cloaca, this
Show that virus has infected the gi system of SPF chickens.Respectively organizing internal organs to collection chicken simultaneously carries out the detection discovery of viral level,
Virus respectively organizes internal organs in chicken, and reproducible has been bred, wherein liver content highest, next to that duodenum, jejunum and blind
Intestines, brain content is minimum (see Fig. 6).
Embodiment described above only expresses embodiments of the present invention, and its description is more specific and detailed, but can not
Therefore it is interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
Enclose.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>China Agriculture Academe Shanghai Veterinary Institute
<120>The primer pair of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus, probe, kit and method
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
ccagaggcgc aactttat 18
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
atgtactcgt aggtggtagg 20
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
tgcccgaccg ttacaagttt agca 24
<210> 4
<211> 28
<212> DNA
<213>Artificial sequence
<400> 4
caactacatc gggttcaggg ataacttc 28
<210> 5
<211> 27
<212> DNA
<213>Artificial sequence
<400> 5
ccagtttctg tggtggttga aggggtt 27
Claims (8)
1. a kind of primer pair of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus, it is characterised in that the primer pair has such as
SEQ ID No:1 and SEQ ID No:Base sequence shown in 2.
2. a kind of probe of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus, it is characterised in that the probe has such as SEQ
ID No:Base sequence shown in 3.
3. the probe of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus according to claim 2, it is characterised in that institute
State the 5 ' of probe and be combined with FAM, the 3 ' of the probe is combined with BHQ1.
4. a kind of kit of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus, it is characterised in that the kit is included such as
The probe described in primer pair and Claims 2 or 3 described in claim 1.
5. the kit of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus according to claim 4, it is characterised in that
The kit also includes：Contain FAdV-4 type aviadenovirus ON1 strain hexon the 852nd~1518 nucleotide sequence of gene
Positive recombinant plasmid standard items.
6. a kind of method of fluorescence quantitative PCR detection FAdV-4 type aviadenovirus, it is characterised in that comprise the following steps：With sample
Product DNA is template, SEQ ID No:1 and SEQ ID No:2 is primer, SEQ ID No:3 is probe, carries out quantitative fluorescent PCR
Amplification, gathers fluorescence signal.
7. the method for fluorescence quantitative PCR detection FAdV-4 type aviadenovirus according to claim 6, it is characterised in that institute
The reaction system for stating fluorescent quantitative PCR is：Premix Ex Taq 10μL、ROX Reference Dye II 0.2μL、
The μ L of DNA profiling 2, SEQ ID No:The μ L of 1 primer 0.4, SEQ ID No:The μ L of 2 primer 0.4, SEQ ID No:The μ L of 3 probe 0.8, plus
Sterile purified water is to 20 μ L.
8. the method for fluorescence quantitative PCR detection FAdV-4 type aviadenovirus according to claim 6, it is characterised in that institute
The reaction condition for stating fluorescent quantitative PCR is：95 DEG C of predegeneration 30sec；95 DEG C of denaturation 5sec, 60 DEG C of annealing extend 34sec,
40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710173030.6A CN106811551A (en) | 2017-03-22 | 2017-03-22 | The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710173030.6A CN106811551A (en) | 2017-03-22 | 2017-03-22 | The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106811551A true CN106811551A (en) | 2017-06-09 |
Family
ID=59115479
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710173030.6A Pending CN106811551A (en) | 2017-03-22 | 2017-03-22 | The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106811551A (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315479A (en) * | 2018-05-10 | 2018-07-24 | 佛山科学技术学院 | Ankara real-time fluorescence quantitative PCR primer pair and kit |
| CN108531651A (en) * | 2018-04-13 | 2018-09-14 | 华南农业大学 | A kind of specific primer and its application for detecting and differentiating FAdV-4 and FAdV-8b |
| CN108676921A (en) * | 2018-07-18 | 2018-10-19 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | A kind of LAMP primer group and detection method and its application for aviadenovirus detection |
| CN109207637A (en) * | 2018-09-30 | 2019-01-15 | 河南牧业经济学院 | A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method |
| CN110373496A (en) * | 2019-06-13 | 2019-10-25 | 温氏食品集团股份有限公司 | Kit and detection method for the detection of 8 type aviadenovirus of I subgroup serum |
| CN111926116A (en) * | 2020-08-12 | 2020-11-13 | 广东省农业科学院动物卫生研究所 | Primer and probe for rapidly and quantitatively detecting duck adenovirus type 4, detection method and application thereof |
| CN112048570A (en) * | 2020-08-12 | 2020-12-08 | 广东省农业科学院动物卫生研究所 | PCR primer for detecting duck adenovirus type 4 and detection method and application thereof |
| CN112195289A (en) * | 2020-11-19 | 2021-01-08 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel E-type avian adenovirus Fiber fluorescent quantitative PCR detection kit and application thereof |
| CN112646933A (en) * | 2021-01-21 | 2021-04-13 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and kit for duck type 4 adenovirus |
| CN112725533A (en) * | 2021-01-21 | 2021-04-30 | 福建省农业科学院畜牧兽医研究所 | Primer group for real-time fluorescent quantitative PCR identification of duck type 1 adenovirus and duck type 4 adenovirus |
| CN113293237A (en) * | 2021-07-01 | 2021-08-24 | 广西壮族自治区兽医研究所 | Double-fluorescence LAMP (loop-mediated isothermal amplification) primer group for FAdV-4 variant strains and non-variant strains, kit and application |
| CN114196786A (en) * | 2021-11-11 | 2022-03-18 | 佛山科学技术学院 | Poultry adenovirus type 4 and 8 dual fluorescent quantitative PCR rapid detection kit and method |
| CN114875023A (en) * | 2022-04-13 | 2022-08-09 | 广东省华晟生物技术有限公司 | Primer, probe, gene chip and kit for detecting avian adenovirus type 4 and application thereof |
| CN114990262A (en) * | 2022-06-20 | 2022-09-02 | 云南省畜牧兽医科学院 | Primer group for LAMP (loop-mediated isothermal amplification) detection of avian adenovirus serotype 4 and application of primer group |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160619A (en) * | 2013-04-10 | 2013-06-19 | 北京博海通达生物科技有限公司 | Adenovirus multi-fluorescent quantitative PCR (polymerase chain reaction) detection kit and using method thereof |
| CN103215380A (en) * | 2013-04-16 | 2013-07-24 | 武汉珈创生物技术有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting mouse adenoviruses and application |
| CN105483292A (en) * | 2016-01-20 | 2016-04-13 | 河北农业大学 | Avian adenovirus type 4 PCR detection kit and detection method thereof |
-
2017
- 2017-03-22 CN CN201710173030.6A patent/CN106811551A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160619A (en) * | 2013-04-10 | 2013-06-19 | 北京博海通达生物科技有限公司 | Adenovirus multi-fluorescent quantitative PCR (polymerase chain reaction) detection kit and using method thereof |
| CN103215380A (en) * | 2013-04-16 | 2013-07-24 | 武汉珈创生物技术有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting mouse adenoviruses and application |
| CN105483292A (en) * | 2016-01-20 | 2016-04-13 | 河北农业大学 | Avian adenovirus type 4 PCR detection kit and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 袁万哲等: "禽腺病毒4 型PCR 检测方法的建立与应用", 《中国动物检疫》 * |
| 陈珍等: "禽腺病毒４型的分离与初步鉴定", 《福建农业学报》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531651A (en) * | 2018-04-13 | 2018-09-14 | 华南农业大学 | A kind of specific primer and its application for detecting and differentiating FAdV-4 and FAdV-8b |
| CN108315479A (en) * | 2018-05-10 | 2018-07-24 | 佛山科学技术学院 | Ankara real-time fluorescence quantitative PCR primer pair and kit |
| CN108676921A (en) * | 2018-07-18 | 2018-10-19 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | A kind of LAMP primer group and detection method and its application for aviadenovirus detection |
| CN109207637A (en) * | 2018-09-30 | 2019-01-15 | 河南牧业经济学院 | A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method |
| CN110373496A (en) * | 2019-06-13 | 2019-10-25 | 温氏食品集团股份有限公司 | Kit and detection method for the detection of 8 type aviadenovirus of I subgroup serum |
| CN112048570A (en) * | 2020-08-12 | 2020-12-08 | 广东省农业科学院动物卫生研究所 | PCR primer for detecting duck adenovirus type 4 and detection method and application thereof |
| CN111926116A (en) * | 2020-08-12 | 2020-11-13 | 广东省农业科学院动物卫生研究所 | Primer and probe for rapidly and quantitatively detecting duck adenovirus type 4, detection method and application thereof |
| CN112195289A (en) * | 2020-11-19 | 2021-01-08 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel E-type avian adenovirus Fiber fluorescent quantitative PCR detection kit and application thereof |
| CN112646933A (en) * | 2021-01-21 | 2021-04-13 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and kit for duck type 4 adenovirus |
| CN112725533A (en) * | 2021-01-21 | 2021-04-30 | 福建省农业科学院畜牧兽医研究所 | Primer group for real-time fluorescent quantitative PCR identification of duck type 1 adenovirus and duck type 4 adenovirus |
| CN113293237A (en) * | 2021-07-01 | 2021-08-24 | 广西壮族自治区兽医研究所 | Double-fluorescence LAMP (loop-mediated isothermal amplification) primer group for FAdV-4 variant strains and non-variant strains, kit and application |
| CN114196786A (en) * | 2021-11-11 | 2022-03-18 | 佛山科学技术学院 | Poultry adenovirus type 4 and 8 dual fluorescent quantitative PCR rapid detection kit and method |
| CN114875023A (en) * | 2022-04-13 | 2022-08-09 | 广东省华晟生物技术有限公司 | Primer, probe, gene chip and kit for detecting avian adenovirus type 4 and application thereof |
| CN114990262A (en) * | 2022-06-20 | 2022-09-02 | 云南省畜牧兽医科学院 | Primer group for LAMP (loop-mediated isothermal amplification) detection of avian adenovirus serotype 4 and application of primer group |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106811551A (en) | The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method | |
| Chen et al. | Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duck hepatitis A virus type 1 and type 3 in ducklings | |
| CN107299155A (en) | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection | |
| CN107841575B (en) | Nano multiplex PCR method for distinguishing four serotype avian adenovirus group I | |
| CN105331742A (en) | Multiplex-PCR (polymerase chain reaction) kit for detecting six viruses of sheep and goats simultaneously | |
| WO2016078553A1 (en) | Kit for detecting and typing dengue viruses by reverse transcription pcr and detection method thereof | |
| CN108950068B (en) | Kit for identifying and detecting QX-type strains of chicken infectious bronchitis viruses | |
| Abdel-Alim et al. | Characterization of Egyptian field strains of infectious bursal disease virus | |
| Guionie et al. | Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus | |
| CN108315483A (en) | A kind of combination for distinguishing the primer and probe of duck tembusu virus street strain and vaccine strain | |
| CN112391497A (en) | Primer probe set, application thereof and kit for detecting African swine fever virus and porcine epidemic diarrhea virus | |
| CN113652505A (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| Yue et al. | Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR | |
| CN115478120A (en) | Method for simultaneously detecting nodavirus and decapod iridovirus 1 of macrobrachium rosenbergii | |
| CN103484568B (en) | Method and kit for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II | |
| CN110592278A (en) | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV | |
| CN106754911A (en) | Primer sets and its application for identifying Mycoplasma bovis, bovine viral diarrhea virus and infectious bovine rhinotrachetis virus | |
| CN110205405B (en) | Kit, primer and probe for detecting and identifying O, A and Asial types of Seneca Valley virus, foot-and-mouth disease virus | |
| CN113337643A (en) | RT-LAMP primer group and kit for detecting bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus | |
| CN105176980A (en) | Multiple PCR method for detecting seven fish viruses synchronously | |
| US20230250497A1 (en) | One-step nested pcr primers set and kit modified with locked nucleic acid for detecting african swine fever virus | |
| CN112746135A (en) | Primer probe combination and kit for detecting I group 4 avian adenovirus based on RAA technology | |
| Majo et al. | Molecular characterization of Spanish infectious bursal disease virus field isolates | |
| CN116426690A (en) | Primer composition for detecting gene causing boundary disease virus and application thereof | |
| CN108998575A (en) | The foundation of chicken parvovirus and newcastle disease virus duplex PCR detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chen Hongjun Inventor after: Wang Kai Inventor after: Li Zejun Inventor after: Liu Qinfang Inventor after: Yang Jianmei Inventor after: Teng Qiaoyang Inventor after: Li Xuesong Inventor before: Chen Hongjun Inventor before: Wei Xiaofeng Inventor before: Cai Xiaoyao Inventor before: Xiong Wei Inventor before: Hu Jianhua Inventor before: Zhang Quan |
|
| CB03 | Change of inventor or designer information | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170609 |
|
| WD01 | Invention patent application deemed withdrawn after publication |