CN108707695A - A kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit - Google Patents
A kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit Download PDFInfo
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- CN108707695A CN108707695A CN201810476127.9A CN201810476127A CN108707695A CN 108707695 A CN108707695 A CN 108707695A CN 201810476127 A CN201810476127 A CN 201810476127A CN 108707695 A CN108707695 A CN 108707695A
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- 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
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention relates to a kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kits, belong to disease of poultry field.The present invention includes the design of specific primer and probe sequence, the structure of standard plasmid, the foundation of real-time fluorescence quantitative PCR amplification method and optimization, sample DNA extraction and result detection judgement.The method that the present invention establishes the primer and probe of detection parrot young bird disease virus real-time fluorescence quantitative PCR, in the upper high sensitivity of detection parrot young bird disease virus, high specificity, reproducible, minimum detectable 30.7 copies/μ L can be used for detecting the infection of parrot young bird disease virus in clinical sample.
Description
Technical field
The present invention relates to a kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kits, belong to disease of poultry field.
Background technology
Real time fluorescence quantifying PCR method（Real time PCR）Be one kind in DNA amplification reaction, with fluorescence chemical object
The each PCR of quality detection（PCR）The method of product total amount after cycle.Test sample is treated by internal reference or outer ginseng method
The method that specific dna sequence in product carries out quantitative analysis.Real-time fluorescence quantitative PCR is to pass through fluorescence during PCR amplification
Signal is measured in real time PCR processes.Due to the exponential time base in PCR amplification, the starting of the Ct values and the template of template is copied
There are linear relationships for shellfish number.Fluorescence probe method is to detect product, the appearance of sonde method with the fluorescence labeling probe of sequence specific
So that the specificity of quantitative PCR technique is greatly improved than Standard PCR technology.What is more often referred at present has TaqMan probe, FRET
Hybridization probe (fluorescence resonance energy transmission probe) and molecular beacon（molecular Beacon）.TaqMan probe method refers to
A specific fluorescence probe is additionally incorporated when PCR amplification while pair of primers is added, the probe is only special with template
It combines to property, binding site is between two primers.5 ' ends of probe are marked with fluorescent reporter group (Reporter, R),
Such as FAM, VIC, JOE, 3 ' ends are marked with fluorescent quenching group, such as Eclipse, TAMRA.When probe is complete, 5 '
The fluorescence that end reporter group is excited through light source for instrument is just quenched by 3 ' end fluorophors of short distance, and instrument can't detect 5 ' ends
The fluorescence signal that reporter group is excited is (that is the launch wavelength of 5 ' fluorophors is exactly the absorption wave of 3 ' fluorophors
It is long, thus energy is absorbed and is transmitted to 3 ' fluorophors and sends out other fluorescence).With the progress of PCR, Taq enzyme is in chain extension
The probe combined with template is encountered in the process, and (this activity is double-stranded specific to 5 ' -3 ' 5 prime excision enzyme activity, and free is single-stranded
Probe is unaffected) probe will be cut, 5 ' end reporter group of release is free in reaction system, far from 3 ' end fluorescent quenching bases
The shielding of group, 5 ' end reporter groups emitted fluorescence signals that are stimulated can be detected by probe.That is it often expands
As soon as DNA chain is formed there are one fluorescent molecular, realizes the accumulation of fluorescence signal and PCR product forms fully synchronized, report
The intensity of signal just represents the copy number of template DNA.
Parrot young bird disease virus（Budgerigar fledgling disease polyomavirus, BFPyV）Belong to more
Tumor virus section（Family: Polyomavirida）, γ-Polyomavirus（Genus: Gammapolyomavirus）, fowl it is more
1 type of tumor virus（Species: Aves polyomavirus 1）.The cause of disease can infect multi items parrot nestling（1-3 weeks）And it leads
Cause high lethality（80% or so）, infection symptoms show as becoming thin, have difficulty in breathing, subcutaneous hemorrhage and diarrhea.BFPyV belongs to no capsule
Film, circular double stranded DNA virus, full-length genome is about 5.0 kb, encodes 5 main open reading frame（open reading
Frame, ORF）：Structural proteins（capsid protein）3（VP1, VP2 and VP3）With T antigen proteins（T antigen）2
It is a（Large T antigen and T antigen, large T antigen and small T antigens）.It, can be by 80% polyomavirus based on large T antigen
Section member（More than 100 plant）It is further divided into 4 categories.BFPyV is most earlier than America & Canada is reported in, and then Japan, meaning are big
The report for having parrot infection BFPyV is seen by profit, Hungary, Germany, Australia and Poland.China 1994 in Hubei Yunmeng and
BFPyV is reported in Qingdao for the first time, and the subsequent disease, which continues to exist in two places, distributes prevalence.The study found that birds infection polyoma disease
Poison can observe cellular damage in multiple histoorgans, but there is not yet birds infection polyomavirus can cause animal body to go out
There is very big difference in the characteristics of existing tumour, this and mammalian infections polyomavirus.Currently both at home and abroad there is not yet being based on TaqMan
Primer, probe and its method correlative study report of probe for real-time fluorescence quantitative PCR detection parrot young bird disease virus, it is of the invention
Foundation can fill up domestic and international related field blank.
Invention content
The purpose of the present invention is fill up the primer and probe of existing real-time fluorescence quantitative PCR detection parrot young bird disease virus
The blank of method correlative study report, provides a kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit and its inspection
Survey method.This method high sensitivity, stability is good, high specificity, reproducible, and minimum detectable 30.7 copies/μ L can be used for
To the Molecule Epidemiology Investigation of parrot young bird disease virus in clinical sample, for the Molecular Epidemic of clear parrot young bird disease virus
Feature provides detection method and means.
To achieve the above object, following methods are provided：
A kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit, the kit include pair of primers and probe,
The primer and probe design is as follows：
According to the parrot young bird disease virus of identification（WF-GM01 plants, GenBank accession number is GU452537）Large T antigen（Large
T antigen）The gene order segment of coding, design specificity is for the primer and probe of parrot young bird disease virus, sequence
It is as follows：
Sense primer BFPyV-F：5 '-CCATCTAAGAGTTCCTGAA -3 ',
Downstream primer BFPyV-R：5'- CTCACTATTTCTGCACATC -3'；
Probe sequence BFPyV-the P are：5 '-AACGCCTATGCCAATGTGCTG-3 ', 5 '-end mark fluorescents report base
Group FAM, 3 '-end mark fluorescent quenching group Eclipse.
PCR optimal reaction systems are：12.5 μ L of Probe qPCR Mix mixed liquors, 10 μm of ol/L up/down swim primer
（BFPyV-F and BFPyV-R）Each 0.6 μ L, 10 μm of ol/L probes（BFPyV -P）1 μ L, 1 μ L of DNA profiling, water
（Nuclease-free Water）Complement to 25 μ L.Optimum reaction condition is：95 DEG C of 1 min pre-degeneration；95℃ 5 s,60
DEG C 30s, totally 40 cycles.
Advantageous effect
The present invention carries out parrot young bird disease virus using the primer and probe of parrot young bird disease virus real-time fluorescence quantitative PCR detection
Detection, has the following advantages and effect：
1, detection is quick, efficient：The detection method can lead to after reaction without carrying out conventional agarose gel electrophoresis detection
It crosses the program carried with real-time fluorescence quantitative PCR machine and carries out result judgement.2h, and one are only needed from nucleic acid extraction to result judgement
It is secondary to be carried out at the same time 96 sample detections.It is found after being detected to 17 parts of parrot source pathological material of diseases that clinic is collected, detects 3 portions of parrots
Nautilus young bird disease virus infection is positive, positive rate 17.65%（3/17）.
2, quantitative accurate：By preparing standard items, drawing standard curve, according to parrot young bird disease disease in detection measuring samples
The Cq values of poison directly infect it parrot young bird disease virus and carry out accurate quantitative analysis.
3, high sensitivity：Minimum detectable 30.7 copies/μ L.
4, high specificity：With parrot common transmittable disease（Such as Escherichia coli, salmonella, Pasteurella, parrot annulus disease
Poison, avian influenza virus）Only there is fluorescence signal to parrot young bird disease viral diagnosis in reactionless signal.
5, reproducible：The real-time fluorescence quantitative PCR detection method of foundation carries out in the group of parrot young bird disease viral diagnosis
The coefficient of variation is 0.54%-1.79%, between-group variation coefficient 0.62%-2.33%, shows the real-time fluorescence quantitative PCR detection side established
Method is reproducible.
Description of the drawings
Fig. 1 real time fluorescent quantitatives detect the amplification curve of the PCR method of parrot young bird disease virus.1：3.07×107It copies
Shellfish/μ L；2：3.07×106Copy/μ L；3：3.07×105Copy/μ L；4：3.07×104Copy/μ L；5：3.07×103
Copy/μ L；6：3.07×102Copy/μ L；7：3.07×101Copy/μ L.
Fig. 2 real time fluorescent quantitatives detect the standard curve of the PCR method of parrot young bird disease virus.
Fig. 3 real time fluorescent quantitatives detect the specificity of the PCR method of parrot young bird disease virus.1：Parrot young bird disease virus；
2：Experimental control（Escherichia coli, salmonella, Pasteurella, parrot circovirus, avian influenza virus and blank control, due to
It does not expand, there is no Positive fluorescence signal, cannot be distinguished.）.
Specific implementation mode
The present invention will be further described for following example.
Embodiment 1
1, correlation test cause of disease
The experiment sick virus of cause of disease parrot young bird, Escherichia coli, salmonella, Pasteurella, parrot circovirus, avian flu
Poison is identified and is preserved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.
The identification of parrot young bird disease virus
2.1 sample treatment
The dove pathological material of disease of dying of illness of the somewhere of Fujian in 2016 clinic inspection carefully acquires its liver and spleen tissue, and sterilizing PBS is added
（Volume ratio is 1: 5）After milled processed, multigelation 3 times, 4 000 r/min centrifuge 20 min, and it is spare to draw supernatant.
The extraction and detection of 2.2 nucleic acid DNAs
It draws the supernatant obtained in 200 μ L 2.1 to be added in 1.5 mL centrifuge tubes, 200 μ L Proteinase Ks and 200 μ L BB5 is added
Buffer solution（It needs that 5.6 μ g Carrier RNA are added before use）, after shaking mixing, it is placed in after 56 DEG C of 30 min of water-bath with reference to north
Jing Quanshijin Bioisystech Co., Ltd EasyPure Viral DNA/RNA Kit kit specifications carry out operation and obtain disease
The nucleic acid DNA of material, and experimental control strain nucleic acid is extracted simultaneously according to the method for kit, placing -20 DEG C saves backup.Profit
With BFPyV detection primers, primer sequence is：Sense primer BFPyV-F1：5 '-CAGGCCTTATATCCTGTTTGCGTC-3 ', under
Swim primer BFPyV-R1：5 '-GATATCAAGACTGCCTATCGTCGC-3 ', it is contemplated that amplified fragments size is 298 bp.PCR is anti-
Answer 50 μ L of system：2 × TransTaq-T PCR SuperMix reaction solutions, 25 μ L, up/down swim primer（BFPyV-F1/ BFPyV-
R1,10 μm of mol/L）Each 1 μ L, 1 μ L of template, sterile deionized water are mended to 50 μ L.PCR reaction conditions：95 DEG C of pre-degenerations 5
min；94 DEG C of 50 s of denaturation, 55 DEG C of 30 s of annealing, 72 DEG C of 45 s of extension, totally 35 recycle；72 DEG C extend 10 min eventually.Instead
It is identified according to conventional agarose gel electrophoresis after answering, as a result detects that BFPyV infection is positive（It is denoted as BFPyV FJ-
2016 plants）.
, parrot young bird disease virus (PRRSV) TaqMan real-time fluorescence quantitative PCR detection method foundation
3.1 primer and probe
According to the gene order segment of parrot young bird disease virus Large T codings, the sick for parrot young bird disease of specificity is designed
The primer and probe of poison, sequence are as follows：
Sense primer BFPyV-F：5 '-CCATCTAAGAGTTCCTGAA -3 ',
Downstream primer BFPyV-R：5'- CTCACTATTTCTGCACATC -3'；
Probe sequence BFPyV-the P are：5 '-AACGCCTATGCCAATGTGCTG-3 ', 5 '-end mark fluorescents report base
Group FAM, 3 '-end mark fluorescent quenching group Eclipse.Primer and probe cures biotechnology in precious day（Beijing）Co., Ltd
Synthesis.
The Primer-BLAST analyses of primer are carried out through ncbi database.Primer-BLAST analysis shows, the present invention is set
The relevant primer high specificity of meter, without cross jamming, Pass Test expected design.
The structure of 3.2 positive criteria products
Using the gene expression characteristics of large T antigen, 7 primer-design software design primers of Oligo, sense primer LTF1 are utilized：5 '-
TGATCGATAGCGCATCCTAGT-3 ', downstream primer LTR1：5 '-AATGCAGTATTGATATGAATGTT-3 ', it is contemplated that amplification
Clip size is 649 bp.Use PCR amplification reagent（2×PCR Master MIX）The 50 μ L systems recommended are expanded,
In 2 × PCR Master Mix reaction solutions, 25 μ L, up/down swim primer（LTF1/ LTR1）（Primer concentration is 10 μm of olL-1）Each 1 μ L, extraction 1 μ L of nucleic acid (FJ-2016 plants of BFPyV) template DNA, supplement sterile deionized water is to end reaction system
50 μL.PCR amplification is carried out after mixing, amplification condition enters cycle, 94 DEG C of 50 s of denaturation, 53 after being 95 DEG C of 5 min of pre-degeneration
DEG C annealing 35s, 72 DEG C extension 45s, 30 are after circulation terminates, 72 DEG C eventually extend 10 min.
PCR after reaction, identifies PCR product with 1.0% agarose gel electrophoresis, utilizes Ago-Gel
QIAquick Gel Extraction Kit carries out gel extraction to specific target fragment.Connect according to pEASY-T1 Simple Cloning Kit clones
Kit specification is connect by the Hexon gene fragment clones to pEASY-T1 carriers of parrot young bird disease virus, random picking 8
Single bacterium falls within ampicillin（Content is 100 μ g/mL）After 14 h of LB liquid medium culture of resistance, rapid plasmid is utilized
Small extraction reagent kit extracts corresponding plasmid.Primer when using PCR amplification（LTF1/ LTR1）With condition to the plasmid of extraction into
Row PCR identifications, send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced the positive recombinant plasmid filtered out.Through
After Blast analyses, meet expected standard items of the positive recombinant plasmid as this research of experiment（P-LT）.Utilize spectrophotometer
After measuring its concentration, it is 3.07 × 10 to calculate corresponding copy number7Copy/μ L.After linearized digestion, continuous 10 times are carried out
The concentration of doubling dilution, acquisition is respectively 3.07 × 106Copy/μ L, 3.07 × 105Copy/μ L, 3.07 × 104Copy/μ L
、3.07×103Copy/μ L, 3.07 × 102Copy/μ L, 3.07 × 101Copy/μ L.
The foundation of 3.3 TaqMan real-time fluorescence quantitative PCRs reaction condition optimizations and standard curve
The real-time fluorescence quantitative PCR that 25 μ L are prepared according to Premix Ex Taq (Probe qPCR) kit specification is anti-
System is answered, in different annealing temperature（52,54,56,58,60,62 and 64 DEG C）, primer concentration（1.25,2.5,5.0,10 and 20
μmol/L）And concentration and probe concentration（1.25,2.5,5.0,10 and 20 μm of ol/L）Under, reaction condition is optimized.Respectively with standard
Product（P-LT）Content is 3.07 × 107Copy/L-3.07 × 10 μ1The standard items of copy/μ L are as template, with anti-after optimization
It answers condition to be expanded, obtains amplification kinetic curve.Using the common logarithm of standard items starting copy number as abscissa, with cycle
Number threshold value（Cycle threshold, Ct values）For ordinate, standard linear regression equation is derived（Standard curve）.
Optimal reaction system is：12.5 μ L of Probe qPCR Mix mixed liquors, up/down swim primer（BFPyV-F and
BFPyV-R）（10 μmol/L）Each 0.6 μ L, probe（BFPyV -P）（10 μmol/L）1 μ L, 1 μ L of DNA profiling, water
（Nuclease-free Water）Complement to 25 μ L.Optimum reaction condition is：95 DEG C of 1 min pre-degeneration；95℃ 5 s,60
DEG C 30s, totally 40 cycles.
From amplification kinetic curve（Fig. 1）As can be seen that the real time fluorescence quantifying PCR method lowest detection that the present invention establishes
It is limited to 30.7 copies/μ L.With the common logarithm of copy number in each concentration standards template（lgC）For abscissa, with recurring number
Threshold value（Cycle threshold, Ct values）For ordinate, parrot young bird disease virus (PRRSV) TaqMan real-time fluorescence quantitative PCR mark is obtained
Directrix curve（See Fig. 2）Linear equation slope be -3.198, related coefficient 1, amplification efficiency 105%, show establish reality
When fluorescence quantifying PCR method standard curve have good linear relationship.
3.4 specific detection
With the TaqMan real-time fluorescence quantitative PCRs condition after optimization respectively to parrot young bird disease virus and parrot other encountered pathogenics
（Such as Escherichia coli, salmonella, Pasteurella, parrot circovirus, avian influenza virus）It is detected.
As a result as it can be seen that other encountered pathogenics of parrot（Such as Escherichia coli, salmonella, Pasteurella, parrot annulus disease
Poison, avian influenza virus）Only there is fluorescence signal to parrot young bird disease viral diagnosis in reactionless signal.
3.5 repetitive test
It is respectively 3.07 × 10 to plasmid content with the real time fluorescence quantifying PCR method of foundation7、3.07×106 、3.07×105、
3.07×104 、3.07×103、3.07×102 、3.07×101The standard items of copy/μ L are detected, each plasmid content
It is repeated 3 times, in calculating group（intra-group）The coefficient of variation.The standard items of above-mentioned different plasmid contents are dispensed into postposition respectively
It in -20 DEG C of preservations, takes out every 7d, is detected with the real time fluorescence quantifying PCR method of foundation, detect 3 times altogether, calculating group
Between（inter-group）The coefficient of variation.The real-time fluorescence quantitative PCR detection method of foundation carries out parrot young bird disease viral diagnosis
The coefficient of variation is 0.54%-1.79%, between-group variation coefficient 0.62%-2.33% in group, shows the real-time fluorescence quantitative PCR established inspection
Survey method is reproducible.
Clinical application
Using foundation parrot young bird disease virus (PRRSV) TaqMan real-time fluorescence quantitative PCR detect primer and probe detection method,
It is found after being detected to 17 parts of parrot source pathological material of diseases that clinic is collected, detects that 3 parts of parrot young bird disease virus infection are positive, it is positive
Rate is 17.65%（3/17）.By the product utilization Ago-Gel of related positive real-time fluorescence quantitative PCR after reaction
QIAquick Gel Extraction Kit carries out gel extraction to specific target fragment.Connect according to pEASY-T1 Simple Cloning Kit clones
It connects kit specification target gene fragment is cloned on pEASY-T1 carriers, 8 single bacteriums of random picking fall within ammonia benzyl mould
Element（Content is 100 μ g/mL）After 14 h of LB liquid medium culture of resistance, phase is extracted using the small extraction reagent kit of rapid plasmid
The plasmid answered.The plasmid of primer pair extraction when being expanded using real-time fluorescence quantitative PCR carries out PCR identifications, the sun that will be filtered out
Property recombinant plasmid send raw work bioengineering（Shanghai）Limited liability company is sequenced.Sequencing result is carried out on NCBI
BLAST analysis verifications are the sick virus Large T genetic fragments of corresponding parrot young bird.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>A kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit
<130> 7
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
ccatctaaga gttcctgaa 19
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
ctcactattt ctgcacatc 19
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
aacgcctatg ccaatgtgct g 21
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence
<400> 4
caggccttat atcctgtttg cgtc 24
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
gatatcaaga ctgcctatcg tcgc 24
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
tgatcgatag cgcatcctag t 21
<210> 7
<211> 23
<212> DNA
<213>Artificial sequence
<400> 7
aatgcagtat tgatatgaat gtt 23
Claims (1)
1. a kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit, it is characterised in that：The kit includes one
To primer and probe, sequence is as follows：
Sense primer BFPyV-F：5 '-CCATCTAAGAGTTCCTGAA -3 ',
Downstream primer BFPyV-R：5'- CTCACTATTTCTGCACATC -3'；
Probe sequence BFPyV-the P are：5 '-AACGCCTATGCCAATGTGCTG-3 ', 5 '-end mark fluorescents report base
Group FAM, 3 '-end mark fluorescent quenching group Eclipse.
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| CN201810476127.9A CN108707695A (en) | 2018-05-17 | 2018-05-17 | A kind of parrot young bird disease virus real-time fluorescence quantitative PCR detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551847A (en) * | 2019-08-21 | 2019-12-10 | 青岛立见诊断技术发展中心 | Avian polyoma virus detection primer, detection kit, virus detection method and application |
| CN111733294A (en) * | 2020-07-27 | 2020-10-02 | 广州动物园 | Identification primer, identification method and kit for type 4 of Borna psittaci virus |
-
2018
- 2018-05-17 CN CN201810476127.9A patent/CN108707695A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| KATOH 等: ""Development of novel real-time PCR assays for detecting DNA virus infections in psittaciform birds"", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 庄青叶 扥G: ""山东一株鹦鹉幼雏病病毒全序列测定与分析"", 《现代生物医学进展》 * |
| 蒋文明 等: ""青岛即墨鹦鹉幼雏病的诊断及病毒VP1基因序列分析"", 《中国动物检疫》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551847A (en) * | 2019-08-21 | 2019-12-10 | 青岛立见诊断技术发展中心 | Avian polyoma virus detection primer, detection kit, virus detection method and application |
| CN111733294A (en) * | 2020-07-27 | 2020-10-02 | 广州动物园 | Identification primer, identification method and kit for type 4 of Borna psittaci virus |
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