CN109593882A - For detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method - Google Patents
For detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method Download PDFInfo
- Publication number
- CN109593882A CN109593882A CN201710918669.2A CN201710918669A CN109593882A CN 109593882 A CN109593882 A CN 109593882A CN 201710918669 A CN201710918669 A CN 201710918669A CN 109593882 A CN109593882 A CN 109593882A
- Authority
- CN
- China
- Prior art keywords
- kit
- pcv3
- virus
- pcr
- pcr primer
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of PCR primer pair of specific detection PCV3 virus and contain the kit of the PCR primer pair.The PCR primer provides foundation to that can detect PCV3 viral DNA sample specifically, in high sensitivity, for the pig progress early prevention of 3 type of pig circular ring virus infection.
Description
Technical field
The invention belongs to field of virus detection, it is related to the primer pair and kit for detecting 3 type of pig circular ring virus.
Background technique
Pig circular ring virus (Porcine circoviruses, PCV) is the cricoid DNA virus of sub-thread, and genome length is about
It is one of the smallest animal DNA virus for 1.7kb.Have determined the PCV there are two types of type, i.e. 1 type of pig circular ring virus (PCV1)
With porcine circovirus 2 type (PCV2).PCV1 was sent out in PK cell culture as a kind of pollutants identification for the first time in 1974
It is existing, it is not pathogenic to pig.PCV2 was reported for the first time in 1998, can cause the pig circular ring virus 2 of pig in the clinical setting
Malicious related disease (Porcine circovirus associated diseases, PCVAD), mainly causes piglet multisystem to decline
Syndrome, pneumonia, pigskin inflammation and nephrotic syndrome and breeding difficulty are exhausted, is mainly shown as breathing, uropoiesis, enteron aisle, lymph, painstaking effort
The dysfunction of pipe, nerve, propagating system and skin causes weight huge economic loss to global pig-breeding.
However, it is separated to the circovirus that a strain virus genome is 2.0kb together in the breeding difficulty case of pig,
It confirms through follow-up test, is respectively less than with the homology of known circovirus either nucleotide or amino acid sequence
50%, according to the standard of the international virology classification committee, same virus should have >'s 75% in Circovirus
The homology of genome nucleotide sequence, the homology of amino acid sequence of the Cap protein with > 70%, therefore can affirm this
It is a kind of new pig circular ring virus.Currently, the identification for the new pig circular ring virus is mainly carried out by PCR, however, utilizing
Method in the prior art is such as special using circovirus kind when the pathological material of disease to different geographical source morbid pig detects
Property primer pair different geographical source 3 type of doubtful pig circular ring virus morbidity pathological material of disease identified missing inspection and false positive often occur,
Meanwhile the primer pair according to disclosed in the prior art is checked, and still has site morbidity pathological material of disease not identified accurately, it is existing
Also without developing the PCR detection kit at product in technology, therefore, the high detection side of a kind of high specificity, sensibility is established
Method is significant.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology with it is insufficient, a kind of detection 3 type of pig circular ring virus is provided
PCR primer pair, the PCR primer is to being SEQ for upstream primer and sequence that sequence is nucleotide sequence shown in SEQ ID NO.5
The downstream primer of nucleotide sequence shown in ID NO.10.
3 type of primer pair energy specific detection pig circular ring virus of the invention, and virus can be detected in high sensitivity.The present invention
A further object be to provide the kit of the PCR primer pair comprising detection 3 type of pig circular ring virus.
Kit of the invention is fast and convenient, high specificity, sensibility is high, testing cost is low, detection efficiency is high, false positive
It is low.Kit of the present invention can specifically detect PCV3, especially early diagnose, and be monitoring, the prevention and control of PCV3 epidemic situation
Strong technical support is provided.
Specific embodiment
Hereinafter, embodiments of the present invention will be described.
3 type of pig circular ring virus be genome 2.0kb circovirus, with known circovirus no matter nucleotide also
It is that the homology of amino acid sequence is respectively less than 50%, is a kind of new pig circular ring virus, the dermatitis nephrosis of pig can be caused comprehensive
The inflammatory reaction of sign, breeding difficulty and heart and multisystem.
The purpose of the present invention is to provide a kind of PCR primer pair for detecting PCV3 virus, the PCR primer is to for sequence
The upstream primer and sequence of nucleotide sequence shown in SEQ ID NO.5 are that the downstream of nucleotide sequence shown in SEQ ID NO.10 is drawn
Object.3 type of primer pair energy specific detection pig circular ring virus of the invention, and virus can be detected in high sensitivity.
The present invention also provides a kind of kits of specific detection PCV3 virus, wherein the kit includes institute
The PCR primer pair for the detection PCV3 virus stated, the PCR primer is to being nucleotide sequence shown in SEQ ID NO.5 for sequence
Upstream primer and sequence are the downstream primer of nucleotide sequence shown in SEQ ID NO.10.
As one embodiment of the present invention, kit of the invention further includes enzyme mixation, the enzyme mixation by
Taq archaeal dna polymerase, Tris-HCl, EDTA, DTT and glycerol composition, Taq archaeal dna polymerase, Tris-HCl, EDTA, DTT and sweet
Oil content is respectively 0.5U/ μ l, 20mM, 0.1mM, 50%V/V.
As one embodiment of the present invention, kit of the invention further includes PCR reaction solution, the PCR reaction solution
By the detection PCV3 virus PCR primer to, reaction buffer, dNTPs and ddH2O presses the volume ratio mixing group of 1:2:1:4
At.
As a kind of preferred embodiment of the invention, the PCR that PCV3 virus is detected described in kit of the invention draws
Object is 10 μM to concentration, and the reaction buffer is 5 × Tris-HCl (pH8.3), and the dDTPs concentration is 2.5mM.
As one embodiment of the present invention, kit of the invention further includes positive control and negative control.
As a kind of preferred embodiment of the invention, positive control is PCV3DNA template, yin in kit of the invention
Property control be reaction buffer.
As one embodiment of the present invention, kit of the invention further includes the examination for extracting sample to be tested DNA profiling
Agent.
As a kind of preferred embodiment of the invention, sample to be tested DNA template concentration described in kit of the invention
≥1×10-5ng/μl。
As one embodiment of the present invention, the PCR reaction system that kit of the invention uses is 20 μ l, is mixed by enzyme
Close 2 μ l of liquid, 16 μ l of PCR reaction solution and 2 μ l of DNA profiling to be measured composition.
The present invention also provides a kind of pcr amplification reaction programs for detecting 3 type of pig circular ring virus: 94 DEG C of initial denaturation 3min；35
A circulation is 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 60sec every recycling；72 DEG C of extension 10min after circulation terminates.
The present invention also provides a kind of PCR methods for detecting 3 type of pig circular ring virus, comprising the following steps:
1) DNA profiling extracts；
2) PCR reaction solution, enzyme mixation, DNA profiling are mixed to form amplification reaction system；
3) amplification system of step 2) is carried out to PCR reaction in PCR amplification instrument；
4) amplified production is subjected to agarose gel electrophoresis, observes testing result.
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more
It is clear.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
It should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form carry out
Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Used chemical reagent is that analysis is pure in the embodiment of the present invention, is purchased from Chinese medicines group.
To keep the present invention easier to understand with reference to specific embodiments the present invention is further explained.It is of the present invention
Experimental method, if being conventional method without specified otherwise；The biomaterial, if without specified otherwise, it can be from business way
Diameter obtains.
The separation of 1 pig circular ring virus of embodiment, 3 type is identified
1, pathological material of disease source
There is the sow death rate and increases by 9.4% compared with history average in a commercialization pig farm at home, pregnancy rate drop
Low 1.2%, the phenomenon that the mummification of fetus increases by 8.2%.In clinical manifestation, impacted sow shows anorexia, in multifocal
Papule, the symptom of spot and surface dermatitis.Containing the mummified fetus of different gestational age in the nest of miscarriage, draw with PCV2 infection
The symptom for playing miscarriage is consistent.Although the overall clinical performance observed in sow and miscarriage symptom and porcine circovirus 2 type
Caused breeding difficulty disease is consistent, but the different tissues of all sows, including kidney, lymph node, lung, skin and stillborn foetus, leads to
Crossing immunohistochemistry and quantitative PCR is feminine gender to PCV2, PRRSV, PPV, CSFV, mycoplasma hyopneumoniae detection.Further to look into
Clear reason chooses each tissue pathological material of disease and carries out pathogen separation.
2, the separation and culture of Strain
DMEM culture solution is added with 1:10 (volume ratio) in pathological material of disease, grinding prepares tissue suspension, tissue suspension is through repeatedly 3
After secondary freeze thawing, it is centrifuged 15min in 8000r/min, collects supernatant, again through 0.22 μm of filter membrane filter, filtrate exists supernatant
It is passed on PK15 cell, 37 DEG C of culture 1h, the DMEM culture solution for containing 2% calf serum is added in replacement, is placed in 37 DEG C and cultivates 5.
Harvest virus harvests the culture solution containing virus after 2 freeze thawing.
3, PCR and sequencing analysis identify viral species
The viral cultures for taking above step extract the nucleic acid of viral sample with nucleic acid extraction kit, use annulus disease
Seed culture of viruses Genus-specific primers carry out PCR amplification identification, the results show that PCR amplification goes out 2000bp purpose band.PCR product send survey
Sequence company carries out nucleotide sequencing, and sequencing results carry out phylogenetic analysis.The Strain full genome as the result is shown
Group sequence and amino acid sequence are with it has been reported that the homology for other circovirus crossed is less than 50%, and according to international virus
The standard of the credit class committee, same virus should have the genome nucleotide sequence of > 75% in Circovirus
Homology, the homology of amino acid sequence of the Cap protein with > 70%, therefore can affirm, it is a kind of new pig annulus
Virus, and the third circovirus found on pig body at present.
The extraction of 2 viral DNA of embodiment
1, sample acquires: the pig for dying of illness or slaughtering takes the lesions such as lung, lymph node and health portion intersection tissue；It is to be checked
Live hog takes blood 5ml with syringe.
2, sample treatment: every part of sample is handled respectively；Tissue sample processing, every part of tissue is respectively from three different positions
Sample about 1g is weighed, takes 0.02g to grind in dismembyator after shredding mixing with operating scissors, 1.5ml physiological saline is added and continues to grind
Mill, goes to after being homogenized in 1.5ml sterile centrifugation tube, and 8000rpm is centrifuged 2min, takes 100 μ l of supernatant to sterilize in 1.5ml and is centrifuged
Guan Zhong adds 200 μ l digestive juices and 20 μ l Proteinase Ks and sets in 56 DEG C of water-baths and digest 1 hour after oscillation mixes；Whole blood sample
Processing, 100 μ l of serum is taken after blood clotting, is set in 1.5ml sterile centrifugation tube.
3, sample cell is taken out, is cooled to room temperature, 300 μ lDNA combination liquid are added, and (Biospin tissue gene group DNA extracts examination
Agent box), it is mixed by inversion, whole liquid is moved into adsorption column, be stored at room temperature 3min, 10000rpm is centrifuged 30s.
4, liquid in collecting pipe is discarded, 500 μ lDNA cleaning solutions are added, 10000rpm is centrifuged 30s.Repeat the step 1 time.
5, liquid in collecting pipe is discarded, 10000rpm is centrifuged 30s, to remove remaining DNA cleaning solution.
6, adsorption column is put into new 1.5ml centrifuge tube, the 50 μ l of DNA eluent of 56 DEG C of preheatings, room is added to center
Temperature places 2min, and 10000rpm is centrifuged 30s, and liquid is template DNA in centrifuge tube.
3 specific primer screening test of embodiment
For the PCV3 virus in specificity identification different geographical source, the present inventor is directed to the multipair PCR amplification primer of PCV3,
By precious bioengineering (Dalian), Co., Ltd is synthesized.Particular sequence is shown in sequence table, and the corresponding title of each sequence, label are shown in Table 1:
PCR of the table 1 for PCV3 screens primer
The PCR reaction system of PCV3 is shown in Table 2, and reaction condition is shown in Table 3.
The PCR reaction system of table 2PCV3
The PCR reaction condition of table 3PCV3
It is any to select a pair of of upstream primer and downstream primer (such as primer pair F1/R1, F1/R2 ... ...), that can obtain simultaneously
The primer sets that specificity is good and sensibility is high are combined into screening criteria, select optimal combination.
By multiplicating, comparative test, PCV3 detection primer pair optimal combination primer pair F5/R5, sequence are selected
Respectively SEQ ID NO.5 and SEQ ID NO.10.
Embodiment 4PCV3PCR detection kit condition optimizing
Commercialization 5U/ μ l Taq enzyme generally adds 1 μ l, final concentration of 0.2U/ μ l, the present embodiment in 25 μ l reaction systems
Under conditions of further decreasing Taq enzyme final concentration, by preparing specific enzyme mixation, Lai Shixian kit testing conditions
It advanced optimizes, substantially increases sensibility and specificity.The PCR reaction system of PCV3 is shown in Table 4 after optimization.Enzyme mixation is 20
2 μ l, final concentration of 0.05U/ μ l are generally added in μ l reaction system.
The PCR reaction system of PCV3 after table 4 optimizes
Embodiment 5PCR detection kit sensibility and specificity comparative test
With the best primer pair that embodiment 3 is screened, kit 1, embodiment are assembled according to component before 3 condition optimizing of embodiment
Component assembles kit 2 after 4 condition optimizings.Compare the sensibility and specificity of two kits.
After the continuous 10 times of dilutions of the PCV3DNA that embodiment 2 is extracted, kit 1 and kit 2 respectively according to before optimization,
Optimize postcondition and carry out PCR sensitivity tests, while measuring all DNA profiling amounts with nucleic acid-protein analyzer.
DNA template concentration is respectively 1 × 10-10ng/μl、1×10-9ng/μl、1×10-8ng/μl、1×10-7ng/μl、1
×10-6ng/μl、1×10-5ng/μl、1×10-4ng/μl、1×10-3ng/μl、1×10-2ng/μl、1×10-1ng/μl.As a result
It has been shown that, 1 susceptibility of kit are minimum up to 1 × 10-5Ng/ μ l, 2 susceptibility of kit are minimum up to 1 × 10-6ng/μl.The knot
Fruit shows that the primer that the present invention screens has very high sensibility, further improves the sensibility of kit after condition optimizing.
Using kit 1 and kit 2 respectively according to before optimization, optimization postcondition to 3 type of pig circular ring virus, pig blue-ear disease
Virus, porcine pseudorabies virus, swine fever virus, pig parainfluenza virus, pig parvoviral, porcine rotavirus, pig transmissible stomach and intestine
Scorching virus carries out augmentation detection, the results show that only 3 type of the pig circular ring virus segment that expands to have obtained about 345bp, passes through sequence
It compares, it was demonstrated that amplified production is PCV3 target fragment；Other viruses are generated without amplified band.Experimental result confirms, of the invention
Primer pair F5, R5 have quite high specificity.
The detection of the doubtful PCV3 samples of embodiment 6 is compared
Take the tissue sample for picking up from the doubtful sick pig on different regions clinical onset pig farm, including 6 parts of pig brain tissue, tonsillotome
7 parts, 10 parts of lungs, 6 parts of kidney, 5 parts of lymph node, 6 parts of spleen, 5 parts of aborted fetus, are pressed respectively using kit 1 and kit 2
According to before optimization, optimization postcondition carry out the wild poison detection of PCV3.Testing result is shown in Table 5.
The detection of the clinical doubtful PCV3 infection pig sample of table 5
The result shows that kit 1 detects 24 parts of PCV3 positive pathological material of diseases, recall rate 53.3% in 45 parts of pathological material of diseases；Reagent
Box 2 can equally detect the positive pathological material of disease that kit 1 is detected, and the two coincidence rate is up to 100%；Kit 2 detects 34 parts of PCV3 sun
Venereal disease material, recall rate 75.6% are higher than the recall rate of kit 1 by 22.3%；Kit 2 detects positive and kit 1 is not examined
Pathological material of disease out is mostly the less tissue of the viral levels such as brain, spleen and kidney.2 detection sensitivity of kit after condition optimizing is bright
It is aobvious to be higher than kit 1, it can detect the lower pathological material of disease of viral level, and cannot be detected similarly for negative sample.
The clinical application of 7 kit of embodiment
It is excellent according to 2 condition of embodiment with the part primer pair (F1/R1, F2/R2, F3/R3, F4/R4) that embodiment 3 designs
Component assembles kit 3, kit 4, kit 5, kit 6 before changing.
The sample that clinical symptoms pig farm does not occur for different regions, including 18 parts of nose swab, 30 parts of serum are picked up from sampling, use
Kit 1, kit 3, kit 4, kit 5, kit 6 carry out the wild poison detection of PCV3 according to optimization precondition respectively, together
When, the wild poison detection review of PCV3 is carried out according to optimization postcondition using kit 2.Testing result is shown in Table 6.
6 kit clinical application testing result of table
The result shows that kit 1 detects 26 parts of PCV3 positive samples, recall rate 54.2% in 48 parts of pathological material of diseases；Reagent
Box 3 detects 11 parts of PCV3 positive samples, recall rate 22.9%；Kit 4 detects 12 parts of PCV3 positive samples, and recall rate is
25.0%；Kit 5 detects 18 parts of PCV3 positive samples, recall rate 37.5%；Kit 6 detects 20 parts of PCV3 positive samples
Product, recall rate 41.7%；Kit 2 can equally detect kit 1, kit 3, kit 4, kit 5,6 institute of kit
The positive pathological material of disease of detection, coincidence rate is up to 100%；Kit 2 detects 37 parts of PCV3 positive pathological material of diseases, and recall rate 77.1% is had a competition
The recall rate of agent box 1 is high by 22.9%, higher than the recall rate of kit 3 by 54.2%, higher than the recall rate of kit 4 by 52.1%, than
The recall rate of kit 5 is high by 39.6%, higher than the recall rate of kit 6 by 35.4%.Kit 2 after condition optimizing detects sensitivity
Property is apparently higher than kit 1, kit 3, kit 4, kit 5, kit 6, can detect the lower sample of viral level,
And it cannot be detected similarly for negative sample.
The detection test of 8 early infection of embodiment
Three are randomly divided into through ELISA detection PCV2, PCV3 antigen, sodium selenite 15 of negative antibody with 28~30 ages in days
Group, 5/group, the 1st group (contains 10 with PCV3SG plants3.0TCID50/ head) poison is attacked, the 2nd group (contains 10 with PCV3SG plants5.0TCID50/
Head) poison, intramuscular injection are attacked, the 3rd group of blank control group is inoculated with DMEM culture medium, each piglet isolated rearing.It is observed continuously after attacking poison
Each group piglet clinical symptoms, start that blood was collected daily carries out viral diagnosis after function poison on the 2nd day, and concrete outcome is shown in Table 7, table 8.
7 early infection of table tests clinical symptoms
The results show that the 1st group of low dosage, which attacks poison, organizes all pig clinical symptoms without exception, the 2nd group of high dose attacks poison group institute
There is pig 40.5 DEG C of duration or more high temperature on the 3rd~5 occur, appetite stimulator, spirit are depressed, coat is thick disorderly, thin and growth
Speed slows down, and the 3rd group of blank control group is without exception.
8 early infection of table tests the viral diagnosis positive time
The results show that the 2nd group of high dose attacks the full group pig of poison group in morbidity state, all kits are on day 2 or the 3rd day
It can detect the positive；Although the 1st group of low dosage attacks malicious group of clinical symptoms without exception, kit 2 after condition optimizing of the present invention
The positive can be detected in third day, and kit 1, kit 3, kit 4, kit 5, kit 6 be then before condition optimizing
At the 6th day to the 15th day, equi-time point did not detected the positive respectively；All kit detections of 3rd group of blank control group are in yin
Property；All positive test symbols, which are further sequenced, shows that attacking poison with the present invention uses strain SG pnca gene sequence consistent.
Illustrating, detection kit sensibility with higher of the present invention can be detected more early in early infection,
Pig for the infection of 3 type of pig circular ring virus carries out early prevention.
The above is only the preferred embodiment of the present invention, not does limitation in any form to the present invention, though
So the present invention is disclosed above with preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this profession
Member, in the range of not departing from technical solution of the present invention, when the technology contents using the disclosure above make a little change or repair
Decorations are the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, technology according to the present invention are real
Matter any simple modification, equivalent change and modification to the above embodiments, still fall within the range of technical solution of the present invention
It is interior.
Sequence table
<110>Luoyang Pu Laikewantai Bioisystech Co., Ltd
<120>for detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> PCV3
<400> 1
gggtgaagta acggctgtgt 20
<210> 2
<211> 19
<212> DNA
<213> PCV3
<400> 2
cygcatawgg gtcgtcttg 19
<210> 3
<211> 20
<212> DNA
<213> PCV3
<400> 3
cgggacataa atgctccaaa 20
<210> 4
<211> 20
<212> DNA
<213> PCV3
<400> 4
cgtgccgtag aagtctgtca 20
<210> 5
<211> 20
<212> DNA
<213> PCV3
<400> 5
acggtggggt catatgtgtt 20
<210> 6
<211> 20
<212> DNA
<213> PCV3
<400> 6
catgaacgtc atttccgttg 20
<210> 7
<211> 19
<212> DNA
<213> PCV3
<400> 7
ccacagragg cgctatgkc 19
<210> 8
<211> 20
<212> DNA
<213> PCV3
<400> 8
gcgctatgcc agaagaagac 20
<210> 9
<211> 20
<212> DNA
<213> PCV3
<400> 9
gcgctatgcc agaagaagac 20
<210> 10
<211> 20
<212> DNA
<213> PCV3
<400> 10
tcattacccg cctaaacgag 20
Claims (8)
1. a kind of PCR primer pair for detecting PCV3 virus, the PCR primer is to being nucleotide shown in SEQ ID NO.5 for sequence
The upstream primer and sequence of sequence are the downstream primer of nucleotide sequence shown in SEQ ID NO.10.
2. a kind of kit of specific detection PCV3 virus, wherein the kit includes detection described in claim 1
The PCR primer pair of PCV3 virus.
3. kit according to claim 2, wherein the kit further includes enzyme mixation, the enzyme mixation by
Taq archaeal dna polymerase, Tris-HCl, EDTA, DTT and glycerol composition, Taq archaeal dna polymerase, Tris-HCl, EDTA, DTT and sweet
Oil content is respectively 0.5u/ μ l, 20mM, 0.1mM, 50%V/V.
4. kit according to claim 2, wherein the kit further includes PCR reaction solution, the PCR reaction solution
By the detection PCV3 virus PCR primer to, reaction buffer, dNTPs and ddH2O presses the volume ratio mixing group of 1:2:1:4
At；Preferably, the PCR primer of the detection PCV3 virus is 10 μM to concentration, and the reaction buffer is 5 × Tris-HCl
(pH8.3), the dDTPs concentration is 2.5mM.
5. kit according to claim 2, wherein the kit further includes positive control and negative control；It is preferred that
Ground, positive control are PCV3DNA template, and negative control is reaction buffer.
6. kit according to claim 2, wherein the kit further includes the examination for extracting sample to be tested DNA profiling
Agent.
7. kit according to claim 6, wherein sample to be tested DNA template concentration >=1 × 10-5ng/μl。
8. kit according to claim 2, wherein the PCR reaction system that the kit uses is 20 μ l, is mixed by enzyme
Close 2 μ l of liquid, 16 μ l of PCR reaction solution and 2 μ l of DNA profiling to be measured composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710918669.2A CN109593882A (en) | 2017-09-30 | 2017-09-30 | For detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710918669.2A CN109593882A (en) | 2017-09-30 | 2017-09-30 | For detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109593882A true CN109593882A (en) | 2019-04-09 |
Family
ID=65956943
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710918669.2A Pending CN109593882A (en) | 2017-09-30 | 2017-09-30 | For detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109593882A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929607A (en) * | 2017-04-25 | 2017-07-07 | 华南农业大学 | A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit |
| CN107012259A (en) * | 2017-04-14 | 2017-08-04 | 华南农业大学 | A kind of PCR primer and detection method and detection kit of the type of Testing and appraisal pig circular ring virus 3 |
| CN107083450A (en) * | 2017-05-27 | 2017-08-22 | 河北农业大学 | The type PCR detection kit of pig circular ring virus 3 and detection method |
-
2017
- 2017-09-30 CN CN201710918669.2A patent/CN109593882A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107012259A (en) * | 2017-04-14 | 2017-08-04 | 华南农业大学 | A kind of PCR primer and detection method and detection kit of the type of Testing and appraisal pig circular ring virus 3 |
| CN106929607A (en) * | 2017-04-25 | 2017-07-07 | 华南农业大学 | A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit |
| CN107083450A (en) * | 2017-05-27 | 2017-08-22 | 河北农业大学 | The type PCR detection kit of pig circular ring virus 3 and detection method |
Non-Patent Citations (2)
| Title |
|---|
| RACHEL PALINSKI等: "A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure", 《J VIROL》 * |
| 徐朋丽等: "猪圆环病毒3型PCR检测方法的建立及应用", 《中国预防兽医学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636012B (en) | One boar lid his virus stain, vaccine combination and its preparation method and application | |
| CN109182278A (en) | Seneca Valley virus strain and its application | |
| CN106591242B (en) | One plant of canine parvovirus poison strain CPV-YH and its application | |
| CN107619822B (en) | Porcine epidemic diarrhea virus low virulent strain, and culture method and application thereof | |
| CN107012261A (en) | Ana 1 aviadenovirus A types and the dual EvaGreen real-time fluorescence quantitative PCRs detection primer of 2 types | |
| CN110093461A (en) | The quadruple RT-PCR detection primer and kit of four boar diarrhea virus | |
| CN103305638A (en) | Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus | |
| CN110144332A (en) | One plant of pig Delta Coronavirus Strain and its application | |
| CN107083450A (en) | The type PCR detection kit of pig circular ring virus 3 and detection method | |
| CN106978511A (en) | A kind of method for detecting feline herpetovirus | |
| CN110564880A (en) | Method for detecting live attenuated Brucella vaccine strain and wild strain and application | |
| CN107815440A (en) | Separation, the preparation and application of inactivated vaccine of pig Delta coronavirus strain | |
| CN109593883A (en) | Kit of the pig circular ring virus multiple real time fluorescence PCR detection primer to, probe and preparation | |
| CN113564133B (en) | Coxsackie virus A16 type strain and immunogenic composition and application thereof | |
| CN101886139A (en) | Double-color fluorescence polymerase chain reaction (PCR) detection method of novel influenza A virus H1N1 subtype and kit thereof | |
| CN109207438A (en) | Porcine pseudorabies virus velogen strain and its preparing the application in inactivated vaccine | |
| CN107574263A (en) | A kind of kit and method for the type of PCR quick detections pig circular ring virus 3 | |
| CN110295254A (en) | Identify the Multiplex real-time PCR primer and probe of detection Rift Valley fever virus | |
| CN107937606B (en) | Reagent and method for identifying rabies virus vaccine strain and wild strain | |
| CN109593882A (en) | For detecting the PCR primer of 3 type of pig circular ring virus to, kit and detection method | |
| CN109022371A (en) | One plant of Porcine epidemic diarrhea virus strain and its preparing the application in pig epidemic diarrhea vaccine | |
| RU2766344C1 (en) | Method for detection and identification of coronaviruses in cattle | |
| RU2607025C1 (en) | Synthetic oligonucleotide primers and method for detecting rna atypical pestivirus of cattle | |
| CN108950085A (en) | It is a kind of for detecting the primer sets and kit of 3 type of pig circular ring virus and porcine pseudorabies virus street strain | |
| CN107893056A (en) | The type vaccine strain of bovid herpesvirus 5 I and its application |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190409 |