CN106086238A - The PCR detection method of porcine circovirus 2 type, the primer and detection kit - Google Patents
The PCR detection method of porcine circovirus 2 type, the primer and detection kit Download PDFInfo
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- CN106086238A CN106086238A CN201610524436.XA CN201610524436A CN106086238A CN 106086238 A CN106086238 A CN 106086238A CN 201610524436 A CN201610524436 A CN 201610524436A CN 106086238 A CN106086238 A CN 106086238A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention provides the PCR detection method of a kind of porcine circovirus 2 type, the primer and detection kit, described PCR detection method is simple to operate, technology requires low, is suitable for Clinical detection.Described PCR detection method, comprises the following steps: (1) carries out PCR amplification with the DNA of extraction in sample for template, and wherein the primer sequence of PCR amplification is: upstream 5 TGGGATGATCTACTGAGAC 3；Downstream 5 ATTTCATATGGAAATTCAG 3；(2) amplified production is carried out electrophoresis；(3) analyze, result of determination, there is 268bp amplified band in sample, and negative control without corresponding amplified band time, result be the positive.
Description
Technical field
The present invention relates to the PCR detection method of a kind of porcine circovirus 2 type, the primer and detection kit.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) is the minimum animal virus having been found that, belongs to annulus
The member of Viraceae Circovirus, for single stranded circle virus.PCV has 1 type (PCV1) and two kinds of genotype of 2 types (PCV2), its
Middle PCV1 is not pathogenic, and PCV2 is one of main pathogen causing pmws (PMWS).PMWS in
Within 1997, being found first in Canada, morbidity pig mainly shows as growth retardation, palor, and dyspnea is with death.
In China, Lang Hong force in 2000 is detected from Beijing by the method for ELISA, Hebei, Shandong, Tianjin, Jiangxi, Jilin, Henan 7 province
The 559 parts of all kinds of pig anteserum samples gathered, find that it detects positive rate 42.9%, and antibody positive rate are along with the growth in age pig year
And raise.Thereafter in November, 2002 in August, 2002, Wang Zhongtian uses the method for PCR 12 to Beijing, Tianjin, Guangdong and other places
55 parts of tissue pathological material of diseases of individual Large-scale pig farm PCV2 infection morbidity group have carried out the detection of PCV2, find in 12 pig farms 11
Morbidity swinery mainly shows as pmws, and 1 is dermatitis and nephrotic syndrome.This disease as can be seen here
Some large-scale pig farms in China generally exist.
The immune organ of PCV2 main damage infected pigs, causes decline and the disorder of body's immunity, and it is the thinnest with pig
The microorganism synergism such as small virus, hyopneumoniae virus, porcine reproductive and respiratory syndrome virus, play it pathogenic.PCV2 draws
The disease risen mainly has pmws, Corii Sus domestica inflammation and the nephrotic syndrome, reproductive and respiratory syndrome, oozes
Going out property dermatitis etc., in whole pathogenic process, PCV2 often plays Main Function.
Owing to there is no the effective Prevention and control measures for this disease at present, and this disease is often deposited with the form of subclinical infection
, so the diagnosis carrying out this disease is particularly important to the monitoring of this disease.
Summary of the invention
Goal of the invention
It is an object of the invention to provide the PCR detection method of a kind of porcine circovirus 2 type, simple to operate, technology requires low, is suitable for
In Clinical detection.
It is a further object to provide the primer of PCR detection method for described porcine circovirus 2 type.
It is a further object to provide detection kit corresponding with above-mentioned detection method.
Summary of the invention
According to the first aspect of the invention, it is provided that the PCR detection method of a kind of porcine circovirus 2 type, comprise the following steps:
(1) carrying out PCR amplification with the DNA of extraction in sample for template, wherein the primer sequence of PCR amplification is:
Upstream 5-TGGGATGATCTACTGAGAC-3；
Downstream 5-ATTTCATATGGAAATTCAG-3；
(2) amplified production is carried out electrophoresis；
(3) analyze, result of determination, there is 268bp amplified band in sample, and negative control without corresponding amplified band time, result
For the positive.
Preferably, condition and the program of PCR amplification is: 95 DEG C of denaturations 5min;94 DEG C of degeneration 30s, 43-56 DEG C of gradient
Annealing 30s, 72 DEG C extend 30s, totally 36 circulations, and last 72 DEG C extend 6min.It is further preferred that PCR amplification condition and
Program is: 95 DEG C of denaturations 5min;94 DEG C of degeneration 30s, 49 DEG C of annealing 30s, 72 DEG C of extensions 30s, totally 36 circulations, last 72
DEG C extend 6min.
Preferably, PCR reaction system is:
PCR buffer；
MgCl2Final concentration 2.5mmol/L；
DNTP final concentration 0.2mmol/L；
Taq DNA polymerase 1 unit；
Upstream and downstream primer final concentration is 0.5 μm ol/l；
Template 2.0 μ l；
Add ddH2O(distilled water) to final volume 50 μ l.
Wherein, final concentration of this area conventional parameter of Taq DNA polymerase, the final concentration of template can be by this area skill
Art personnel are tests determined, can obtain effective pcr amplification reaction as target.Described effective pcr amplification reaction refers to work as
When can carry out pcr amplification reaction containing enough target dna sequences in DNA profiling, the amount of amplified production be enough to be detected, display
Result is positive.
Described negative control can be selected by those skilled in the art, it is preferred that described PCR detection method is pseudo-with pig
Rabies virus is as negative control.
According to the second aspect of the invention, drawing of the PCR detection method for described porcine circovirus 2 type is additionally provided
Thing, its sequence is:
Upstream 5-TGGGATGATCTACTGAGAC-3；
Downstream 5-ATTTCATATGGAAATTCAG-3.
According to the third aspect of the invention we, a kind of PCR detection kit for porcine circovirus 2 type, institute are additionally provided
State test kit to include:
PCR buffer；
Primer pair, sequence is:
Upstream 5-TGGGATGATCTACTGAGAC-3,
Downstream 5-ATTTCATATGGAAATTCAG-3；
MgCl2；
dNTP；
Taq archaeal dna polymerase；
ddH2O；
Negative control；
DNA Ladder。
Preferably, described negative control is porcine pseudorabies virus.
The PCR detection method of porcine circovirus 2 type of the present invention is simple to operate, and technology requires relatively low, it is simple to clinical inspection
Surveying or Epidemiological study, the pathological material of disease of the 67 parts of doubtful PMWS in Taizhou Region detected, wherein 42 parts show as the positive, and it is positive
Rate is up to 62.6%.
Accompanying drawing explanation
Fig. 1 is that the PCR of embodiment 1 expands electrophoretogram, wherein, M:100bp DNA Ladder；1,2:PCV2 amplification is produced
Thing；
Fig. 2 is that the PCR of sensitivity tests expands electrophoretogram, wherein, M:100bp DNA Ladder；1,2,3,4,5,6,7 respectively
Represent dilution 10-1、10-2、10-3、10-4、10-5、10-6、10-7The PCV2 amplified production of multiple.
Detailed description of the invention
Explain below in conjunction with the embodiments.
1.1 main agents and instrument
Genomic DNA Rapid extraction test kit, 100bp DNA Ladder, 6 × DNA Loading Dye, dNTP Mix are
Shanghai Sheng Gong biological engineering company limited product；Taq DNA polymerase is Fermentas product, model EP0404, and concentration is 1u/
μl；25mM MgCl2And 10 × Buffer is Fermentas product；PCR amplification instrument (PTC-200rev), JS-380A coagulate automatically
Glue image analyzer, Mikro200R high-speed refrigerated centrifuge, DYY-7C type electrophresis apparatus, DYCP-31DN Agarose horizontal electrophoresis
Groove, calorstat, micropipettor etc..
The design of 1.2 primers and synthesis
With reference to the PCV2 complete genome sequence JX682407 delivered on GenBank and 5 strain PCV2 sequences of inventor's isolation identification
(GenBank accession number:: KC788750, KF039888, KF039889, KF039890, KF039891), design pair for amplification
The specific primer of PCV2 fragment, amplified fragments is positioned at 717-985bp, a length of 268bp, and the sequence of primer is:
Upstream 5-TGGGATGATCTACTGAGAC-3；
Downstream 5-ATTTCATATGGAAATTCAG-3.
The synthesis of primer is synthesized by Shanghai Sheng Gong biological engineering company limited, and-20 DEG C of Refrigerator stores are standby, and concentration is 25
μmol/L。
The extraction of 1.3 DNA profilings
Gather pathological tissues such as having the PMWS classical symptom morbidity spleen of pig, lymph node and lung, according to genomic DNA Rapid extraction
The description operation of test kit.The DNA profiling-20 DEG C extracted saves backup.
Embodiment 1
With porcine pseudorabies virus as negative control, with 1.3 DNA extracted as template, expand with above-mentioned specific primer,
Reaction system is: the MgCl of 10 × Buffer and 25mM2Each 5 μ l, 2.5mmol/L dNTP 4 μ l, Taq DNA polymerase 1 μ l, on
Downstream primer each 1 μ l, template 2.0 μ l, add ddH2O, to final volume 50 μ l, puts in PCR amplification instrument after mixing.The program of reaction
For: 95 DEG C of denaturations 5min;94 DEG C of degeneration 30s, different temperatures annealing 30s in the range of using 43-56 DEG C, 72 DEG C extend 30s,
Totally 41 circulations, last 72 DEG C extend 6min, find that when annealing temperature is 49 DEG C, expanding effect is best.After reaction terminates, by PCR
Product carries out electrophoresis on 1.5% agarose, and gel imaging system is observed.A specific band is seen at about 268bp, with
Intended in the same size.(1 in Fig. 1 is amplified production result when 49 DEG C for annealing temperature to result such as Fig. 1, and 2 is negative control
Amplified production result).
The requirement that PCR primer reclaims test kit by DNA gel is reclaimed, recovery product is sent to biotech firm and carries out
Checking order, the result of order-checking is compared with the PCV2 sequence in GenBank, and the band of result display amplification is PCV2 sequence.
Embodiment 2 specific assay
With 1.3 extract DNA, swine fever spleen drench Seedling, the DNA of porcine pseudorabies virus, swine escherichia coli DNA as template, use
The specific primer stating designed PCV2 expands according to the method for embodiment 1, electrophoresis, observed result.Result shows, institute
The primer of design is only capable of amplifying the DNA fragmentation of PCV2, about 268bp, and other amplification does not shows band.Wherein swine fever
Spleen drenches Seedling country fair big north agriculture science and technology Group Plc commercially available vaccine, the DNA of porcine pseudorabies virus and swine escherichia coli
DNA is preserved according to prior art isolation identification by applicant.
Embodiment 3 sensitivity testing
The DNA extracted 1.3 makees dilution in various degree, carries out PCR amplification, after amplification terminates by the method for embodiment 1 respectively
Electrophoresis, the sensitivity of detection PCR method is carried out with the PCR primer of 10 μ L.Result is as in figure 2 it is shown, explanation DNA profiling is passing through
10-1-10-7After dilution, the condition amplification of described optimization is used to remain to 10-4Time expand fragment, show that its sensitivity is higher.
The checking test of embodiment 4 PCR detection method
The method using embodiment 1, the 5 strain PCV2(inventor's recent years of (Taizhou, area from Soviet Union to inventor's isolation identification
City and surrounding area) morbidity pig farm is separated to the PCV2 strain of many strains different biological characteristic, and it carried out Cap
Gene sequencing (GenBank accession number: KC788750, KF039888, KF039889, KF039890, KF039891) and entering
Change tree analyze) PK15 cell culture carried out the detection of PCV2, set negative control (porcine pseudorabies virus PK15 is thin simultaneously
Born of the same parents' culture), calculate the coincidence rate with Virus Isolation method testing result.Result shows, 2 kinds of methods detect 5 strain PCV2
And negative control, negative and positive findings complies fully with, and coincidence rate is 100%.
The application of embodiment 5 PCR detection method
The method using embodiment 1, the surrounding area, Taizhou collecting Jiangsu Agri-Animal Husbandry Vocational College's teaching pets hospital is sent out
The doubtful pathological material of disease of sick pig has carried out the detection of PCV2.
Use the amplification condition of embodiment 1, the detection of PCV2 to the pathological material of disease row of the 67 parts of doubtful PMWS adopted.Result shows,
Wherein 42 parts show as the positive, and its positive rate is up to 62.6%.
The full-length genome of PCV2 is 1767bp or 1768bp.PCV2 infringement host after, it is possible to promote lymphocyte apoptosis and
The propagation of suppression lymphocyte, makes the change of lymphoblastic disappearance and subgroup, makes body immune system low, it is impossible in time
Immunogen is carried out effective immunne response, easily cause secondary infection.Therefore often breed and respiratory disorder syndrome virus with pig
The mixing such as poison (PRRSV), porcine pseudorabies virus (PRV), pig parvoviral (PPV), mycoplasma hyopneumoniae, haemophilus parasuis
Infect, increase the weight of its hazardness.Up to now, the control infected for PCV2 still lacks effective measures, and market does not also have
This disease prevented and treated by preferably vaccine and medicine.Therefore, set up diagnostic method quick, easy, to strengthening the monitoring of this disease and pre-
Anti-have extremely important meaning.
Round pcr is used widely in multiple fields, and it has stronger sensitivity, accuracy and specificity,
Diagnosis can be rapidly performed by, compensate for time-consuming, the laborious shortcoming of traditional detection method.The present invention according in GenBank
A pair specific primer of sequential design of the PCV2 issued, by optimization to its reaction condition, establishes the PCR inspection of PCV2
Survey method, has preferable specificity and sensitivity, and is detected 67 parts of clinical pathological material of diseases of Taizhou and surrounding area, its
Positive rate, up to 62.6%, illustrates that PCV2 exists certain hazardness to this area.The foundation of the method is conducive to large-scale pig farm pair
The monitoring of PCV2, the morbidity for prevention PCV2 ahead of time provides basis.
Sequence table
<110>Jiangsu Agri-Animal Husbandry Vocational College
<120>PCR detection method of porcine circovirus 2 type, the primer and detection kit
<160>2
<170>PatentIn version 3.4
<210> 1
<211>19
<212>DNA
<213>artificial sequence
<400> 1
tgggatgatc tactgagac 19
<210> 2
<211>19
<212>DNA
<213>artificial sequence
<400> 2
atttcatatg gaaattcag 19
Claims (8)
1. the PCR detection method of a porcine circovirus 2 type, it is characterised in that order comprises the following steps:
(1) carrying out PCR amplification with the DNA of extraction in sample for template, wherein the primer sequence of PCR amplification is:
Upstream 5-TGGGATGATCTACTGAGAC-3；
Downstream 5-ATTTCATATGGAAATTCAG-3；
(2) amplified production is carried out electrophoresis；
(3) analyze, result of determination, there is 268bp amplified band in sample, and negative control without corresponding amplified band time, result
For the positive.
2. PCR detection method as claimed in claim 1, it is characterised in that described PCR detection method is with porcine pseudorabies virus
As negative control.
3. PCR detection method as claimed in claim 1, it is characterised in that condition and the program of PCR amplification be: 95 DEG C of pre-changes
Property 5min;94 DEG C of degeneration 30s, 43-56 DEG C of Gradient annealing 30s, 72 DEG C extend 30s, totally 36 circulations, last 72 DEG C of extensions
6min。
4. PCR detection method as claimed in claim 3, it is characterised in that condition and the program of PCR amplification be: 95 DEG C of pre-changes
Property 5min;94 DEG C of degeneration 30s, 49 DEG C of annealing 30s, 72 DEG C extend 30s, totally 36 circulations, and last 72 DEG C extend 6min.
5. the PCR detection method as according to any one of claim 1-4, it is characterised in that PCR reaction system is:
PCR buffer；
MgCl2Final concentration 2.5mmol/L；
DNTP final concentration 0.2mmol/L；
Taq DNA polymerase 1 unit；
Upstream and downstream primer final concentration is 0.5 μm ol/l；
Template 2.0 μ l；
Add ddH2O to final volume 50 μ l.
6., for a primer for porcine circovirus 2 type PCR detection method, its sequence is:
Upstream 5-TGGGATGATCTACTGAGAC-3；
Downstream 5-ATTTCATATGGAAATTCAG-3.
7. the PCR detection kit for porcine circovirus 2 type, it is characterised in that described test kit includes:
PCR buffer；
Primer pair, sequence is:
Upstream 5-TGGGATGATCTACTGAGAC-3,
Downstream 5-ATTTCATATGGAAATTCAG-3；
MgCl2；
dNTP；
Taq archaeal dna polymerase；
ddH2O；
Negative control；
DNA Ladder。
8. PCR detection kit as claimed in claim 7, it is characterised in that described negative control is porcine pseudorabies virus.
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Cited By (3)
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| 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 |
| CN109517929A (en) * | 2018-12-21 | 2019-03-26 | 武汉科前生物股份有限公司 | Primer sets and kit for pig circular ring virus detection and 2 type partings |
| CN109913586A (en) * | 2019-03-25 | 2019-06-21 | 新乡学院 | A method of PCV-2 is detected using PCR-ELISA |
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| CN109913586A (en) * | 2019-03-25 | 2019-06-21 | 新乡学院 | A method of PCV-2 is detected using PCR-ELISA |
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