CN108531656A - A kind of the duplex PCR detection primer and kit of quick differentiation porcine circovirus 2 type and 3 types - Google Patents
A kind of the duplex PCR detection primer and kit of quick differentiation porcine circovirus 2 type and 3 types Download PDFInfo
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a kind of the duplex PCR detection primers and kit of quick differentiation porcine circovirus 2 type and 3 types, and the invention belongs to animal virologies and technical field of molecular biology.The kit includes primer sequence shown in ID1 ~ 4 SEQ, further comprises 2 × F8 Fastlong PCR MasterMix, cDNA templates and aqua sterilisa.The duplex PCR detection kit of porcine circovirus 2 type provided by the invention and 3 types is expanded using duplex PCR, two kinds of lesions are similar and are all the viral one-time detection of DNA virus, total time control is detected at 2 hours or so, its detection method is easily operated, convenient and efficient, can achieve the purpose that quickly to detect.
Description
Technical field
The invention belongs to animal virologies and technical field of molecular biology, are specially directed to porcine circovirus 2 type and pig
Detection primer, detection kit and the application of 3 type one-step method duplex PCR of circovirus.
Background technology
Pig circular ring virus（Porcine circovirus, PCV）It is so far for a kind of covalence closed, sub-thread cyclic DNA virus
The present finds minimum animal virus.Two genotype of PCV1 and PCV2 are only found before 2015, in global range.Wherein, PCV1 quilts
It is considered porcine kidney cell PK-15 pollutants, but does not generate cytopathic effect, to pig no pathogenicity.PCV2 can cause pig annulus
Virus associated-diseases（PCVAD）, mainly there is pmws（PMWS）, pigskin is scorching and nephrotic syndrome
（PDNS）, porcine respiratory disease syndrome（PRDC）, breeding difficulty, Hypertrophic necrotizing pneumonia（PNP）With enteritis etc..Clinically
PCV2 is often with porcine reproductive and respiratory syndrome virus (PRRSV), pig parvoviral (PPV) and mycoplasma hyopneumoniae (M. hyo) etc.
Pathogen mixed infection.Meanwhile it being also easy to secondary bacterial infection after infecting PCV2, cause huge economic losses to pig breeding industry.Closely
PCV2 prevalences strain is from PCV2a to PCV2b over year, and then to the transformation of PCV2d, viral pathogenicity gradually becomes strong, to pig raising
The harm of industry aggravates.
PDNS epidemic situations are broken out in June, 2015, North Carolina commercialization pig farm.Illness sow apocleisis, skin
Multifocal papule, spot and shallow dermatitis is presented, produced fetus (including weak tire, stillborn foetus, the mummification of fetus) has similar clinical
Symptom.Palinski etc. is by new-generation sequencing (NGS) technology, and first identified goes out one kind out of illness sow and aborted fetus body
New virus, the virus and circovirus coe virus have similar genome structure and a genetic similarity, but with other circovirus
Capsid egg is less than 70% from amino acid sequence homology, according to International Commission on Virus Classification (ICTV) circovirus criteria for classification
Circle, is classified as a kind of novel circovirus, is named as PCV3.As new animal pathogen, PCV3 it is pathogenic to pig and
Effect and existing commercial PCV2 vaccines need further to be studied to its immune effect etc. in PCVAD.Existing PCV3 exists at present
The U.S., South Korea, Brazil, Italy, Poland and the south China of China, the more provinces and cities in Central China and North China are popular.
Since the 1990s, PCV2 just threatens always the development of pig breeding industry, and PCV2 is gradually presented in recent years
Go out the common popular trend of more hypotypes, keeps PCV2 prevention and control more difficult.Meanwhile the appearance of PCV3 keeps Porcine circovirus desease more multiple
Hydridization, therefore two kinds of viral popular variation tendencies of monitoring are particularly necessary for swine disease prevention and control in time, therefore the present invention is directed to establish
A kind of special, quick PCV2, PCV3 duplex PCR detection method, understands the popularity of PCV2, PCV3, to effective prevention and control
The generation of Porcine circovirus desease and prevalence, have important practical significance.
Invention content
Present invention aim to address above-mentioned technical problem, the two of a kind of quick differentiation porcine circovirus 2 type and 3 types are provided
Weight PCR detection primers and kit, the kit can quickly distinguish 3 type of porcine circovirus 2 type and pig circular ring virus, sensitive sense
High, high specificity, the generation to effective prevention and control Porcine circovirus desease and prevalence.Technology used in purpose to realize the present invention
Scheme is：
A kind of duplex PCR detection primer of quick differentiation porcine circovirus 2 type and 3 types, the duplex PCR detection primer includes pig
3 type PCR detection primers of circovurus type 2 PCR detection primers and pig circular ring virus；The porcine circovirus 2 type PCR detection primers
By with SEQ ID NO:The sense primer of nucleotide sequence shown in 1 and have SEQ ID NO:Nucleotide sequence shown in 2
Downstream primer forms；3 type PCR detection primers of the pig circular ring virus are by with SEQ ID NO:Nucleotide sequence shown in 3 it is upper
It swims primer and there is SEQ ID NO:The downstream primer of nucleotide sequence shown in 4 forms.
Preferably, the porcine circovirus 2 type PCR detection primers are the full genome conserved regions according to porcine circovirus 2 type
Domain is designed, and the target fragment size of amplification is 1155 bp；3 type PCR detection primers of the pig circular ring virus are pig annulus
The full genome conservative region of viral 3 types is designed, and the target fragment size of amplification is 651 bp.
Preferably, the duplex PCR detection primer is in the application for preparing pig circular ring virus parting detecting reagent.
The present invention also provides a kind of duplex PCR detection kits of quick differentiation porcine circovirus 2 type and 3 types, including tool
There are SEQ ID NO:1、SEQ ID NO:The sense primer of nucleotide sequence shown in 3 and have SEQ ID NO:2、SEQ ID
NO:The downstream primer of nucleotide sequence shown in 4.
Preferably, further include 2 × F8 Fastlong PCR MasterMix, cDNA templates and aqua sterilisa.
Preferably, PCR reaction conditions are in the duplex PCR detection kit：94 DEG C of 2 min of pre-degeneration；94 DEG C of changes
Property 10 s, 56.1 DEG C of 10 s of renaturation, 72 DEG C extension 10 s, 30 cycle；3 min of last 72 DEG C of extensions.
Preferably, the SEQ ID NO of the PCR detection kit:1、SEQ ID NO:2、SEQ ID NO:3 and SEQ ID
NO:Primer shown in 4 uses a concentration of 25 μm of ol/L.
Beneficial effects of the present invention are：
The duplex PCR detection kit of porcine circovirus 2 type provided by the invention and 3 types is expanded using duplex PCR, two kinds of lesions
Viral one-time detection that is similar and being all DNA virus, the control of detection total time at 2 hours or so, detection method is easily operated,
It is convenient and efficient, it can achieve the purpose that quickly to detect.The present invention can thoroughly solve similar disease caused by porcine circovirus 2 type and 3 types
Shape but the different diagnosis problem of cause of disease, are suitble to pig farm quickly to detect, and are also suitable for epidemiological survey.
The present invention provides the specific primer that two pairs are directed to porcine circovirus 2 type and 3 types, PCV2-P1/PCV2-P2 is
Porcine circovurus type 2 specific amplimer group, PCV3-P1/PCV3-P2 are 3 type specificity amplimer group of pig circular ring virus,
It can quickly, specifically be detected using its manufactured double kit single in 3 type of porcine circovirus 2 type and pig circular ring virus
There is mono- band of 1155bp in the hybrid virus of virus and the two, porcine circovirus 2 type, and 651bp occurs in 3 type of pig circular ring virus
There are two bands of 1155bp, 651bp in one band, porcine circovirus 2 type, 3 type of pig circular ring virus, two kinds of virus mixing samples.
Detection pole of the duplex PCR detection kit of porcine circovirus 2 type and 3 types of the invention to porcine circovirus 2 type
It is limited to 1.08 × 10-5μ g/ μ L, the duplex PCR detection kit of the porcine circovirus 2 type and 3 types that show the present invention is in detection pig
There is higher sensitivity when circovurus type 2,3 type.The sensitivity of conventional 3 type PCR detection kit of pig circular ring virus is
Most low energy detects 0.1597ng/ μ L, and the sensitivity of porcine circovirus 2 type of the invention and 3 type PCR differential diagnosis kits is about
For 14.8 times of the sensitivity of conventional 3 type PCR detection kit of pig circular ring virus.The porcine circovirus 2 type and 3 types of the present invention
PCR differential diagnosis kits can shorten Diagnostic Time, save diagnostic reagent and reduce use cost.
Porcine circovirus 2 type provided by the invention is consistent with 3 type PCR differential diagnosis kits testing results in 6 months,
Show that porcine circovirus 2 type of the present invention and 3 type PCR differential diagnosis kits have very high stability.Field trial proves this
Porcine circovirus 2 type and the 3 type PCR differential diagnosis kits practicabilities and applicability for inventing gained are good.
Description of the drawings
Fig. 1 is the PCR amplification of target gene as a result, wherein M：DNA molecular quality standard；1：The bis- positive groups of PCV2, PCV3
Knit sample；2：PCV2 assaypositive tissue samples；3：PCV3 assaypositive tissue samples；4：Negative control.
Fig. 2 is the different annealing temperature of PCR as a result, wherein M：DNA molecular quality standard；1：50.0℃；2：50.7℃；3：
51.9℃；4：53.8℃；5：56.1℃；6：58.0℃；7：59.2℃；8：60.0℃.
Fig. 3 is the sensitivity tests of kit PCR of the present invention as a result, wherein M：DNA molecular quality standard；1： 1.08×
10µg/μL；2：1.08×100µg/μL；3：1.08×10-1µg/μL；4：1.08×10-2µg/μL；5：1.08×10-3µg/μL；
6：1.08×10-4µg/μL；7：1.08×10-5µg/μL；8：1.08×10-6µg/μL。
Fig. 4 is the specificity test result of kit PCR of the present invention, wherein M：DNA molecular quality standard；1：PCV2、
The bis- assaypositive tissue samples of PCV3；2：PCV2 assaypositive tissue samples；3：PCV3 assaypositive tissue samples；4：Parvovirus；5：Pseudo- mad dog
Virus；6：Escherichia coli；7：Staphylococcus aureus； 8：Haemophilus parasuis； 9：Actinobacillus pleuropneumoniae；10：It is negative
Control.
Specific implementation mode
1 materials and methods
1.1 viruses and clinical sample
Pig parvoviral (PPV), porcine pseudorabies virus（PRV）, Escherichia coli, staphylococcus aureus, secondary haemophilus, chest
Film Actinobacillus strain or bacterial strain are preserved by this laboratory to be provided.Clinical sample picks up from the doubtful sense in Guangxi various regions pig farm
It contaminates the dying pig of circovirus, sick dead pig or cuts open the main organs for killing pig（Lymph node, spleen, lungs and kidney etc.）, clip group
Tissue samples about 0.5g~1.0g is placed in sterilizing mortar, is fully ground into homogenate, the 1mol/LPBS solution that sterilizing is added is made into
1：5 emulsion suspension liquids, multigelation 3 times.10 000 r/min centrifuge 5min, collect supernatant, are transferred in 1.5mL centrifuge tubes and number confession
DNA is extracted or -20 DEG C save backup.
1.2 main agents
pMD18-T Vector、BamHⅠ、HinD III, DL2000 DNA Marker, DNA gel QIAquick Gel Extraction Kit（DNA Gel
Extraction Kit）It is Dalian treasured bioengineering Co., Ltd product；2 × F8 Fastlong PCR MasterMix are
Beijing Ai Delai biotechnology Products；Virus genom DNA/RNA Rapid extraction kits, which are Axygen biotechnologies, to be had
Limit Products；Small amount plasmid extraction agent box is purchased from Beijing Tiangeng biochemical technology Co., Ltd；E.coli DH5αStrain, by
This laboratory preserves.
The design and synthesis of 1.3 primers
According to the whole genome sequence of the PCV2 and PCV3 that are logged on GenBank, using Meg Align (DNA Star),
7.0 softwares of Primer Premier separately design a pair of of specific primer for the full-length genome conservative region of PCV2 and PCV3
PCV2-P1/ PCV2-P2 and PCV3-P1/PCV3-P2.PCV2 sense primers PCV2-P1：5’-AATTTCCGCGGGCTGGCTG-
3 ', PCV2 downstream primer PCV2-P2：5 '-CCCGCCACCGTTACCGCTG-3 ', PCR amplification target fragment size are 1155bp.
PCV3 sense primers PCV3-P1：5’-TTTTCACTTAGAGAACGGACTTG-3’；PCV3 downstream primers PCV3-P2：5’-
TGAGACACAGAGCTATATTCAG-3 ', PCR amplification target fragment size are 651bp.Primer is limited by Dalian treasured bioengineering
Company synthesizes.
The extraction of 1.4 viruses/DNA of bacteria
By PRV, PPV virus liquid, Escherichia coli bacteria liquid, staphylococcus aureus bacterium solution, secondary haemophilus bacterium solution, pleuropneumonia
Actinobacillus bacterium solution and the tissue pathological material of disease handled well, according to AxyPrep Body Fluid Viral DNA/RNA Miniprep
Kit operation instructions carry out DNA extractions, and PCRs of the DNA obtained for next step reacts.
1.5 PCR（PCR）
25 μ L of PCR reaction systems, 7.5 μ L, 2 × F8 Fastlong PCR MasterMix of aqua sterilisa 12.5 μ L, 25 μ
Each 0.5 μ L of mol/L PCV2, PCV3 upstream and downstream primers, 3.0 μ L of template.It is expanded according to following condition：94 DEG C of pre-degenerations 2
min；94 DEG C of 10 s of denaturation, 56.1 DEG C of 10 s of renaturation, 72 DEG C of 10 s of extension, 30 recycle；Last 72 DEG C of extensions 3 min, PCR are total
Reaction time is about 50 minutes.10 μ L of amplified production electrophoresis in 10 g/L Ago-Gels is taken, and observes result.
1.6 specific test
It is put with the parvovirus of extraction, Pseudorabies virus, Escherichia coli, staphylococcus aureus, secondary haemophilus, pleuropneumonia
Line bacillus DNA and PCV2, PCV3, PCV2/PCV3 mixing positive material DNA are template, are expanded with the method established
Increase, to verify the specificity of this method.
1.7 sensitivity tests
1.7.1 the preparation of PCV2, PCV3 positive template
It after PCV2 and PCV3 PCR products are carried out glue recovery purifying, is cloned into pMD18-T vector, conversion DH5 α impressions
In state cell, 100 μ L bacterium solutions is taken uniformly to be coated on LA tablets, chosen after 12-18 h positive bacteria drop into LA fluid nutrient mediums into
After row expansion culture, extracting plasmid and digestion identification correctly, positive bacterium solution is sent to the sequencing of Shanghai bioengineering Co., Ltd, it will
Sequencing result is placed on progress Blast on NCBI and is determined as PCV2 and PCV3 specific fragments.Finally, positive plasmid concentration is measured,
2 kinds of plasmids are mixed according to corresponding ratio, plasmid concentration is identical after making mixing.
1.7.2 duplex PCR sensitivity tests
Will after measured and mixed two kinds of plasmids do continuous 10 times be serially diluted after, by the mixing of the various concentration after each dilution
Plasmid carries out PCR reactions under the same conditions as PCR reaction templates, measures the quick of established duplex PCR diagnostic method
Perception.
1.8 duplex PCR repetitive tests
With the duplex PCR detection method of foundation, detection 3 times is repeated to the bis- positives of PCV2 and PCV3, with verification result can
By property.
2 results
2.1 PCR amplification
Using PCV2 positives DNA, PCV3 positive DNA, PCV2 and PCV3 positives hybrid dna as template, according to the reaction system in 1.5
Duplex PCR reaction is carried out with response procedures.Electrophoresis result is shown（See Fig. 1）, the duplex PCR method established is capable of detecting when
PCV2 is positive, PCV3 is positive and the bis- positive materials of PCV2 and PCV3, amplifies about 1155 bp of PCV2 target gene, amplifies
About 651 bp of PCV3 target gene, is consistent with expected purpose clip size.
2.2 optimum annealing temperature
To obtain the best expanding effect of the reaction, 8 temperature gradients are set within the temperature range of 50-60 DEG C and carry out duplex PCR expansion
Increase, it is found that two bands are most apparent at 56.1 DEG C, it is determined that optimum annealing temperature is 56.1 DEG C（See Fig. 2）.
2.3 sensitivity tests
The concentration of pMD18-T-PCV2 and pMD18-T-PCV3 plasmids is respectively 1.8 × 10 μ g/ μ L and 2.7 × 10 μ g/ μ after measured
L, by pMD18-T-PCV2 and pMD18-T-PCV3 plasmids according to 3:2 carry out mixings, and the concentration both after mixing is 1.08 ×
10µg/µL.Continuous 10 times of dilutions are carried out using mixed plasmid as initial concentration, are then carried out according to the PCR method of foundation
Detection.The results show that the duplex PCR detection method is 1.08 × 10 to the detectable limit of PCV2, PCV3-5µg/μL（See figure
3）.
2.4 specific test results
Test result is shown（See Fig. 4）, this research establish PCR differential diagnostic methods can specifically detect PCV2, PCV3 or
Person detects PCV2 and PCV3 simultaneously, and bloodthirsty to parvovirus, Pseudorabies virus, Escherichia coli, staphylococcus aureus, pair
Bacillus, Actinobacillus pleuropneumoniae testing result without band, show that established method has preferable specificity.
2.5 duplex PCR repetitive test results
It with the duplex PCR detection method of foundation, is repeated 3 times, as a result at 1155 bp and at 651bp, has obtained high-visible
Purpose band, illustrate establish duplex PCR detection method have good stability.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection domain should be determined by the scope of protection defined in the claims.
Sequence table
<110>Veterinary Institute of Guangxi Zhuang Autonomous Region
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Claims (7)
1. a kind of duplex PCR detection primer of quick differentiation porcine circovirus 2 type and 3 types, which is characterized in that the duplex PCR
Detection primer includes 3 type PCR detection primers of porcine circovirus 2 type PCR detection primers and pig circular ring virus；The pig circular ring virus
2 type PCR detection primers are by with SEQ ID NO:The sense primer of nucleotide sequence shown in 1 and have SEQ ID NO:2 institutes
Show the downstream primer composition of nucleotide sequence；3 type PCR detection primers of the pig circular ring virus are by with SEQ ID NO:Shown in 3
The sense primer of nucleotide sequence and have SEQ ID NO:The downstream primer of nucleotide sequence shown in 4 forms.
2. quickly distinguishing the duplex PCR detection primer of porcine circovirus 2 type and 3 types according to claim 1, feature exists
In the porcine circovirus 2 type PCR detection primers are that the full genome conservative region of foundation porcine circovirus 2 type is designed, and are expanded
The target fragment size of increasing is 1155 bp；3 type PCR detection primers of the pig circular ring virus are the full bases of 3 type of pig circular ring virus
Because conservative region is designed, the target fragment size of amplification is 651 bp.
3. quickly distinguishing the duplex PCR detection primer of porcine circovirus 2 type and 3 types according to claim 1, feature exists
In the duplex PCR detection primer is in the application for preparing pig circular ring virus parting detecting reagent.
4. a kind of duplex PCR detection kit for quickly distinguishing porcine circovirus 2 type and 3 types as described in claim 1, feature
It is, including with SEQ ID NO:1、SEQ ID NO:The sense primer of nucleotide sequence shown in 3 and have SEQ ID
NO:2、SEQ ID NO:The downstream primer of nucleotide sequence shown in 4.
5. quickly distinguishing the duplex PCR detection kit of porcine circovirus 2 type and 3 types according to claim 4, feature exists
In further including 2 × F8 Fastlong PCR MasterMix, cDNA templates and aqua sterilisa.
6. the duplex PCR detection kit of the quick differentiation porcine circovirus 2 type and 3 types according to claim 4 or 5, special
Sign is that PCR reaction conditions are in the duplex PCR detection kit：94 DEG C of 2 min of pre-degeneration；94 DEG C of 10 s of denaturation,
56.1 DEG C of 10 s of renaturation, 72 DEG C of 10 s of extension, 30 recycle；3 min of last 72 DEG C of extensions.
7. the duplex PCR detection kit of the quick differentiation porcine circovirus 2 type and 3 types according to claim 4 or 5, special
Sign is, the SEQ ID NO of the PCR detection kit:1、SEQ ID NO:2、SEQ ID NO:3 and SEQ ID NO:4 institutes
Show that primer uses a concentration of 25 μm of ol/L.
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| CN109182603A (en) * | 2018-09-30 | 2019-01-11 | 广西壮族自治区兽医研究所 | A kind of the multiple PCR detection primer group and kit of quick differentiation PCV2, PCV3 and Mhp |
| CN109234454A (en) * | 2018-09-30 | 2019-01-18 | 广西壮族自治区兽医研究所 | A kind of the duplex PCR detection primer group and kit of quick differentiation PCV3 and Mhp |
| 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 |
| CN109762943A (en) * | 2019-03-22 | 2019-05-17 | 福建省农业科学院畜牧兽医研究所 | For identifying the primer sets and its application of 1,2,3 type of pig circular ring virus |
| CN109897916A (en) * | 2019-03-22 | 2019-06-18 | 福建省农业科学院畜牧兽医研究所 | The universal PCR detection primer of 3 kinds of pig circular ring virus and detection method |
| CN113046472A (en) * | 2019-12-27 | 2021-06-29 | 北京市农林科学院 | Triple PCR primer and kit for detecting porcine circovirus |
| CN113046472B (en) * | 2019-12-27 | 2023-03-28 | 北京市农林科学院 | Triple PCR primer and kit for detecting porcine circovirus |
| CN111154915A (en) * | 2020-01-06 | 2020-05-15 | 咸阳职业技术学院 | PCR differential diagnosis kit for porcine circovirus type 4 and type 3 and detection method thereof |
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