CN108265126A - The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method - Google Patents
The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method Download PDFInfo
- Publication number
- CN108265126A CN108265126A CN201810032076.0A CN201810032076A CN108265126A CN 108265126 A CN108265126 A CN 108265126A CN 201810032076 A CN201810032076 A CN 201810032076A CN 108265126 A CN108265126 A CN 108265126A
- Authority
- CN
- China
- Prior art keywords
- type
- circular ring
- porcine circovirus
- pig circular
- ring virus
- 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.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of while detect the PCR method of 3 type of pig circular ring virus and porcine circovirus 2 type, two pairs of specific primers are designed with the conservative region of PCV3 and PCV2 genomes respectively, duplex PCR detection method detection method is established, to realize the purpose that 3 type of pig circular ring virus and porcine circovirus 2 type are quick and precisely detected and identified.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of 3 type of pig circular ring virus and porcine circovirus 2 type are double
The present invention relates to detection 3 types of pig circular ring virus and the primer of porcine circovirus 2 type for PCR detection method.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) is the animal virus of presently found minimum, is cyclic annular single
Chain DNA virus, can in mammalian cell autonomous replication.PCV includes two genotype [2] of PCV1 and PCV2.PCV1 is
The virus found in PK15 cell cultivation process, research are thought to pig no pathogenicity.PCV2 genomes are by 1,767-1,768 core
Thuja acid forms, and prediction has 11 open reading frame (ORF).Intergenic region (Intergenic Region, IR) is answered containing virus
Starting point (SL) loop-stem structure processed.Genome is separately encoded replicase (Rep) and capsid comprising 2 main ORF, ORF1 and ORF2
Albumen (Cap).There are a glycosylation sites for Cap N-terminals 143-145aa (NYS) positions.Rep includes three potential glycosylations
Site, respectively in 23-25aa (NPS), 256-258aa (NQT) and 286-288aa (NAT).Clinicing aspect, PCV2 master
Domestic pig and wild boar are infected, it is most of to be infected in 4-11 week old.Clinical common PMWS performances include piglet and become thin, breathe and be stranded
The difficult and apparent enlargement of lymph node.PDNS be characterized as it is red to the irregular patch and skin rashes of purple, subcutaneous hemorrhage and oedema,
Enlargement of lymph nodes, bilateral renal expansion, small cortex ecchymosis and renal plevis oedema based on superficial groin etc..
PCV3 is a kind of new gene type of PCV, reports within 2016 that PCV3 is related with myocarditis and glomerulonephritis, grinds for the first time
Study carefully discovery and unique pathogen is detected in PDNS symptoms sow and its aborted fetus body as PCV3.Hubei, Guangdong, Anhui in 2017
Provinces and cities is waited to detect the phenomenon that PCV3 infects swinery successively.PCV3 includes 2 for sub-thread without cyst membrane DNA, Genome Size 2000bp
A main open reading frame (ORF), wherein, ORF1 and ORF2 are separately encoded and form replicase protein Rep by 297 amino acid,
With 214 amino acid composition capsid protein Cap.PCV2 and PCV3Cap Argine Monohydrochloride homologys are only 30%, while studying
Middle discovery, the two mixed infection rate are 42.9%.Since PCV3 in China is newfound epidemic disease, still it is lack of pertinence at present
Detection technique and method can not carry out related aetology and epidemiological study, with the Scientific evaluation disease to the harm of swinery and
Its degree, and work up targetedly prevention and control measure.Therefore, there is an urgent need to develop fast and accurately 3 type of pig circular ring virus and pig
Circovurus type 2 detection method.
Invention content
It is an object of the invention to be directed to lacking for current 3 type of pig circular ring virus and porcine circovirus 2 type detection technique means
It is weary, a kind of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method and detection primer are provided.The detection method
Detection sensitivity respectively reach up to 508copies/ μ L and 412copies/ μ L, be 1.5 for single sample detection time
A hour can be carried out at the same time large batch of pattern detection, have good specificity, sensibility and stability, can realize pair
The purpose that 3 type of pig circular ring virus is quick and precisely detected and identified with porcine circovirus 2 type.
In one aspect of the invention, a kind of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method are provided,
Its feature includes：
(1) specificity that can be detected for 3 type of pig circular ring virus and porcine circovirus 2 type design while detect poison is drawn
Object；
(2) it will be placed in same reaction tube, use for detecting the primer of 3 type of pig circular ring virus and porcine circovirus 2 type
PCR amplification instrument carries out PCR reactions
A kind of primer for being used to detect 3 type of pig circular ring virus and porcine circovirus 2 type simultaneously, sequence are provided in the present invention
For：
PCV3F：TTACTTAGAGAACGGACTTGTAACG
PCV3R：AAATGAGACACAGAGCTATATTCAG
PCV2F：TTTCAGCGATGACGTATCCAAGGAG
PCV2R：TAGTATTCAAAGGGCACAGTGAGGG
In another aspect of this invention, the response procedures of the real-time fluorescence PCR detection of detection duck poxvirus are provided, specifically
For：98 DEG C of pre-degeneration 5min, 35 cycle denaturation；98 DEG C of 10s, 58 DEG C of 20s, 72 DEG C of 20s；72℃5min.
The present invention carries out the specific detection of 3 type of pig circular ring virus and porcine circovirus 2 type using PCR, has following excellent
Point：
(1) specificity is good, with the duplex PCR reaction system, can be amplified with PCV3 and PCV2 positive templates 649bp and
The specific band of 295bp, and using other Prevention of Common Occurrence Porcine Disease poison nucleic acid as template when do not amplify specific band；
(2) high sensitivity, detection technique of fluorescence are extremely sensitive detection techniques, and the detection sensitivity of this method is reachable
508copies/ μ L and 412copies/ μ L；
(3) easy to operate, high degree of automation, amplification and detection short time complete.
Description of the drawings
Fig. 1 is 1PCV3 and PCV2 duplex PCR annealing temperature optimum results of the embodiment of the present invention.1-6：60 DEG C of annealing temperature,
59 DEG C, 58 DEG C, 57 DEG C, 56 DEG C.
Fig. 2 is the specific test result of the test of 2PCV3 and PCV2 duplex PCR detection method of the embodiment of the present invention.
1.PCV2；2.PCV3；3.PCV3+PCV2；4~7：It is PRRSV, JEV, PRV, FMDV respectively；8：Negative control.
Fig. 3 is the sensitivity tests result of the dual PCR detection method of 3PCV3 and PCV2 of the embodiment of the present invention.1：5.08
×1010copies/μLPCV3+4.12×1010copies/μL PCV2；2：5.08×109copies/μL PCV3+4.12×
109copies/μL PCV2；3：5.08×108copies/μL PCV3+4.12×108copies/μL PCV2；4：5.08×
107copies/μL PCV3+4.12×107copies/μLPCV2；5：5.08×106copies/μL PCV3+4.12×
106copies/μL PCV2；6：5.08×105copies/μLPCV3+4.12×105copies/μLPCV2；7：5.08×
104copies/μLPCV3+4.12×104copies/μL PCV2；8：5.08×103copies/μL PCV3+4.12×
103copies/μL PCV2；9：5.08×102copies/μL PCV3+4.12×102copies/μL PCV2；10：It is negative right
According to
Specific embodiment
The present invention will be further described in detail with reference to the accompanying drawings and examples.It should be understood that these embodiments are only used
In illustrating the present invention rather than limit the scope of the invention.Test method without specific conditions in the following example is led to
Normal routinely condition.
Embodiment 1：
PCV3 and PCV2 duplex PCR annealing temperature optimum results
1st, the specific primer of PCV3 and PCV2 can be detected for the design of PCV3 and PCV2 genes：
PCV3F：TTACTTAGAGAACGGACTTGTAACG
PCV3R：AAATGAGACACAGAGCTATATTCAG
PCV2F：TTTCAGCGATGACGTATCCAAGGAG
PCV2R：TAGTATTCAAAGGGCACAGTGAGGG
Upstream and downstream primer is synthesized by Jilin Ku Mei biotech firms.
2nd, viral DNA is extracted
PCV3 porcine tissue positive pathological material of disease tissue samples are taken, sterile saline is added to be ground into tissue suspension, NM plants of bases of PCV2
Because a group extraction TakaRa MiniBEST Viral DNA/RNA Extraction Kit (Dalian treasured biotech firm) extract sample
Product DNA.
3rd, PCR is detected
By the optimization to reaction system and reaction condition, duplex PCR system is finally determined.Each reaction total volume is 25
μL：1 μ LDNA, 20 μ L gold medals Mix, 1 μ L each primer (PCV3-F/PCV3-R, PCV2-F/PCV2-R) (10 μM)：Reaction condition
For：98 DEG C of pre-degeneration 5min, 35 cycle denaturation；98 DEG C of 10s, 58 DEG C of 20s, 72 DEG C of 20s；72℃5min.
4th, result of the test
1-6：60 DEG C, 59 DEG C, 58 DEG C, 57 DEG C, 56 DEG C of annealing temperature.Show real-time fluorescence PCR detection result and expected phase
Symbol shows primer energy the specific detection PCV3 and PCV2 of design.
Embodiment 2：
The specific test of PCV3 and PCV2 duplex PCR detection methods
1st, experiment virus used and sample
PCV3 tissue samples acquire, and are provided by Guangxi animal epidemic prevention and control center veterinary laboratories.PCV2NM plants by
Military veterinary institute provides.
Other pig sources virus：Porcine reproductive and respiratory syndrome virus (Procine reproductive and
Respirator syndrome, PRRS), encephalitis B virus (Japanese encephalitis virus, JEV), pseudoabies
Viral (Pseudorabies virus, PRV), foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV) are equal
It is preserved by military veterinary institute.
2nd, viral nucleic acid extracts
PCV3 tissue samples are ground into tissue suspension.Then TakaRa MiniBEST Viral DNA/RNA are used
Extraction Kit (Dalian treasured biotech firm) extract the disease in the samples such as tissue suspension, viral cultures or vaccine respectively
Malicious nucleic acid.
3rd, the specific test of PCR detection method
The nucleic acid samples of extraction are detected respectively using the PCR detection method provided in embodiment 1, to determine side
The specificity of method.
4th, result of the test
Specific test can be expanded the results show that with the duplex PCR reaction system with PCV3 and PCV2 positive templates
Go out the specific band of 649bp and 295bp, and using other Prevention of Common Occurrence Porcine Disease poison nucleic acid as template when does not amplify specific item
Band.
Embodiment 3
The sensitivity experiment of PCV3 and PCV2 duplex PCR detection methods
1st, sample needed for experiment
Using the positive plasmid of structure as template, it is respectively 5.08 × 10 to calculate PCV3 and PCV2 plasmid copy numbers10copies/
μ L and 4.12 × 1010Two positive template plasmids are carried out 10 times of gradient dilutions by copies/ μ L respectively, double using what is established
Weight PCR method is expanded respectively.2nd, the sensitivity experiment of real-time fluorescence PCR detection method
Gradient plasmid to have diluted is examined as sample using the fluorescence PCR detecting method provided in embodiment 1
It surveys, to determine the sensitivity of method.
3rd, result of the test
This method is respectively 508copies/ μ L and 412copies/ μ L (figures to the minimal detectable concentration of PCV3 and PCV2
3)。
Attachment 1：
Attachment 2：
Claims (4)
1. PCR method that is a kind of while detecting 3 type of pig circular ring virus and porcine circovirus 2 type, feature include：
(1) it can be detected for 3 type of pig circular ring virus and porcine circovirus 2 type design while detect the specific primer of poison；
(2) it will be placed in same reaction tube for detecting the primer of 3 type of pig circular ring virus and porcine circovirus 2 type, expanded using PCR
Increase instrument and carry out PCR reactions.
2. the PCR detection method of 3 type of pig circular ring virus according to claim 1 and porcine circovirus 2 type, it is characterised in that
Specific primer described in step (1) is to sequence：
PCV3F：TTACTTAGAGAACGGACTTGTAACG
PCV3R：AAATGAGACACAGAGCTATATTCAG
PCV2F：TTTCAGCGATGACGTATCCAAGGAG
PCV2R：TAGTATTCAAAGGGCACAGTGAGGG
3. the PCR detection method of 3 type of pig circular ring virus according to claim 1 and porcine circovirus 2 type, it is characterised in that
Step (1) is described reaction PCV3649bp, PCV2295bp are carried out at the same time, according to of different sizes in same reaction tube
It distinguishes.
4. the PCR detection method of 3 type of circovirus according to claim 1 and porcine circovirus 2 type, it is characterised in that step
Suddenly the response procedures of real-time fluorescence PCR detection described in (3) are：98 DEG C of pre-degeneration 5min, 35 cycle denaturation；98 DEG C of 10s, 58
DEG C 20s, 72 DEG C of 20s；72℃5min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032076.0A CN108265126A (en) | 2018-01-12 | 2018-01-12 | The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032076.0A CN108265126A (en) | 2018-01-12 | 2018-01-12 | The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108265126A true CN108265126A (en) | 2018-07-10 |
Family
ID=62775557
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810032076.0A Withdrawn CN108265126A (en) | 2018-01-12 | 2018-01-12 | The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108265126A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998577A (en) * | 2018-09-18 | 2018-12-14 | 上海市动物疫病预防控制中心 | A kind of kit for detecting porcine circovirus 2 type and 3 types, primer pair, probe and method |
| CN109055619A (en) * | 2018-10-12 | 2018-12-21 | 华南农业大学 | For detecting double PCR primer, method and the kit of 3 type of pig circular ring virus and non-typical swine fever virus |
| CN109735665A (en) * | 2019-03-22 | 2019-05-10 | 福建省农业科学院畜牧兽医研究所 | For detecting real-time fluorescence quantitative PCR-HRM primer of PCV1 and PCV3 |
| CN113718064A (en) * | 2021-10-22 | 2021-11-30 | 贵州傲农七环畜牧养殖有限公司 | Probe primer combination, kit and application for identifying PCV2 and PCV3 |
| CN115478118A (en) * | 2022-06-28 | 2022-12-16 | 四川农业大学 | Taqman multiplex fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107338331A (en) * | 2017-08-30 | 2017-11-10 | 江西农业大学 | The multiple PCR detection primer pair and kit of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 |
-
2018
- 2018-01-12 CN CN201810032076.0A patent/CN108265126A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107338331A (en) * | 2017-08-30 | 2017-11-10 | 江西农业大学 | The multiple PCR detection primer pair and kit of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 |
Non-Patent Citations (1)
| Title |
|---|
| 徐朋丽等: ""猪圆环病毒2/3型双重PCR检测方法的建立及初步应用"", 《中国畜牧兽医学会动物传染病学分会第十七次全国学术研讨会论文集》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998577A (en) * | 2018-09-18 | 2018-12-14 | 上海市动物疫病预防控制中心 | A kind of kit for detecting porcine circovirus 2 type and 3 types, primer pair, probe and method |
| CN109055619A (en) * | 2018-10-12 | 2018-12-21 | 华南农业大学 | For detecting double PCR primer, method and the kit of 3 type of pig circular ring virus and non-typical swine fever virus |
| CN109735665A (en) * | 2019-03-22 | 2019-05-10 | 福建省农业科学院畜牧兽医研究所 | For detecting real-time fluorescence quantitative PCR-HRM primer of PCV1 and PCV3 |
| CN113718064A (en) * | 2021-10-22 | 2021-11-30 | 贵州傲农七环畜牧养殖有限公司 | Probe primer combination, kit and application for identifying PCV2 and PCV3 |
| CN115478118A (en) * | 2022-06-28 | 2022-12-16 | 四川农业大学 | Taqman multiplex fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108265126A (en) | The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method | |
| CN101260442B (en) | Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus | |
| CN105331742B (en) | It is a kind of for detecting six kinds of viral multiple PCR reagent kits of sheep and goat simultaneously | |
| CN101307367B (en) | Technology for rapidly detecting porcine circovirus type2 | |
| Seah et al. | Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens | |
| CN108531656A (en) | A kind of the duplex PCR detection primer and kit of quick differentiation porcine circovirus 2 type and 3 types | |
| CN107557494A (en) | Differentiate the primer of detection diarrhea of pigs coronavirus and multiple RT PCR methods | |
| CN107236824A (en) | A kind of fluorescent quantitation RT PCR kits and application for porcine reproductive and respiratory syndrome virus specific detection | |
| CN106947834B (en) | Multiplex PCR method for detecting six duck susceptibility viruses | |
| CN110592278A (en) | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV | |
| CN113122655A (en) | TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for African swine fever virus EP402R gene | |
| Qiu et al. | The prevalence of porcine teschovirus in the pig population in northeast of China | |
| CN114015805A (en) | Fluorescent RT-RAA primer and kit for detecting Getavirus and application of fluorescent RT-RAA primer and kit | |
| CN113174446A (en) | One-step double RT-PCR detection method for bovine viral diarrhea virus typing | |
| CN105602991B (en) | Japanese encephalitis pseudovirus and preparation method thereof | |
| CN104263852B (en) | Fluorescence quantitative PCR detection pig breathes breeding difficulty disease association virus kit | |
| CN106435032A (en) | Double RT-PCR (reverse transcription-polymerase chain reaction) primer, kit and method for amplifying North America and European porcine blue ear disease viruses | |
| CN110592285A (en) | RAA primer probe for detecting Nipah virus and detection method | |
| CN103255234B (en) | Kit for detecting orf virus of sheep and detection method thereof | |
| Zhu et al. | Identification and rapid diagnosis of the pathogen responsible for haemorrhagic disease of the gill of Allogynogenetic crucian carp | |
| CN103215389B (en) | Porcine reproductive and respiratory syndrome and porcine Japanese B encephalitis dual one-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) diagnosis kit | |
| CN109182596A (en) | A kind of the reverse transcription polymerase spiral response isothermal detection reagent and detection method of Porcine epidemic diarrhea virus | |
| CN108588275A (en) | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings | |
| CN110735005B (en) | SIV and PRRSV multiple RT-PCR rapid detection kit and primer | |
| CN109517929B (en) | Primer group and kit for porcine circovirus detection and type2 typing |
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 | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180710 |
|
| WW01 | Invention patent application withdrawn after publication |