CN109706267A - A kind of universal pig parvoviral nest-type PRC primer sets and its application - Google Patents
A kind of universal pig parvoviral nest-type PRC primer sets and its application Download PDFInfo
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Abstract
The present invention provides a kind of universal pig parvoviral nest-type PRC primer sets and its applications, including Outside primer PPV-O-F, PPV-O-R and inner primer PPV-I-F, PPV-I-R；The method that the present invention carries out nest-type PRC detection pig parvoviral using above-mentioned nest-type PRC primer sets includes the following steps: sample to be tested extraction DNA obtaining sample DNA；The sample DNA is subjected to twice PCR amplification, first time PCR amplification uses Outside primer PPV-O-F and PPV-O-R, and second of PCR amplification uses inner primer PPV-I-F and PPV-I-R；Each PCR product carries out electrophoresis detection pig parvoviral.Nest-type PRC primer sets provided by the present invention are two pairs of specific primers that base sequence is designed in the Viral structural protein VP2 for pig parvoviral, and are expanded to a variety of virus causing diseases, and only pig parvoviral can amplify specific band, have specificity.Nested PCR detection method high specificity provided by the invention, high sensitivity, good in anti-interference performance, and highly reliable, it is low to overcome regular-PCR detection sensitivity, poor specificity problem.
Description
Technical field
The present invention relates to molecular diagnostic techniques fields, and in particular, to a kind of universal pig parvoviral nest-type PRC primer
Group and its application.
Background technique
Pig parvoviral (porcine circovirus 2, PPV) is the breeding difficulty disease that can cause pig, is to threaten at present
One of the important pathogen of world's pig breeding industry.The sow of PPV main infection first half period of gestation, causes Sow abortion, embryonic death, fetus
Deformity, mummification, weak tire and the harm such as infertile, while can also cause the dermatitis and diarrhea of piglet.PPV can infect each stage
Pig, but in addition to farrowing sow and piglet, without obvious clinical symptoms after the pig infection in other stages.Mainly pass through respiratory tract and disappears
Change road infection, fetus can also be transmitted to by placenta.Spermatid, spermatic cord, epididymis and the sexual accessory gland of infection boar may separate out disease
Poison, when breeding, are easily transmitted to susceptible sow.Once this occurs after being ill, pig farm may constantly occur sow breeding for successive years and lose
It loses.The generation of the disease brings weight huge economic loss to pig breeding industry, therefore the diagnosis and the superseded of sick pig of PPV infection are become
The emphasis of prevention and treatment.
Pig parvoviral (PPV) belongs to Parvoviridae parvovirus category, which is sub-thread threadiness minus-strand dna virus, nothing
Cyst membrane, appearance are made of in hexagon or circle, icosahedron axis cubic symmetry, core clothing 32 capsomeres.Genome Size is about.
Only one current serotype of PPV.PPV genome is sub-thread minus-strand dna, size about 5000bp, wherein there is 2 main openings to read
Frame is separately encoded structural proteins VP and non-structural protein NS.In recent years, domestic and foreign scholars establish hemagglutination test, in serum
And test, enzyme-linked immunosorbent assay, immunofluorescent test, regular-PCR diagnosis, nucleic acid probe, SYBRGreen real-time fluorescence are fixed
Measure the detection that the technologies such as PCR carry out PPV.Cost is relatively low for regular-PCR method, easy to operate, is widely applied.But it is common
There are the lower such problems of sensitivity for PCR diagnosis, and viral level is very low in PPV subclinical infection swinery, and regular-PCR is sensitive
It spends poor, easily causes missing inspection.
Summary of the invention
The purpose of the invention is to overcome the defect of regular-PCR detection pig parvoviral of the existing technology, provide
A kind of universal pig parvoviral nest-type PRC primer sets and its application, nest-type PRC primer sets provided by the present invention are for pig
Two pairs of specific primers that base sequence is designed in the Viral structural protein VP2 of parvovirus, and a variety of pathogens are tested
Card, only pig parvoviral can amplify specific band, have specificity.
To achieve the goals above, in the embodiment on basis, the present invention provides a kind of universal pig parvoviral nest
Formula PCR primer group, including Outside primer PPV-O-F, PPV-O-R and inner primer PPV-I-F, PPV-I-R；Wherein, described outer
The sequence of side primer PPV-O-F is as shown in SEQ ID NO:1, the sequence of the Outside primer PPV-O-R such as SEQ ID NO:2 institute
Show, the sequence of the inner primer PPV-I-F is as shown in SEQ ID NO:3, the inner primer PPV-I-R such as SEQ ID NO:
Shown in 4.
Another aspect of the present invention provides above-mentioned pig parvoviral nest-type PRC primer sets in detection pig parvoviral and system
Application in standby detection pig parvoviral reagent.
In a preferred embodiment, nest-type PRC is carried out using above-mentioned nest-type PRC primer sets detect pig parvoviral
Method, include the following steps:
1) sample to be tested extraction DNA is obtained into sample DNA；
2) sample DNA is subjected to twice PCR amplification, first time PCR amplification uses Outside primer PPV-O-F and PPV-
O-R, second of PCR amplification use inner primer PPV-I-F and PPV-I-R；Each PCR product carries out the tiny disease of electrophoresis detection pig
Poison.
In a preferred embodiment, it is 25 μ L that the PCR reaction system of first round amplification, which includes: total volume, in which:
Each 1 μ L of gold medal mix12.5 μ L, PPV-O-F and PPV-O-R, DNA profiling 1 μ L, ddH2O supplies surplus.
In a preferred embodiment, it is 25 μ L that the PCR reaction system of the second wheel amplification, which includes: total volume, in which:
Each 1 μ L of gold medal mix12.5 μ L, PPV-I-F and PPV-I-R, using first round amplified production as template 1 μ L, ddH2O supplies surplus.
In a preferred embodiment, the reaction condition of the first time PCR includes: 94 DEG C of initial denaturation 5min, and 94
DEG C denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle, 72 DEG C of extension 6min.
In a preferred embodiment, the reaction condition of the first time PCR includes: 94 DEG C of initial denaturation 5min, and 94
DEG C denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle, 72 DEG C of extension 6min.
In a preferred embodiment, the sample to be tested is piggy serum sample or spleen, lungs, colostrum, essence
The tissue samples of liquid, lochia.
In a preferred embodiment, first time pcr amplification product carries out electrophoresis inspection with 0.8% Ago-Gel
It surveys；The purpose band of first time PCR amplification is 716bp.
In a preferred embodiment, the product after second of PCR amplification carries out electrophoresis detection, 286bp item occurs
Band can determine whether that there are pig parvovirals in the sample.
Through the above technical solutions, nest-type PRC detection primer group provided by the present invention and detection method are thin for pig
Two pairs of specific primers that base sequence is designed in the Viral structural protein VP2 of small virus, and a variety of virus causing diseases are expanded
Increase, only pig parvoviral can amplify specific band, have specificity.Nested PCR detection method provided by the present invention
Specific band can be amplified from hybrid template, display detection method anti-interference is preferable.Nest-type PRC detection side of the invention
Method can reach 10 to the minimum copy number of existing popular strain detection1A order of magnitude, minimum copy number are 100, examined than regular-PCR
Survey method 3-5 order of magnitude of high sensitivity.
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure of 2 nest-type PRC Outside primer specific detection of the embodiment of the present invention；
Fig. 2 is the gel electrophoresis figure of 2 nest-type PRC inner primer specific detection of the embodiment of the present invention；
Fig. 3 is the gel electrophoresis figure of 3 nest-type PRC Outside primer sensitivity Detection of the embodiment of the present invention；
Fig. 4 is the gel electrophoresis figure of 3 nest-type PRC inner primer sensitivity Detection of the embodiment of the present invention；
Fig. 5 is the gel electrophoresis figure of 3 nest-type PRC Outside primer interference of embodiment of the present invention detection；
Fig. 6 is the gel electrophoresis figure of 3 nest-type PRC inner primer interference of embodiment of the present invention detection.
Specific embodiment
In order to better understand the above technical scheme, being done below by specific embodiment to technical scheme detailed
Explanation, it should be understood that the specific features in the embodiment of the present application and embodiment be to technical scheme specifically
It is bright, rather than the restriction to technical scheme, in the absence of conflict, the skill in the embodiment of the present application and embodiment
Art feature can be combined with each other.It should be understood that term "and/or" used herein above includes listed by one of them or more
Any and all combinations of associated item out.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The defect of regular-PCR detection pig parvoviral of the existing technology, the main thought of the embodiment of the present invention is:
A kind of universal pig parvoviral nest-type PRC primer sets, including Outside primer PPV-O-F, PPV-O-R and inside are drawn
Object PPV-I-F, PPV-I-R；Wherein, the sequence of the Outside primer PPV-O-F as shown in SEQ ID NO:1, draw by the outside
The sequence of object PPV-O-R as shown in SEQ ID NO:2, the sequence of the inner primer PPV-I-F as shown in SEQ ID NO:3,
The inner primer PPV-I-R is as shown in SEQ ID NO:4.
The method for carrying out nest-type PRC detection pig parvoviral using above-mentioned nest-type PRC primer sets, includes the following steps:
1) sample to be tested extraction DNA is obtained into sample DNA；
2) sample DNA is subjected to twice PCR amplification, first time PCR amplification uses Outside primer PPV-O-F and PPV-
O-R, second of PCR amplification use inner primer PPV-I-F and PPV-I-R；Each PCR product carries out the tiny disease of electrophoresis detection pig
Poison.
Nested PCR detection method high specificity provided by the invention, high sensitivity, good in anti-interference performance, and it is highly reliable,
It is low to overcome regular-PCR detection sensitivity, the problems such as poor specificity, and with existing indirect immunofluorescence, genetic chip etc.
For detection method compared to cost is relatively low, detection cycle is short, practical.In addition, present invention method result is reliable, certain is extracted
More parts of blood serum samples of farm detect by two-wheeled nested PCR amplification rear electrophoresis, the sample of tests positive are sequenced,
Sequencing result shows to be pig parvoviral gene, it was demonstrated that the reliability of the method for the present invention.Present invention method is practical
Property it is strong, can be used for the detection to tissue samples, serum, blood plasma and sperm equal samples, be suitable for any laboratory and base it is at different levels
Prevention and control unit, veterinary station and large, medium and small farm etc..
Below with reference to specific embodiment, the present invention will be described in detail.Material used in embodiment can pass through city
Sell channel acquisition.
Embodiment 1: the foundation of pig parvoviral nested PCR detection method
(1) sampling is extracted with DNA
A: serum sample processing: vena cava anterior blood sampling, 1500r/min are centrifuged 30 minutes, are collected sample serum, are used Kang Wei
DNA extraction kit extracts DNA, and measurement concentration is spare.
B: tissue sample processing: the histoorgan of pig sample to be detected is taken, is mentioned after shredding with Kang Wei DNA extraction kit
DNA is taken, measurement concentration is spare.
(2) PCR amplification and electrophoresis detection
A: first time PCR amplification is carried out with outside detection primer PPV-O-F, PPV-O-R.PCR reaction system (25 μ l) are as follows:
Each 1 μ L of gold medal mix12.5 μ L, PPV-O-F and PPV-O-R, DNA profiling 1 μ L, ddH2O supplies surplus；PCR response procedures are as follows: 94
DEG C initial denaturation 5min, 94 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 circulations, 72 DEG C of extension 6min.PCR is produced
Object carries out electrophoresis detection with 0.8% Ago-Gel, and second is carried out if without 716bp band and is expanded.
B: second is carried out to first time amplified production template with inside detection primer PPV-I-F, PPV-I-R and is expanded.PCR
Reaction system (25 μ l) are as follows: each 1 μ L of gold medal mix12.5 μ L, PPV-I-F and PPV-I-R, DNA profiling 1 μ L, ddH2O supplies remaining
Amount；PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle,
72 DEG C of extension 6min.PCR product carries out whether electrophoresis detection has 286bp band with 0.8% Ago-Gel.
(3) interpretation of result
According to nested PCR amplification result judgement: detecting that target stripe is then reported as sun in first time PCR amplification rear electrophoresis
Property, and it is higher to detect sample band poison amount；Second of PCR amplification rear electrophoresis detects that being also judged as target stripe is positive, shows
Sample band poison amount is relatively low；It is detected after two-wheeled nest-type PRC and is then reported as feminine gender without purpose band.
Embodiment 2: recombination positive plasmid pMD20-T-VP2 building
(1) clone of VP2 gene
The genomic DNA for extracting pig parvoviral (PPV) is drawn using the genomic DNA as template using nest-type PRC outside
Guard VP2 gene in object PPV-O-F and PPV-O-R amplification part.PCR reaction system (25 μ l) are as follows: gold medal mix12.5 μ L, PPV-
O-F and each 1 μ L of PPV-O-R, DNA profiling 1 μ L, ddH2O supply surplus；PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C
It is denaturalized 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 circulations, 72 DEG C of extension 6min, PCR product is in 0.8% agarose
Electroresis appraisal is carried out in gel, as a result as shown in Figure 1, obtaining the specific DNA band of a treaty 716bp, with target dna piece
Duan great little is consistent.In Fig. 1: M be DL2000 DNA Marker, 1-8 in template distinguish PPV antigen, swine fever virus (CSFV),
Porcine circovirus 2 type (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome (PRRSV) Escherichia coli are golden yellow
Color staphylococcus, the negative control (ddH of no DNA, RNA2O)。
(2) pMD20-T-VP2 positive plasmid constructs
The above-mentioned PCR product plastic recovery kit of Omega company is recycled, then with the pMD20-T of Takara company
Cloning vector is attached, linked system are as follows: 31 μ L, Steri Vized water of μ L, Vector (PUC19 DNA) of DNA, 1 μ
5 μ L of L, Solution.PCR recovery product 716bp；Conversion bacillus coli DH 5 alpha competence is thin after 16 DEG C of constant temperature connection 30min
Born of the same parents are coated on the LB culture medium of Amp resistance；37 DEG C of overnight incubations go out correct recon by resistance screening.
(3) pMD20-T-VP2 positive recombinant is identified
Bacterium colony PCR identification is carried out to the positive colony selected with primer PPV-O-F and PPV-O-R, identifies correctly recombination
Son is named as pMD20-T-VP2.
(4) extraction of recombinant plasmid pMD20-T-VP2
Sequencing is identified uses the high purity plasmid of Kang Wei company is small to propose examination after correct recon carries out increasing bacterium with LB meat soup
Agent box PurePlasmid Mini Kit extracts plasmid and measures concentration, calculates copy number.
Embodiment 3: nest-type PRC primer Characteristics Detection
(1) PPV cause of disease, swine fever virus (CSFV), porcine circovirus 2 type (PCV2), pseudorabies specific test: are extracted
Viral (PRV), porcine reproductive and respiratory syndrome (PRRSV), Escherichia coli, staphylococcus aureus, negative control (no DNA,
The ddH of RNA2O the specific detection for) carrying out nested PCR method, examines the specificity of the detection method.As a result such as Fig. 1,2 institutes
Show, two pairs of primers all have good specificity and higher amplification efficiency.In Fig. 2, M is DL2000 DNA Marker, 1-8
In template be respectively PPV antigen, swine fever virus (CSFV), porcine circovirus 2 type (PCV2), porcine pseudorabies virus (PRV), pig
Reproductive and respiratory syndrome (PRRSV), Escherichia coli, staphylococcus aureus, the negative control (ddH of no DNA, RNA2O)。
(2) 10 times of doubling dilutions sensitivity tests: are carried out to units copy number to the positive plasmid obtained of embodiment 2
(4copy) chooses each dilution plasmid as template and carries out first time PCR amplification with primer PPV-O-F, PPV-O-R, and PCR is produced
Object is detected with 0.8% agarose gel, and as a result as shown in figure 3, first time PCR amplification electrophoresis result is shown, Outside primer is detectable
It is 10 to gene copy number1, and specific amplification is good.In Fig. 3: M be DL2000 DNA Marker, 1-9 in template copy numbers according to
Secondary is 1.33 × 108, 1.33 × 107, 1.33 × 106, 1.33 × 105, 1.33 × 104, 1.33 × 103, 1.33 × 102, 1.33
×101, 1.33 × 100.Amplified production is second of PCR with primer PPV-I-F, PPV-I-R as the template of corresponding gradient and expands
Increase, amplified production is detected with 0.8% agarose gel, as a result as shown in figure 4, can examine after nest-type PRC expands twice
Minimum 10 measured0A copy number.Wherein 1-9 is respectively using first time PCR product as corresponding template, and 1.33 × 108, 1.33 ×
107, 1.33 × 106, 1.33 × 105, 1.33 × 104, 1.33 × 103, 1.33 × 102, 1.33 × 101, 1.33 × 100。
(3) interference test: the PPV genome used in specific test takes equivalent to be mixed with pseudo- mad dog genome,
PCR amplification is carried out respectively with primer PPV-O-F, PPV-O-R and PPV-I-F, PPV-I-R, examines the anti-interference of primer, it can
Purpose band is amplified from mixutre genome template, as a result as shown in Figure 5 and Figure 6, which can be from hybrid template
In amplify specific band, display primer anti-interference is preferable.Wherein, M is DL2000 DNA Marker, 1-5PPV in Fig. 5
It is the negative control (ddH of no DNA, RNA with porcine pseudorabies virus (PRV) mixing sample, 62O).M is DL2000 DNA in Fig. 6
Marker, 1-5PPV and porcine pseudorabies virus (PRV) mixing sample, 6 be the negative control (ddH of no DNA, RNA2O)。
(4) coincidence rate is tested: the sensibility detected for verifying nested primer to each department prevalence parvovirus is extracted
The genome of the strains such as JN872448, KF429255, KF913345, JX992846, U44978, FJ822038 and EU790642,
Copy number is calculated after measurement concentration, gradient dilution to units copy number, after two-wheeled nested PCR amplification, each genoid
Group can detect 101The gene copy number of a order of magnitude, it was demonstrated that the method for the present invention is practical.
Embodiment 4: nest-type PRC reliability test
1, clinical sample acquires: in October, 2018 acquires 20 parts of piggy blood sample from Hubei large-scale pig farm, 1500r/
Min is centrifuged 30 minutes, collects sample serum, extracts DNA according to the pillar blood sample DNA extraction kit of Kang Wei company.
2, PCR amplification and electrophoresis detection: the step of pressing embodiment 1 (2) carries out two-wheeled PCR amplification.To the product after amplification
Electrophoresis detection is carried out, 20 parts of serum samples detect 15 parts of positives altogether, and sequencing result shows that 15 parts of positive products are tiny disease
Virus gene, it was demonstrated that the result of this method detection has reliability.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
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Claims (10)
1. a kind of universal pig parvoviral nest-type PRC primer sets, it is characterised in that: including Outside primer PPV-O-F, PPV-O-
R and inner primer PPV-I-F, PPV-I-R；Wherein, the sequence of the Outside primer PPV-O-F is as shown in SEQ ID NO:1, institute
The sequence of Outside primer PPV-O-R is stated as shown in SEQ ID NO:2, the sequence of the inner primer PPV-I-F such as SEQ ID
Shown in NO:3, the inner primer PPV-I-R is as shown in SEQ ID NO:4.
2. pig parvoviral nest-type PRC primer sets as described in claim 1 are thin in detection pig parvoviral and preparation detection pig
Application in small virus reagent.
3. the application of pig parvoviral nest-type PRC primer sets according to claim 2, it is characterised in that: use the nido
The method that PCR primer group carries out nest-type PRC detection pig parvoviral, includes the following steps:
1) sample to be tested extraction DNA is obtained into sample DNA；
2) sample DNA being subjected to twice PCR amplification, first time PCR amplification uses Outside primer PPV-O-F and PPV-O-R,
Second of PCR amplification uses inner primer PPV-I-F and PPV-I-R；Each PCR product carries out electrophoresis detection pig parvoviral.
4. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: first round amplification
PCR reaction system includes: gold medal mix12.5 μ each 1 μ L of L, PPV-O-F and PPV-O-R, DNA profiling 1 μ L, ddH2O supplies surplus；
Total volume is 25 μ L.
5. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: the second wheel amplification
PCR reaction system includes: gold medal mix12.5 μ each 1 μ L of L, PPV-I-F and PPV-I-R, using first round amplified production as 1 μ of template
L, ddH2O supplies surplus, and total volume is 25 μ L.
6. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: the first time PCR
Reaction condition include: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 circulations, 72
DEG C extend 6min.
7. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: second of PCR
Reaction condition include: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 circulations, 72
DEG C extend 6min.
8. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: the sample to be tested
For piggy serum sample or spleen, lungs, colostrum, sperm, lochia tissue samples.
9. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: first time PCR amplification
Product carries out electrophoresis detection with 0.8% Ago-Gel；The purpose band of first time PCR amplification is 716bp.
10. the application of pig parvoviral nest-type PRC primer sets according to claim 3, it is characterised in that: second of PCR expands
Product after increasing carries out electrophoresis detection, 286bp band occurs i.e. and can determine whether that there are pig parvovirals in the sample.
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| CN102071257A (en) * | 2009-11-25 | 2011-05-25 | 河南农业大学 | Dual SYBR Green I real-time fluorescence PCR detection primer and method for porcine parvovirus and porcine circovirus type 2 |
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| CN102071257A (en) * | 2009-11-25 | 2011-05-25 | 河南农业大学 | Dual SYBR Green I real-time fluorescence PCR detection primer and method for porcine parvovirus and porcine circovirus type 2 |
| CN107686833A (en) * | 2016-04-18 | 2018-02-13 | 华南农业大学 | A kind of pig parvoviral strain and its application |
| CN107312875A (en) * | 2017-08-29 | 2017-11-03 | 福建省农业科学院畜牧兽医研究所 | Detect the primer sets of the loop-mediated isothermal amplification method of the type of pig circular ring virus 3 |
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