CN109825640A - A kind of African swine fever virus nested PCR detection method - Google Patents
A kind of African swine fever virus nested PCR detection method Download PDFInfo
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Abstract
The invention discloses a kind of nested PCR detection methods of African swine fever virus, include the following steps: S1, are designed to obtain two pairs of detection primers, respectively Outside primer and inner primer according to the base sequence in African swine fever virus capsid protein p72；The extraction of S2, DNA to be detected；S3, first time amplification is carried out using Outside primer；S4, second of amplification is carried out using inner primer；Two step amplified production agarose gel electrophoresis are to detect African swine fever virus.Nested PCR detection method high specificity provided by the invention, high sensitivity, good in anti-interference performance, and cost is relatively low, detection cycle is short, has a good application prospect.
Description
Technical field
The present invention relates to molecular diagnostic techniques field, specially a kind of African swine fever virus nested PCR detection method.
Technical background
African swine fever (African swine fever, ASF) is by African swine fever virus (African swine fever
Virus, ASFV) infection domestic pig and wild boar caused by a kind of deadly infectious disease, acute clinical signs be high fever, it is depressed, detest
Food, skin cyanosis, each internal organs bleeding, disease incidence and case fatality rate may be up to 100%.World Organization for Animal Health (OIE) is arranged
For statutory report animal epidemic, China is classified as a kind of animal epidemic, is one of exotic animals epidemic disease of China's guard key.
African Territories, Italian Sardinia, Caucasus region and Russia and Eastern Europe Countries stream of the ASF on the south the Sahara
Row, causes huge economic loss, and the international trade of serious impact livestock products to the pig breeding industry of epidemic-stricken area country.In August, 2018,
ASF is passed to China for the first time, and then a wide range of sprawling rapidly, constitutes significant threat to China's pig breeding industry, prevention and control situation is extremely severe.
As economic globalization develops, ASF is in Global prevalence situation, and the risk for being constantly incoming China is high.In view of current commercial-free
ASF vaccine, needing research and development can be achieved the early diagnosis technology that quickly detects, accomplish to epidemic situation early discovery, early control.Due to
ASFV has huge genome structure and complicated Immune escaping mechanism, so that it is very difficult to develop effective vaccine.At present
Inactivated vaccine, subunit vaccine and the nucleic acid vaccine of development cannot provide immunoprotection or be only capable of providing part protection, and be attenuated
Live vaccine and gene-deleted vaccine can induce completely with source protection and partial cross protection.Future needs deeply parsing disease
Malicious virulence associated gene and immune protective related antigen, and put forth effort to develop gene-deleted vaccine and attenuated vaccine, solve its peace
The problems such as Quan Xing, stability and immune efficacy.
African swine fever virus category Iridoviridae iridescent virus, the virus are large-scale DNA virus, and genome is bifilar
Linear DNA, 170~190kb of size.Virion diameter is 175~225ms, symmetrical in 20 face bodies, there is cyst membrane.In pig body,
African swine fever virus can especially replicate in reticuloendothelial cell and mononuclear macrophage in the cytoplasm of several types.It should
Virus can be proliferated in blunt edge bee.
In recent years, domestic and foreign scholars to it establishes hemadsorption test, isothermal amplification technique, fluorescent antibody test,
Regular-PCR diagnosis, the technologies such as SYBRGreen real-time fluorescence quantitative PCR carry out the detection of ASF.Cost is relatively low for regular-PCR method,
It is easy to operate, it is widely applied.But there are the lower such problems of sensitivity for regular-PCR diagnosis, and ASF can exist centainly
Incubation period, regular-PCR sensitivity is poor, easily causes missing inspection.
Nest-type PRC is a kind of polymerase chain reaction of variation, expands target fragment using two pairs of PCR primers.By twice
PCR amplification, can be with the sensitivity of the raising regular-PCR of high degree, and the probability of non-specific binding occurs for secondary PCR primer
It is extremely low.The target of current African swine fever virus ASF detection is mostly P72 gene, contains virulent conserved sequence, decides African pig
The phyletic evolution of pestivirus and hemagglutination activity, tissue tropism and the host range of virus, therefore the P72 of encoding capsid protein
Gene order can be used as the diagnosis target of detection ASF.
Summary of the invention
The purpose of the invention is to provide a kind of fast and convenient African swine fever virus (ASF) nested PCR detection method,
It has the characteristics that high specificity, high sensitivity, and cost is relatively low, and detection cycle is short, there is good application prospect.
The present invention is achieved by the following technical solutions:
A kind of nested PCR detection method of African swine fever virus, which comprises the following steps:
S1, according to the base sequence in African swine fever virus capsid protein P72, design obtains two pairs of primers, respectively outer
Side primer ASF-O-F, ASF-O-R and inner primer ASF-I-F, ASF-I-R；
The extraction of S2, DNA to be detected；
S3, the DNA extracted using S2 carry out first round amplification, PCR reaction system as template, using Outside primer described in S1
(25 μ l) are as follows: each 1 μ L of Ex Taq12.5 μ L, ASF-O-F and ASF-O-R, DNA profiling 1 μ L, ddH2O supplies surplus；PCR reaction
Program are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 51 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 circulations, 72 DEG C extend
6min, PCR product carry out electrophoresis detection with 0.8% Ago-Gel, and second is carried out if without purpose band and is expanded；
S4, using S3 amplified production as template, utilize inner primer described in S1 carry out second wheel amplification, PCR reaction system (25
μ l) are as follows: each 1 μ L of Ex Taq12.5 μ L, ASF-I-F and ASF-I-R, DNA profiling 1 μ L, ddH2O supplies surplus；PCR response procedures
Are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle, 72 DEG C of extension 6min,
PCR product carries out electrophoresis detection with 0.8% Ago-Gel.
Further, the sequence of described Outside primer ASF-O-F, ASF-O-R and inner primer ASF-I-F, ASF-I-R point
Not are as follows:
ASF-O-F:5'-TCGCCGAAGGGAATG-3'(SEQ ID NO.1)；
ASF-O-R:5'-CTGGGACAACCAAACACC-3'(SEQ ID NO.2)；
ASF-I-F:5'-ATACTGAGGGAATAGCAAGG-3'(SEQ ID NO.3)；
ASF-I-R:5'-CCGCACCAAAGCAAA-3'(SEQ ID NO.4).
Further, DNA to be detected described in step S2 is from the tissue such as the serum of pig, colostrum, lymph node, spleen or lungs
It extracts.
Further, purpose band described in step S3 refers to 633bp band.
Further, in step S4, electrophoresis detection is carried out to the product after the amplification, 295bp band occur can sentence
Breaking in the sample, there are African swine fever viruses.
Compared with prior art, the invention has the benefit that (1) nest-type PRC detection primer provided by the present invention is
It is adopted for 4 specific primers that base sequence in the capsid protein P72 of African swine fever virus is designed by experimental verification
With the nested PCR detection method of the primer its with very strong specificity and preferable anti-interference.(2) nido of the invention
PCR detection method can reach 10 to the minimum copy number of existing popular strain detection1A order of magnitude, minimum copy number are 101, than
Regular-PCR detection method 3-5 order of magnitude of high sensitivity.(3) this method high reliablity, it is practical, it can be used for tissue sample
Feedstuff in sheet, serum, blood plasma and farm, the detection for drawing cotton swab equal samples in pig vehicle compartment are suitable for any experiment
Room and base prevention and control unit at different levels, veterinary station and large, medium and small farm etc..
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure of nest-type PRC primer specificity detection；Wherein M is DL2000DNA Marker, in 1-8
Template distinguish African swine fever (ASF), swine fever virus (CSFV), porcine circovirus 2 type (PCV2), porcine pseudorabies virus (PRV),
Porcine reproductive and respiratory syndrome (PRRSV), Escherichia coli, staphylococcus aureus, the negative control (ddH of no DNA, RNA2O)。
Fig. 2 is the gel electrophoresis figure of nest-type PRC primer specificity detection；Wherein M is DL2000DNA Marker, in 1-8
Template be respectively African swine fever (ASF), swine fever virus (CSFV), porcine circovirus 2 type (PCV2), porcine pseudorabies virus
(PRV), porcine reproductive and respiratory syndrome (PRRSV), Escherichia coli, staphylococcus aureus, negative control be (no DNA, RNA's
ddH2O)。
Fig. 3 is the gel electrophoresis figure of nest-type PRC Outside primer sensitivity Detection；Wherein M is DL2000DNA Marker, 1-
Template copy numbers are followed successively by 8, and 1.89 × 107, 1.89 × 106, 1.89 × 105, 1.89 × 104, 1.89 × 103, 1.89 ×
102, 1.89 × 101, 1.89 × 100.
Fig. 4 is the gel electrophoresis figure of nest-type PRC inner primer sensitivity Detection；Wherein M is DL2000DNAMarke, 1-8
It is middle respectively using first time PCR product as corresponding template: 1.89 × 107, 1.89 × 106, 1.89 × 105, 1.89 × 104, 1.89 ×
103, 1.89 × 102, 1.89 × 101, 1.89 × 100。
Fig. 5 is the gel electrophoresis figure of nest-type PRC Outside primer interference detection；M is DL2000DNA Marker in Fig. 5,
1-5 is the mixing samples such as ASF and porcine pseudorabies virus (PRV), and 6 be the negative control (ddH of no DNA, RNA2O)。
Fig. 6 is the gel electrophoresis figure of nest-type PRC inner primer interference detection；M is DL2000DNAMarker, 1- in Fig. 6
5 be the mixing samples such as ASF and porcine pseudorabies virus (PRV), and 6 be the negative control (ddH of no DNA, RNA2O)。
Specific embodiment
Below in conjunction with embodiment, the present invention will be further described, but is not to limit the scope of the invention, and only makees
It illustrates.
Embodiment 1: the foundation of African swine fever virus nested PCR detection method
(1) design of primers
By the base sequence in coding African swine fever virus capsid protein P72, design obtains two pairs of primers, respectively outer
Side primer ASF-O-F, ASF-O-R and inner primer ASF-I-F, ASF-I-R, sequence are as follows:
ASF-O-F:5'-TCGCCGAAGGGAATG-3'(SEQ ID NO.1)；
ASF-O-R:5'-CTGGGACAACCAAACACC-3'(SEQ ID NO.2)；
ASF-I-F:5'-ATACTGAGGGAATAGCAAGG-3'(SEQ ID NO.3)；
ASF-I-R:5'-CCGCACCAAAGCAAA-3'(SEQ ID NO.4).
(2) 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 taken 100 μ L
DNA is extracted with DNA extraction kit, measurement concentration is spare.
B: tissue sample processing: the histoorgan of pig sample to be detected is taken, is extracted after shredding with DNA extraction kit
DNA, measurement concentration are spare.
(2) PCR amplification and electrophoresis detection
A: first time PCR amplification is carried out with outside detection primer ASF-O-F, ASF-O-R.PCR reaction system (25 μ l) are as follows:
Each 1 μ L of Ex Taq12.5 μ L, ASF-O-F and ASF-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, 51 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 633bp band and is expanded.
B: second is carried out to first time amplified production template with inside detection primer ASF-I-F, ASF-I-R and is expanded.PCR
Reaction system (25 μ l) are as follows: each 1 μ L of Ex Taq12.5 μ L, ASF-I-F and ASF-I-R, DNA profiling 1 μ L, ddH2O supplies surplus；
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle, and 72
DEG C extend 6min.PCR product carries out whether electrophoresis detection has 295bp 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, two-wheeled
It is detected after nest-type PRC and is then reported as feminine gender without purpose band.
Embodiment 2: recombination positive plasmid pMD20-T-P72 building
(1) clone of P72 gene
The genomic DNA for extracting African swine fever virus (ASF), using the genomic DNA as template, using on the outside of nest-type PRC
Guard P72 gene in primer ASF-O-F and ASF-O-R amplification part.PCR reaction system (25 μ l) are as follows: Ex Taq12.5 μ L, ASF-
O-F and each 1 μ L of ASF-O-R, DNA profiling 1 μ L, ddH2O supplies surplus；PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C
It is denaturalized 30s, 51 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 633bp, with target dna piece
Duan great little is consistent.
(2) pMD20-T-P72 positive plasmid constructs
The above-mentioned PCR product plastic recovery kit of Omega company is recycled, then with the pMD20-T of TaRaKa company
Cloning vector is attached, linked system are as follows: 31 μ L, Steri Vized water of μ L, Vector (PUC19DNA) of DNA, 1 μ
5 μ L of L, Solution.PCR recovery product 633bp；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-P72 positive recombinant is identified
Bacterium colony PCR identification is carried out to the positive colony selected with primer ASF-O-F and ASF-O-R, identifies correctly recombination
Son is named as pMD20-T-P72.
(4) extraction of recombinant plasmid pMD20-T-P72
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) it specific test: extracts African swine fever virus (ASFV), swine fever virus (CSFV), porcine circovirus 2 type
(PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome (PRRSV), Escherichia coli, staphylococcus aureus is worked as
Template carries out specific detection to two couples of primers ASF-O-F, ASF-O-R and ASF-I-F used in nest-type PRC, ASF-I-R,
Examine the specificity of two pairs of primers.As a result as shown in figures 1 and 2, two pairs of primers all have good specificity and higher amplification effect
Rate.
(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 ASF-O-F, ASF-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 number2, and specific amplification is good.Template primer ASF-I-F, the ASF- of amplified production as corresponding gradient
I-R does second of PCR amplification, and amplified production is detected with 0.8% agarose gel, as a result as shown in figure 4, by nest-type PRC
Minimum 10 can be detected after expanding twice1A copy number.
(3) interference test: the viral genome used in specific test takes equivalent to be mixed with African swine fever genome
It closes, carries out PCR amplification respectively with primer ASF-O-F, ASF-O-R and ASF-I-F, ASF-I-R, examine the anti-interference of primer,
Purpose band can be amplified from mixutre genome template, as a result as shown in Figure 5 and Figure 6, two pairs of primers can be from hybrid guided mode
Specific band is amplified in plate, display primer anti-interference is preferable.
(4) coincidence rate is tested: the sensibility detected for verifying nested primer to each department prevalence African swine fever virus is extracted
OURT88/3 plants, NH/P68 plants, 2007 plants of Armenia, the strains such as 2007/1 plant and BA71V plants of Georgia genome,
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 November, 2018 acquires 20 parts of piggy blood sample from Hubei large-scale pig farm, 1500r/
Min is centrifuged 30 minutes, collects sample serum, cotton swab and totally 20 parts of feedstuff in pig compartment is drawn, according to the column of Kang Wei company
Formula blood sample DNA extraction kit extracts DNA.
2, PCR amplification and electrophoresis detection: the step of pressing embodiment 1 (3) carries out two-wheeled PCR amplification.To the product after amplification
Electrophoresis detection is carried out, 20 parts of serum samples draw cotton swab and feedstuff in pig compartment to be detected as feminine gender, and the pig farm is really African
Swine fever feminine gender field, it was demonstrated that the result of this method detection has reliability.
For those skilled in the art, it can make other each according to the above description of the technical scheme and ideas
Kind is corresponding to be changed and deforms, and all these change and deform the protection model that all should belong to the claims in the present invention
Within enclosing.
Sequence table
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Claims (5)
1. a kind of nested PCR detection method of African swine fever virus, which comprises the following steps:
S1, according to the base sequence in African swine fever virus capsid protein P72, design obtains two pairs of primers, and respectively outside is drawn
Object ASF-O-F, ASF-O-R and inner primer ASF-I-F, ASF-I-R；
The extraction of S2, DNA to be detected；
S3, the DNA extracted using S2 carry out first round amplification, PCR reaction system (25 μ l) as template, using Outside primer described in S1
Are as follows: each 1 μ L of Ex Taq12.5 μ L, ASF-O-F and ASF-O-R, DNA profiling 1 μ L, ddH2O supplies surplus；PCR response procedures are as follows:
94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 51 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle, 72 DEG C of extensions 6min, PCR
Product carries out electrophoresis detection with 0.8% Ago-Gel, and second is carried out if without purpose band and is expanded；
S4, using S3 amplified production as template, utilize inner primer described in S1 carry out second wheel amplification, PCR reaction system (25 μ l)
Are as follows: each 1 μ L of Ex Taq12.5 μ L, ASF-I-F and ASF-I-R, DNA profiling 1 μ L, ddH2O supplies surplus；PCR response procedures are as follows:
94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 45s, 32 recycle, 72 DEG C of extensions 6min, PCR
Product carries out electrophoresis detection with 0.8% Ago-Gel.
2. a kind of nested PCR detection method of African swine fever virus according to claim 1, which is characterized in that described to draw
The gene order of object are as follows:
ASF-O-F:5'-TCGCCGAAGGGAATG-3'(SEQ ID NO.1)；
ASF-O-R:5'-CTGGGACAACCAAACACC-3'(SEQ ID NO.2)；
ASF-I-F:5'-ATACTGAGGGAATAGCAAGG-3'(SEQ ID NO.3)；
ASF-I-R:5'-CCGCACCAAAGCAAA-3'(SEQ ID NO.4).
3. a kind of nested PCR detection method of African swine fever virus according to claim 1, which is characterized in that the S2
In, DNA to be detected is extracted from the tissue such as the serum of pig, colostrum, lymph node, spleen or lungs.
4. a kind of African swine fever virus nested PCR detection method according to claim 1, it is characterised in that: in the S3,
Purpose band refers to 633bp band.
5. a kind of African swine fever virus nested PCR detection method according to claim 1, it is characterised in that: in the S4,
Electrophoresis detection is carried out to the product after amplification, 295bp band occurs i.e. and can determine whether that there are African swine fever viruses in the sample.
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| CN110804677A (en) * | 2019-10-14 | 2020-02-18 | 华南农业大学 | Nested duplex PCR (polymerase chain reaction) detection primer and kit for distinguishing African swine fever virus wild strain and gene deletion strain |
| CN111074000A (en) * | 2019-11-18 | 2020-04-28 | 华南农业大学 | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain and double-gene deletion strain |
| CN112760416A (en) * | 2020-12-29 | 2021-05-07 | 肇庆大华农生物药品有限公司 | African swine fever virus fluorescent PCR detection reagent, kit and detection method |
| CN113215328A (en) * | 2021-03-24 | 2021-08-06 | 黄婉秋 | Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit |
| CN113584227A (en) * | 2021-08-03 | 2021-11-02 | 长江大学 | Nested PCR detection primer group and method for identifying African swine fever gene deletion strain |
| CN114774581A (en) * | 2022-03-11 | 2022-07-22 | 山东舜丰生物科技有限公司 | PCR primer and probe composition for detecting African swine fever virus |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804677A (en) * | 2019-10-14 | 2020-02-18 | 华南农业大学 | Nested duplex PCR (polymerase chain reaction) detection primer and kit for distinguishing African swine fever virus wild strain and gene deletion strain |
| CN110804677B (en) * | 2019-10-14 | 2023-08-22 | 华南农业大学 | Nested double PCR detection primer and kit for distinguishing wild strain and gene deletion strain of African swine fever virus |
| CN111074000A (en) * | 2019-11-18 | 2020-04-28 | 华南农业大学 | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain and double-gene deletion strain |
| CN111074000B (en) * | 2019-11-18 | 2023-08-22 | 华南农业大学 | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain from double gene deletion strain |
| CN112760416A (en) * | 2020-12-29 | 2021-05-07 | 肇庆大华农生物药品有限公司 | African swine fever virus fluorescent PCR detection reagent, kit and detection method |
| CN113215328A (en) * | 2021-03-24 | 2021-08-06 | 黄婉秋 | Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit |
| CN113584227A (en) * | 2021-08-03 | 2021-11-02 | 长江大学 | Nested PCR detection primer group and method for identifying African swine fever gene deletion strain |
| CN114774581A (en) * | 2022-03-11 | 2022-07-22 | 山东舜丰生物科技有限公司 | PCR primer and probe composition for detecting African swine fever virus |
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