CN105695634B - Detect PCR primer, kit and its application of African swine fever virus - Google Patents
Detect PCR primer, kit and its application of African swine fever virus Download PDFInfo
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Abstract
The invention discloses PCR primer, kit and its applications of detection African swine fever virus, belong to the detection field of African swine fever virus.The present invention devises 4 pairs of primers according to the conservative region of ASFV p72 gene on GenBank first, is screened out from it the high primer of high specificity, sensibility, and primer nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 forms.The present invention further discloses the kit for the detection African swine fever virus being prepared with the primer and corresponding PCR detection method is established, two kinds of African swine fever virus PCR detection methods that the detection method ratio OIE that the present invention establishes recommends are more special, more sensitive；Clinical sample testing result shows that PCR detection method of the present invention is easy to operate, at low cost, and specificity is good, and sensibility is high, can be effectively applied to the screening and quick diagnosis of African swine fever.
Description
Technical field
The present invention relates to the PCR for detecting African swine fever virus (African swine fever virus, ASFV) to draw
Object further relates to kit and its application in African swine fever virus detection that the PCR primer is prepared, belongs to African pig
The detection field of pestivirus.
Background technique
African swine fever (African swine fever, ASF) is by African swine fever virus (African swine fever
Virus, ASFV) caused by domestic pig and wild boar deadly infectious disease, for World Organization for Animal Health (OIE) regulation must be notifiable
A kind of animal epidemic, to epidemic-stricken area country cause huge economic loss (Costard, S., Mur, L., Lubroth, J.,
Sanchez-Vizcaino,J.M.,Pfeiffer,D.U.,2013.Epidemiology of African swine fever
virus.Virus Res.173,191–197.).ASFV is a big double-stranded DNA virus for having cyst membrane, belongs to African swine fever disease
Malicious section (Asfarviridae) African swine fever virus category (Asfivirus) member (Dixon, L.K., Chapman, D.A.,
Netherton,C.L.,Upton,C.,2013.African swine fever virus replication and
genomics.Virus Res.173,3–14.)。
ASFV can cause the Hemorrhagic fever and high mortality that infect domestic pig and aper, and to red river hog, such as warthog
(warthogs) and African bush pig (bushpigs) then in subclinical infection (Denyer, M.S., Wilkinson, P.J.,
1998.African Swine Fever.In:Encyclopedia of Immunology.pp.54–56.).ASFV there is also
In tampan tick, which is the natural reservoir (of bird flu viruses) of ASFV.
It experienced and are passed from the African continent to several long distances in Europe and the U.S. sixties in last century, the seventies and the eighties
After broadcasting, since nearly 30 years, in addition to the Sardinia of Italy, ASF is confined to African Territories.Until 2007, disease outburst in
Georgia (Rowlands, R.J., Michaud, V., Heath, L., Hutchings, G., Oura, C., Vosloo, W.,
Dwarka,R.,Onashvili,T.,Albina,E.,Dixon,L.K.,2008.African swine fever virus
isolate,Georgia,2007.Emerg.Infect.Dis.14,1870–1874.；Costard,S.,Mur,L.,
Lubroth,J.,Sanchez-Vizcaino,J.M.,Pfeiffer,D.U.,2013.Epidemiology of African
Swine fever virus.Virus Res.173,191-197.), subsequent epidemic situation spread to rapidly entire Caucasus region and
The Russian Federation, Byelorussia and Ukraine, passed within most finally 2014 European Union (Gogin, A., Gerasimov, V.,
Malogolovkin,A.,Kolbasov,D.,2013.African swine fever in the North Caucasus
region and the Russian Federation in years 2007-2012.Virus Res.173,198–203.；
OIE.2014.World Animal Health Information Database(WAHID).http://www.oie.int/
wahis_2/public/wahid.php/Diseaseinformation/Immsumma ry.；Sánchez-Vizcaíno,
J.M.,Mur,L.,Gomez-Villamandos,J.C.,Carrasco,L.,2015.An update on the
epidemiology and pathology of African swine fever.J.Comp.Pathol.152,9–21).Mirror
In the continuous expansion of various known risk factors and China nearest economy and international trade that ASF is propagated, generation is passed to for the disease
The risk of pig raising China, the first big country, boundary can not be ignored.
Since currently without the vaccine and effective treatment means for being directed to ASF, fast and reliable diagnosis is to prevention and control measure
Implement in time, it is vital for preventing the sprawling of the destructiveness disease.In addition, it is contemplated that with other pig infectious disease such as swine fevers
(CSF) a possibility that clinical symptoms are similar and nonspecific clinical performance occurs, early stage and accurate detection to ASFV
It is the key that high-speed decision (Oura, C.A., Edwards, L., Batten, C.A., 2013.Virological diagnosis
of African swine fever—comparative study of available tests.Virus Res.173,
150–158.).Virology is detected, the method that OIE recommends includes virus purification, fluorescent-antibody test (FAT), Standard PCR
With real-time quantitative PCR (quantitative fluorescent PCR) (Ag ü ero, M., Fern á ndez, J., Romero, L., S á nchez
Mascaraque,C.,Arias,M.,Sánchez-Vizcaíno,J.M.,2003.Highly sensitive PCR assay
for routine diagnosis of African swine fever virus in clinical
samples.J.Clin.Microbiol.41,4431–4434.；King,D.P.,Reid,S.M.,Hutchings,G.H.,
Grierson,S.S.,Wilkinson,P.J.,Dixon,L.K.,Bastos,A.D.,Drew,T.W.,
2003.Development of a TaqMan PCR assay with internal amplification control
for the detection of African swine fever virus.J.Virol.Methods 107,53–61；
Oura,C.A.L.,Arias,M.,2012.African swine fever.In:OIE Biological Standards
Commission(ed),OIE Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals,7th ed.Office International des Epizooties,Paris,France,pp.1069–
1082.；Oura,C.A.,Edwards,L.,Batten,C.A.,2013.Virological diagnosis of African
swine fever—comparative study of available tests.Virus Res.173,150–158.；Sá
nchez-Vizcaíno,J.M.,Mur,L.,Gomez-Villamandos,J.C.,Carrasco,L.,2015.An update
on the epidemiology and pathology of African swine fever.J.Comp.Pathol.152,9–
21.).Virus purification is considered as the goldstandard of ASF diagnosis, however this method takes time and effort, and is usually only made in reference laboratory
For confirmatory diagnostic method (Oura, C.A., Edwards, L., Batten, C.A., 2013.Virological diagnosis
of African swine fever—comparative study of available tests.Virus Res.173,
150–158.).PCR method is the detection means being most widely used at present because it is with very high sensitivity and specificity, special
The detection of the tissue of virus purification Shi Yongyu be not suitable for.The PCR method that OIE recommends is used for the routine diagnosis of reference laboratory
(Agüero,M.,Fernández,J.,Romero,L.,Sánchez Mascaraque,C.,Arias,M.,Sánchez-
Vizcaíno,J.M.,2003.Highly sensitive PCR assay for routine diagnosis of
African swine fever virus in clinical samples.J.Clin.Microbiol.41,4431–4434.；
King,D.P.,Reid,S.M.,Hutchings,G.H.,Grierson,S.S.,Wilkinson,P.J.,Dixon,L.K.,
Bastos,A.D.,Drew,T.W.,2003.Development of a TaqMan PCR assay with internal
amplification control for the detection of African swine fever
virus.J.Virol.Methods 107,53–61.；Oura,C.A.L.,Arias,M.,2012.African swine
fever.In:OIE Biological Standards Commission(ed),OIE Manual of Diagnostic
Tests and Vaccines for Terrestrial Animals,7th ed.Office International des
Epizooties,Paris,France,pp.1069–1082.).The fluorescent quantitative PCR detection method established in recent years is shown
Higher sensitivity (Fern á ndez-Pinero, J., Gallardo, C., Elizalde, M., Robles, A., G ó mez,
C.,Bishop,R.,Heath,L.,Couacy-Hymann,E.,Fasina,F.O.,Pelayo,V.,Soler,A.,Arias,
M.,2013.Molecular diagnosis of African swine fever by a new real-time PCR
using universal probe library.Transbound.Emerg.Dis.60,48–58.；Gallardo,C.,
Nieto,R.,Soler,A.,Pelayo,V.,Fernández-Pinero,J.,Markowska-Daniel,I.,
Pridotkas,G.,Nurmoja,I.,Granta,R.,Simón,A.,Pérez,C.,Martín,E.,Fernández-
Pacheco,P.,Arias,M.,2015.Assessment of African swine fever diagnostic
techniquesas a response to the epidemic outbreaks in Eastern European Union
countries:how to improve surveillance and control
programs.J.Clin.Microbiol.53,2555–2565.).Standard PCR is more commonly suitable for not having quantitative fluorescent PCR
The laboratory of instrument.However two parts of nearest research reports show that the Standard PCR sensibility and specificity that OIE recommends reduces respectively,
Supposition may be caused by being mismatched as the nucleotide between primer and viral target gene (Gallardo, C., Nieto, R.,
Soler,A.,Pelayo,V.,Fernández-Pinero,J.,Markowska-Daniel,I.,Pridotkas,G.,
Nurmoja,I.,Granta,R.,Simón,A.,Pérez,C.,Martín,E.,Fernández-Pacheco,P.,Arias,
M.,2015.Assessment of African swine fever diagnostic techniques as a response
to the epidemic outbreaks in Eastern European Union countries:how to improve
surveillance and control programs.J.Clin.Microbiol.53,2555–2565.；Petrovan,V.,
Buburuzan,L.,Zaulet,M.,2015.False positive results using PCR detection method
for African swine fever virus in wild boars from northern Romanian hunting
zones.Turk.J.Vet.Anim.Sci.39,287–294.).Therefore, current PCR method is updated to ensure to currently a popular
All ASFV strains carry out reliable detection and be very important.
It would therefore be highly desirable to establish a kind of new ASFV conventional PCR method, enable the method to realize to currently a popular institute
There is ASFV strain reliably to be detected.
Summary of the invention
One of the object of the invention is to provide the PCR primer of detection African swine fever virus, which has high specificity, sensitivity
The advantages that property is high；
The second object of the present invention is to providing a kind of kit for detecting African swine fever virus.
In order to achieve the above objectives, the present invention is achieved through the following technical solutions:
The present invention discloses the PCR primer of detection African swine fever virus first, any one in following four pairs of primers
It is right:
Primer 1: the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 forms；
Primer 2: the nucleotide sequence shown in SEQ ID No.3 and SEQ ID No.4 forms；
Primer 3: the nucleotide sequence shown in SEQ ID No.5 and SEQ ID No.6 forms；
Primer 4: the nucleotide sequence shown in SEQ ID No.7 and SEQ ID No.8 forms；
Preferably, PCR primer nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 forms.
4 pairs of primers are devised according to the conservative region of ASFV p72 gene on GenBank, i.e., P72-F/P72-R is (by SEQ
The composition of nucleotide sequence shown in ID No.1 and SEQ ID No.2), P72-2F/P72-2R is (by SEQ ID No.3 and SEQ ID
Nucleotide sequence shown in No.4 composition), the P72-3F/P72-3R (nucleosides as shown in SEQ ID No.5 and SEQ ID No.6
Acid sequence composition) and P72-4F/P72-4R (nucleotide sequence shown in SEQ ID No.7 and SEQ ID No.8 forms), it is right
Its sensibility and specificity carries out PCR detection, the results showed that primer P72-2F/P72-2R, P72-3F/P72-3R and P72-4F/
The PCR sensibility of P72-4R is substantially less than primer P72-F/P72-R, and primer P72-2F/P72-2R has non-specific band
Amplification, therefore, has finally chosen the high primer P72-F/P72-R of PCR high specificity, sensibility, the primer is by SEQ ID No.1
It is formed with nucleotide sequence shown in SEQ ID No.2.
The invention also discloses a kind of kits for detecting African swine fever virus, comprising: primer, Ex Taq buffer, Ex
Taq Hot-start archaeal dna polymerase, MgCl2, dNTP, the primer be above-mentioned primer 1, primer 2, primer 3 or primer 4,
Preferably the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 forms primer 1.
The invention also discloses application of the kit in detection African swine fever virus, comprising the following steps: (1) mentions
Take the DNA of sample to be tested；(2) using the DNA of sample to be tested as template, PCR reaction is established as PCR primer using primer 1
System carries out PCR amplification；(3) amplified production is analyzed；If the band of 326bp occurs in amplified production, illustrate to be detected
Sample in contain African swine fever virus.
The present invention is to the annealing temperature (48~60 DEG C) in PCR reaction, PCR each component (primer, dNTPs and Mg2+Deng)
Concentration optimizes, final to determine the PCR reaction system are as follows: total volume is 25 μ l:10 × Ex Taq buffer, 2.5 μ
L, 0.25 μ l of 5U/ μ l Ex Taq Hot-start archaeal dna polymerase, 10 μM of 1 μ l of primer, 2.5 μ l of 2.5mM dNTPs, 25mM
MgCl22 μ l, 3 DNA μ l and ddH2O 16.75μl；
The PCR response procedures are as follows: 95 DEG C of initial denaturations 5min, 40 amplification cycles (95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C
30s), 72 DEG C of extension 10min.
The present invention is further to the PCR detection method for the African swine fever virus established with primer 1 (P72-F/P72-R)
Conservative, specificity, sensibility and the testing result for ASFV difference strain are analyzed, in which:
Conservative detection shows: the primer P72-F/P72-R designed according to ASFV p72 gene order in GenBank, than
OIE verifying and the method recommended are more conservative, have more versatility.Tool is compared to design using online NCBI BLAST
Primer sequence search determines that primer for the high conservative region of ASFV p72 gene in GenBank, does not find primer and target sequence
Column mismatch, however, it was found that there are a few place's nucleotide and target sequence to mismatch in the PCR primer that OIE is verified and is recommended, show
The new PCR established can be used as the universal test method of ASFV.
Specific detection shows: the PCR detection method that the present invention establishes can detect the DNA sample of all 14 ASFV strains
Product, and do not deposited without any positive signal when detecting other cause of disease (including CSFV, PRRSV, PRV, PCV2 and the PPV) of correlation
In cross reaction.This method can effectively distinguish ASF and other clinically relevant swine diseases, it is ensured that the Accurate Diagnosis of ASF and facilitate
Timely prevention and control.The sequencing result of PCR product also turns out that the PCR method is special.
Sensitivity Detection shows: PCR of the present invention can detect that the ASFV p72DNA of 60 DNA copies.With OIE verifying
PCR primer PPA-1/2 is compared with OIE-F/R, and the amplified band of PCR of the present invention is stronger.
The detection of ASFV difference strain shows: the PCR detection method that the present invention establishes has compared with the PCR that OIE had previously recommended
Have 10 times same or below it detection limit (PCR of the present invention is to Burkina Faso 2007 and 81 plants of Port-au-Prince
Detection limit be respectively 10-4With 10-5Diluted genomic DNA；The PCR detection limit that OIE had previously recommended is respectively 10-3With 10-4It is dilute
The genomic DNA released).In addition, the PCR that PCR detection method of the present invention and Ag ü ero etc. (2003) are established, verified recently by OIE
Detection method is compared, and is had lower than its 10 times (81 plants of Burkina Faso 2007 and Port-au-Prince) or 100 times
Detection limit (Mozambique 1964, Pontevedra1970, Badajoz 1971 and 1988 plants of Sardinia).It is i.e. of the invention
PCR method shows lower detection limit compared with the OIE PCR previously recommended and the OIE PCR method verified, and shows its sensibility more
It is high.The primer that PCR method of the present invention is related to is directed to the highly conserved region of ASFV p72 gene order, does not find primer and target
Sequence mismatches, and there are nucleotide and the unmatched situation of target sequence in the PCR primer that OIE recommends, and show PCR ratio of the present invention
Both methods is more special, has more versatility.
Clinical sample testing result shows: 62 parts of whole blood samples for picking up from Uganda to 2010 to 2015 years have carried out ASF
Diagnosis, 8 samples are detected as the ASFV positive with PCR method of the present invention, consistent with fluorescence quantitative PCR detection result, these sun
Property sample Ct value≤35.The coincidence rate of PCR method of the invention and the fluorescence quantifying PCR method delivered is 90.3%
(56/62), wherein there are 6 samples of higher Ct value (39.79,38.33,38.11,38.05,38.02 and 37.95) to be sent out with this
Bright PCR is detected as feminine gender.Two methods of remaining 48 sample detection is feminine gender.Wherein, 1 part (Ct value is 28.85) and 2 parts
(Ct value is respectively 28.85 and 31.06) ASFV positive sample with the previous OIE PCR method recommended and Ag ü ero etc. establish and by
The PCR method detection of OIE verifying is feminine gender, and the PCR method for showing that the present invention establishes is more reliable, is suitable for clinical use.
Technical solution of the present invention has the advantages that compared with prior art
The present invention is based on the highly conserved region design primers of ASFV p72 gene, sieve to the primer of Preliminary design
Choosing finally screens to obtain the primer 1 with high susceptibility and specificity；Further the African pig of detection is established with the primer 1
The PCR detection method of pestivirus；The confirmation of the experimental results such as specificity, sensibility, PCR detection method ratio established by the present invention
Two kinds of African swine fever virus PCR methods that OIE recommends are more special, more sensitive；Clinical sample testing result shows: PCR of the present invention
Detection method is easy to operate, at low cost, more reliable, compensate for PCR method primer that current OIE recommends and target sequence mismatch,
The low defect of specificity, can be effectively applied to ASF screening and quick diagnosis.
Term definition according to the present invention
Unless otherwise defined, otherwise all technologies used herein and scientific term all have with it is of the art
Those of ordinary skill usually understands identical meaning.
Term " primer " means a bit of single stranded DNA, as the starting point of DNA replication dna, in nucleic acid synthesis reaction, as
Starting point that each polynucleotide chain is extended and the polynucleotide chain to work.
Term " polymerase chain reaction ", abbreviation PCR, it is intended that a kind of Protocols in Molecular Biology, it is specific for amplifying amplification
DNA fragmentation, PCR is that 95 DEG C of high temperature time variations will become single-stranded in vitro using DNA, and whens low temperature (often 60 DEG C or so) draws
Object is in conjunction with the single-stranded principle by base pair complementarity, then temperature regulating is to archaeal dna polymerase optimal reactive temperature (72 DEG C or so),
Archaeal dna polymerase along phosphoric acid to pentose (5' → 3') direction composition complementary strand.
Detailed description of the invention
The sensibility and specificity of Figure 1A SFV PCR primer compares；Wherein, (A) 1-7:10 times of continuous gradient dilutions
CSFV p72 gene DNA standard items (starting copy number 6.0 × 104)；8: negative control (ddH2O)；(B) 1-7:10 times of continuous ladders
Spend diluted CSFV p72 gene DNA standard items (starting copy number 6.0 × 1010)；8: negative control (ddH2O)。
Fig. 2 is the position of 35 ASFV p72 gene global alignments and primer in GenBank；(A) PCR primer of the present invention
The position P72-F/P72-R；(B) position PCR primer OIE-F/OIE-R that OIE had previously recommended；(C)Agüero et al.(2003)
Design, the position primer PPA-1/PPA-2 of nearest OIE verifying；() indicates consistent base.The position of black box expression primer
It sets.
Fig. 3 is the specificity of ASFV PCR method of the present invention；Wherein, 1:CSFV Shimen plants；HuN4 plants of 2:PRRSV；
BQ plants of 3:PPV；TJ plants of 4:PRV；5:PCV2JXL plants；E75 plants of 6:ASFV；7: negative control (ddH2O)；M:DNA
marker。
Fig. 4 is ASFV PCR method (primer P72-F/P72-R) of the present invention and two kinds of PCR methods that OIE is verified and recommended
The comparison of (primer PPA-1/2, OIE-F/OIE-R) sensibility；Wherein, the CSFV p72 gene of 1-7:10 times of continuous gradient dilutions
DNA standard items (starting copy number 6.0 × 104)；8: negative control (ddH2O)。
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1, virus and cell
14 plants of ASFV genomic DNAs of regions of the world are isolated from, Swedish National veterinary institute (SVA) (table is stored in
1).The virus of other clinically relevant pigs is used to evaluate the specificity of new PCR method, including high-pathogenicity porcine reproductive and breathing
The CH-1a strain of HuN4 plants of syndrome virus (PRRSV) and PRRSV classics, JXL plants of porcine circovirus 2 type (PCV2), swine fever virus
(CSFV) Shimen plants (1.1 genotype), HLJ plants (2.1 genotype) and hog cholera lapinised virus vaccine strain (HCLV, 1.1 genes
Type), pseudorabies virus (PRV) TJ plants (variants) and SC plants (classical strain), BQ plants of pig parvoviral (PPV).PRRSV exists
It is cultivated in MARC-145 cell, CSFV, PRV, PPV and PCV2 are cultivated on PK-15 cell.
Table 1 is used for the virus of ASFV PCR method specificity of the present invention detection
The screening of 1 PCR primer of experimental example
1, experimental method
1.1PCR design of primers
According to 35 complete sequences (Fig. 2) of ASFV p72 gene in GenBank and 158 partial sequences (data are not shown)
Sequence alignment result, design four couples of specific primer P72-F/P72-R (as shown in SEQ ID No.1 and SEQ ID No.2
Nucleotide sequence composition), P72-2F/P72-2R (the nucleotide sequence group as shown in SEQ ID No.3 and SEQ ID No.4
At), P72-3F/P72-3R (nucleotide sequence shown in SEQ ID No.5 and SEQ ID No.6 forms) and P72-4F/
P72-4R (nucleotide sequence shown in SEQ ID No.7 and SEQ ID No.8 forms) (table 2).
2 PCR primer sequence of table
The foundation of 1.2PCR reaction condition
1.2.1DNA/RNA it extracts and cDNA is synthesized
It is mentioned from cell culture or blood sample using DNeasy blood&tissue kit (German Qiagen company)
Take genomic DNA.For the RNA virus of specific detection, using QIAamp viralRNAkit (German Qiagen company) from
RNA is extracted in virocyte culture.The reverse transcription of RNA according to before research carry out (Zhang, X.J., Han, Q.Y.,
Sun,Y.,Zhang,X.,Qiu,H.J.,2012.Development of a triplex TaqMan real-time RT-
PCR assay for differential detection of wild-type and HCLV vaccine strains of
classical swine fever virus and bovine viral diarrhea virus 1.Res Vet Sci.92,
512-518.)。
1.2.2DNA standard items
Design one couple of PCR primers P72-Sd-F (5 '-GAA GAA GAA GAA AGT TAATAG-3 ') and P72-Sd-R
(5 '-CAT TAT ATA TGG CAT CAG GAG-3 ') expands ASFV p72 partial gene fragments.PCR reaction system (50 μ l)
Include: 1 × Ex Taq buffer, 2.5U Ex Taq Hot-Start archaeal dna polymerase (Japanese TaKaRa company), 0.4 μM draw
Object, 0.2mM (dNTPs) and 3 μ l genomic DNAs.PCR reaction carries out in TaKaRa PCR instrument (Japan), and program is as follows: 95 DEG C
Initial denaturation 5min, 35 amplification cycles (95 DEG C of 30s, 51 DEG C of 1min, 72 DEG C of 2min), 72 DEG C of extension 10min.PCR product clone
Recombinant plasmid pMD-p72 is obtained to pMD-18T carrier (TaKaRa).The nucleotide sequence of product is determined by automatic sequencer
(ABIPRISM 3100Genetic Analyzer,USA).Recombinant plasmid is quantified by formula: Y (copies/ μ l)=(6.02
×1023Copies) × (plasmid concentration g/ μ l)/[(base number) × (660daltons/ base)] (Zhao, J.J., Cheng,
D.,Li,N.,Sun,Y.,Shi,Z.,Zhu,Q.H.,Tu,C.,Tong,G.Z.,Qiu,H.J.,2008.Evaluation of a
multiplex real-time RT-PCR for quantitative and differential detection of
wild-type viruses and C-strain vaccine of Classical swine fever virus.Vet
Microbiol.126,1–10.)。
1.2.3 the optimization of PCR method of the present invention
In order to establish a sensitive PCR method, we detect the multipair primer of design one by one, while to reaction
Parameter be optimized, including annealing temperature, PCR each component (primer, dNTPs and Mg2+Deng) concentration.PCR reaction system
It is determined as (25 μ l): 10 × Ex Taq buffer2.5 μ l, 0.25 μ l of 5U/ μ l Ex Taq Hot-start archaeal dna polymerase, 10
μM 1 μ l of primer, 2.5 μ l of 2.5mM dNTPs, 25mM MgCl22 μ l, 3 DNA μ l and ddH2O 16.75μl；PCR reaction condition
Are as follows: 95 DEG C of initial denaturation 5min, 40 amplification cycles (95 DEG C of 30s, 48~60 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of extension 10min.It is logical
It crosses 2% agarose gel electrophoresis and determines PCR product.The optimal pair of primers of sensibility and specificity is selected to carry out subsequent reality
It tests.
2, experimental result
PCR testing result shows based on primer P72-2F/P72-2R, P72-3F/P72-3R and P72-4F/P72-4R
PCR sensibility is substantially less than primer P72-F/P72-R (Figure 1A), and primer P72-2F/P72-2R has non-specific band to expand
Increase (Figure 1B).Therefore, it is subsequent to have finally chosen the optimal primer P72-F/P72-R progress of PCR sensibility and specificity for we
Experiment.
Optimum reaction condition determines are as follows: includes 1 × Ex Taq buffer, 1.25U Ex in 25 μ l PCR reaction systems
Taq Hot-start archaeal dna polymerase (Japanese TaKaRa company), 0.4 μM of primer, 0.25mM dNTP, 2mM MgCl2With 3 μ l
DNA.PCR response procedures are as follows: 95 DEG C of initial denaturation 5min, 40 amplification cycles (95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s), 72 DEG C are prolonged
Stretch 10min.
The confirmatory experiment of 2 primer P72-F/P72-R of experimental example
1, experimental method
The test of 1.1 conservatives
When PCR primer designed for molecular diagnosis, the region for selecting viral genome highly conserved is very important,
To guarantee to detect the virus of all known types.
Therefore according to 35 complete sequences (Fig. 2) of ASFV p72 gene in GenBank and 158 partial sequences, (data are not
Display) sequence alignment result (with DNASTAR sequence alignment program analyze), for region highly conserved in gene order design
Specific primer.Tool is compared using online NCBI BLAST to search for the primer sequence of design, determines what the present invention designed
High conservative region of the primer for ASFV p72 gene in GenBank.
1.2 specific test
The specificity of PCR method of the present invention, including the gene of CSFV 1.1 and 2.1 are evaluated by detecting relevant swine disease poison
The highly pathogenic strain of type, PRRSV and classical strain, PRV variant and classical strain, PCV2 and PPV, while 14 plants of Parallel testing separation
ASFV from different regions.The specificity of sequencing confirmation this method is carried out to the amplified production of expected size (326bp).
1.3 sensitivity tests
In order to determine the minimum detection limit of PCR method of the present invention, by ASFV DNA standard items (2.0 × 104copies/μl)
Continuous 10 times of gradient dilutions are detected, and further the PCR method with two OIE verifying compares, and one previous by OIE
Recommend (Wilkinson, P.J., 2000.African swine fever.In:Manual of Standards for
Diagnostic Test and Vaccines,4th ed.Office International des Epizooties,
Paris, France, pp.189-198.), another by Ag ü ero etc. establish (Ag ü ero, M., Fern á ndez, J., Romero,
L.,Sánchez Mascaraque,C.,Arias,M.,Sánchez-Vizcaíno,J.M.,2003.Highly sensitive
PCR assay for routine diagnosis of African swine fever virus in clinical
Samples.J.Clin.Microbiol.41,4431-4434.), recently through OIE verifying (Oura, C.A.L., Arias, M.,
2012.African swine fever.In:OIE Biological Standards Commission(ed),OIE
Manual of Diagnostic Tests and Vaccines for Terrestrial Animals,7th ed.Office
International des Epizooties,Paris,France,pp.1069–1082.)).PCR reaction system (25 μ l)
Are as follows: 1 × PCR buffer II (50mM KCl, 10mM Tris-HCl, pH 8.3), 2mM MgCl2、0.2mM dNTP、0.2mM
Primer (PPA-1/2 or OIE-F/R), 0.625U Taq Gold archaeal dna polymerase (Applied Biosystems) and 3 μ l samples
DNA.When using primer PPA-1/2, PCR response procedures are as follows: i) 95 DEG C of initial denaturation 10min；Ii) 40 amplification cycles: 95
DEG C 15s, 62 DEG C of 30s, 72 DEG C of 30s；Iii) 72 DEG C of extension 7min.When using primer OIE-F/R, PCR is carried out as follows:
I) 95 DEG C of initial denaturation 10min；Ii) 35 amplification cycles: 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s；Iii) 72 DEG C of extension 7min.Benefit
Amplified production is analyzed with 2% agarose gel electrophoresis containing ethidium bromide.
The comparison of 1.4 different PCR methods
To 14 ASFV strains (continuous ladder of 4 genotype (I, V, IX and VIII) of representative from different parts of the world
Degree dilution) (table 1) analyzed with PCR method of the present invention, and the PCR method of the above-mentioned two kinds of OIE verifying of utilization simultaneously with
And Haines etc. (Haines, F.J., Hofmann, M.A., King, D.P., Drew, T.W., Crooke, H.R.,
2013.Development and validation of a multiplex,real-time RT PCR assay for the
simultaneous detection of classical and African swine fever viruses.PLoS One
8, e71019.) quantitative fluorescent PCR established has carried out Parallel testing.Quantitative fluorescent PCR is delivered according to Haines etc. (2013)
Primer and probe is carried out using Ag Path-ID one-step RT-PCR kit (Life Technologies).Reaction system
ASFV probe, the 1 μ l enzymatic mixture (enzyme marked including 1 × PCR buffer, 0.8 μM of ASFV primer, 0.2 μM of Cy5
) and 3 μ l viral nucleic acids mix.PCR response procedures are as follows: 95 DEG C of 10min, 48 amplification cycles (45 DEG C of 15s and 60 DEG C of 45s).
2, experimental result
The conservative of 2.1 selection primers
As can be seen from Figure 2 the primer P72-F/P72-R designed according to ASFV p72 gene order in GenBank, than
Method (Wilkinson, P.J., 2000.African swine fever.In:Manual of OIE verifying and recommended
Standards for Diagnostic Test and Vaccines,4th ed.Office International des
Epizooties,Paris,France,pp.189–198.；Agüero,M.,Fernández,J.,Romero,L.,Sánchez
Mascaraque,C.,Arias,M.,Sánchez-Vizcaíno,J.M.,2003.Highly sensitive PCR assay
for routine diagnosis of African swine fever virus in clinical
samples.J.Clin.Microbiol.41,4431–4434.；Oura,C.A.L.,Arias,M.,2012.African
swine fever.In:OIE Biological Standards Commission(ed),OIE Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals,7th ed.Office
International des Epizooties, Paris, France, pp.1069-1082.) it is more conservative, with more general
Property.It compares tool using online NCBI BLAST to search for the primer sequence of design, what determining PCR method of the present invention was related to draws
Object does not find that primer and target sequence mismatch for the high conservative region of ASFV p72 gene in GenBank, however, it was found that
There are a few place's nucleotide and target sequence to mismatch in OIE verifying and the PCR primer recommended, and shows PCR of the present invention than both
Method is more special, has more versatility, can be used as the universal test method of ASFV.
The specificity of 2.2 PCR methods of the present invention
In order to confirm the specificity of PCR of the present invention, other related swine disease cause of diseases are had detected with this method.As expected, the PCR
Method can detect the DNA sample of all 14 ASFV strains, and detect related other cause of diseases (including CSFV, PRRSV, PRV,
PCV2 and PPV) when without any positive signal, that is, be not present cross reaction (representative result is shown in Fig. 3).This method can be distinguished effectively
ASF and other clinically relevant swine diseases, it is ensured that the Accurate Diagnosis of ASF and facilitate timely prevention and control.The sequencing result of PCR product
Prove that the PCR method is special.
The sensibility of 2.3 PCR methods of the present invention
PCR of the present invention can detect that the ASFV p72DNA (Fig. 4) of 60 DNA copies.With the PCR primer PPA- of OIE verifying
1/2(Agüero,M.,Fernández,J.,Romero,L.,Sánchez Mascaraque,C.,Arias,M.,Sánchez-
Vizcaíno,J.M.,2003.Highly sensitive PCR assay for routine diagnosis of
African swine fever virus in clinical samples.J.Clin.Microbiol.41,4431–4434.；
Oura,C.A.L.,Arias,M.,2012.African swine fever.In:OIE Biological Standards
Commission(ed),OIE Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals,7th ed.Office International des Epizooties,Paris,France,pp.1069–
And OIE-F/R (Wilkinson, P.J., 2000.African swine fever.In:Manual ofStandards 1082.)
for Diagnostic Test and Vaccines,4th ed.Office International des Epizooties,
Paris, France, pp.189-198.) it compares, the amplified band of PCR of the present invention is stronger (Fig. 4).
The detection of 2.4ASFV difference strain
As shown in table 3, detection of the PCR of the present invention compared with the PCR that OIE had previously recommended with 10 times same or below it limits
(PCR of the present invention is respectively 10- to Burkina Faso 2007 and 81 plants of Port-au-Prince of detection limit4And 10-5Dilution
Genomic DNA；The PCR detection limit that OIE had previously recommended is respectively 10-3And 10-4Diluted genomic DNA).In addition, of the invention
Compared with PCR is with the foundation such as Ag ü ero (2003), recently by the PCR of OIE verifying, have same or below its 10 times of (Burkina
Faso 2007 and 81 plants of Port-au-Prince) or 100 times detection limit (Mozambique 1964, Pontevedra
1970, Badajoz 1971 and 1988 plants of Sardinia).PCR method i.e. of the present invention is tested with the OIE PCR previously recommended and OIE
The PCR method of card is compared, and is shown lower detection limit, is shown that its sensibility is higher.
The different PCR methods of table 3 detect 14 plants of ASFV strains for being isolated from different regions
The detection of 3 clinical sample of experimental example
62 parts of whole blood samples for picking up from Uganda to 2010 to 2015 years have carried out ASF diagnosis.These samples are from clinic
Infection or clinically healthy domestic pig, be sent to the diagnosis of Uganda's National Animal Disease and epidemiology center (NADDEC) into
Row detection.Nucleic acid extraction is carried out to 50 μ l blood samples using DNeasy blood&tissue kit (German Qiagen company),
Nucleic acid samples are sent to SVA and are detected by quantitative fluorescent PCR as described above.Miniprep dna sample is sent to Chinese Kazakhstan
Your shore veterinary institute (HVRI), and to all samples using PCR method of the present invention and two kinds of PCR methods of OIE verifying into
Row detection.
In 62 clinical samples, 8 samples are detected as the ASFV positive with PCR method of the present invention, with fluorescence quantitative PCR detection
As a result consistent, the Ct value of these positive samples is ≤35.PCR method of the present invention and the fluorescence quantifying PCR method delivered
Coincidence rate is 90.3% (56/62), wherein has higher Ct value (39.79,38.33,38.11,38.05,38.02 and 37.95)
6 samples are detected as feminine gender with PCR of the present invention.Two methods of remaining 48 sample detection is feminine gender.Wherein, 1 part of (Ct value
28.85) and 2 parts of (Ct value is respectively 28.85 and 31.06) ASFV the positive samples previous OIE PCR method recommended and Ag ü for
Ero etc. is established, the PCR method detection of OIE verifying is negative (table 4), and it is more reliable to show PCR method of the present invention, is suitable for
Clinical use.
Detection case of the PCR method that the PCR method of the present invention of table 4 and two kinds of OIE are verified to epidemic-stricken area clinical sample
+: it is positive；: it is negative.
Claims (3)
1. detecting the PCR primer of African swine fever virus (African swine fever virus), it is characterised in that: described to draw
Object nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 forms.
2. a kind of kit for detecting African swine fever virus, comprising: primer, Ex Taq buffer, Ex Taq Hot-start
Archaeal dna polymerase, MgCl2, dNTP and aqua sterilisa, it is characterised in that: the primer is PCR primer described in claim 1.
3. application of the PCR primer described in claim 1 in the reagent of preparation detection African swine fever virus.
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| CN107663550A (en) * | 2016-07-29 | 2018-02-06 | 中国动物卫生与流行病学中心 | African swine fever virus pyrosequencing method |
| CN106521027B (en) * | 2016-11-03 | 2019-12-06 | 河北出入境检验检疫局检验检疫技术中心 | Real-time isothermal recombinase polymerase amplification detection kit for African swine fever virus |
| CN106957927B (en) * | 2017-04-20 | 2020-11-20 | 中国检验检疫科学研究院 | African swine fever fluorescent PCR detection reagent, African swine fever fluorescent PCR detection kit and application thereof |
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| CN107937624A (en) * | 2018-01-17 | 2018-04-20 | 石河子大学 | The RPA primers and preparation method and kit of quick detection African swine fever virus nucleic acid |
| WO2020073810A1 (en) * | 2018-10-09 | 2020-04-16 | 福又达生物科技股份有限公司 | Method for detecting african swine fever virus |
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| CN109781981B (en) * | 2019-01-30 | 2022-04-01 | 河南中泽生物工程有限公司 | Molecular probe for detecting African swine fever virus and Realtime-PCR detection method |
| CN110551851A (en) * | 2019-09-06 | 2019-12-10 | 中国科学院过程工程研究所 | CAMP primer group for amplifying ASFV, kit and application |
| CN110878377B (en) * | 2019-11-06 | 2023-08-22 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Fluorescent quantitative PCR differential diagnosis kit for strong and weak viruses of African swine fever viruses |
| CN111363852A (en) * | 2020-04-20 | 2020-07-03 | 叶繁全 | African swine fever virus detection kit |
| CN111575404B (en) * | 2020-05-08 | 2023-03-14 | 中国兽医药品监察所 | Gene chip for differential diagnosis of swine fever wild virus and vaccine thereof, african swine fever virus and detection method |
| CN111621603A (en) * | 2020-06-23 | 2020-09-04 | 广州欧密伽畜牧有限公司 | Specific primer for identifying African swine fever virus, and identification method and detection kit thereof |
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