CN110093457A - A kind of African swine fever virus ASFV-LAMP detection primer group and kit - Google Patents
A kind of African swine fever virus ASFV-LAMP detection primer group and kit Download PDFInfo
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Abstract
The present invention relates to a kind of African swine fever virus LAMP detection primer group, kit and its detection methods.For the reply 2018 African swine fever epidemic situations in China's discovery, the primer sets that the present invention uses African swine fever virus P72 gene to detect as LAMP, primer sets specific detection African swine fever virus gene P72, ASFV can effectively be detected, new technological means is provided for African swine fever prevention and control, is conducive to strain detection and entry and exit rapid screening.The quick detection of African swine fever virus may be implemented in the LAMP detection method, testing result can be estimated directly, only need heating that entire reaction can be realized, get rid of dependence of traditional nucleic acid detection technique for PCR instrument, and the method detection sensitivity height, high specificity, it is reproducible, detection speed it is fast, can be as effective African swine fever on-site test means.
Description
Technical field:
The invention belongs to field of biotechnology, and in particular to a kind of African swine fever virus LAMP detection primer group, kit
And its detection method.
Background technique:
African swine fever (African swine fever, ASF) is by African swine fever virus (African swine fever
Virus, ASFV) caused by one kind is acute, hot, highly contagious disease, disease time is short, and case fatality rate is high.ASF is to feeding
Pig industry endangers epidemic disease the most serious, is classified as statutory report epidemic disease by World Organization for Animal Health (OIE), is classified as one kind by China
Infectious disease.ASFV belongs to double-stranded DNA virus mesh African swine fever virus section African swine fever virus category.The category only has an ASFV at present
Virus kind.The genome of ASFV is unimolecule linear DNA, length about 170~190kb.ASFV individual is larger, and diameter is reachable
200nm, surface be icosahedral structure of virus and one layer of cyst membrane containing lipoid, therefore high temperature and it is some destroy cyst membrane disinfectant
Effectively kill ASFV.But due to its complicated molecular structure, ASFV to the more other togavirus of the resistance of disinfectant slightly
By force, especially the time-to-live is longer in some pork products or blood.It is reported that ASFV is in blood, excrement and tissue
Time-to-live is up to half a year, and in raw meat or in not well-done pork product, the time-to-live, this was resulted in up to 3 months
ASFV, often in plague area long-term existence, causes secondary or secondary infection once being passed to.The disease incidence and case fatality rate of African swine fever
It can reach 100%, and there is no effective vaccine to come out in world wide at present, diffusion and prevalence may cause pig raising industry
Crushing blow, resulting indirect loss can not then be estimated.
African swine fever virus is found in Kenya in nineteen twenty-one for the first time, and nineteen fifty-seven ASF is spread out of from the African continent, and in
Portuguese Lisbon area epidemic outbreaks, then spread to Spain, Italy, France, Belgium, Holland, Malta etc.
European countries.On August 3rd, 2018, Shenyang City, pig farm, Shenbeixin District confirm that ASF epidemic situation occurs.This is that China occurs for the first time
ASF, molecule epidemic disease-ology research the result shows that, be passed to China African swine fever virus category gene II type, with Georgia, Russia sieve
This, Poland announce strain whole genome sequence homology be 99.95% or so.By on December 3rd, 2018, the whole nation shared 21
A province occurs 79 and builds up schweineseuche feelings, 2 wild boar epidemic situations, the whole nation is accumulative slaughter live pig 63.1 ten thousand (source: agriculture rural area portion,
The www.xinhuanet.com), direct economic loss reaches tens of Yu Yiyuan.Due to not can be carried out immunization campaign and effectively treatment, prevention and control to ASF
Work must be taken and slaughter control techniques, and the normal of severe jamming aquaculture produces and orders of life, and great economy is caused to damage
It loses.Therefore, African swine fever has become grave danger of China's pig breeding industry, and research achievement has great political economy meaning.
Existing laboratory testing method mainly has serological method and aetology method.Serological method mainly has indirectly
Enzyme-linked immunosorbent assay blocks enzyme-linked immunosorbent assay and indirect fluorescent antibody test etc., and wherein Enzyme-linked Immunosorbent Assay tries
Testing has had the kit of commercialization to sell.The aetology method of ASFV also has very much, such as viral cell culture and separation, directly
Connect immunofluorescence technique, double antibody sandwich ELISA, regular-PCR and fluorescence PCR method etc., wherein OIE recommend regular-PCR and
Fluorescence PCR method is the most commonly used.3 kinds of ASFV pathogeny detections are referred in China's " African swine fever Prevention Technique specification (tentative) "
Method: double antibody enzyme-linked immunosorbent assays, polymerase chain reaction and real-time fluorescent polyase chain reaction (OIE official
Square website).In addition to this, ASF Control Technology development work dynamics is also increased, develops simple, efficient, special diagnosis as early as possible
Reagent and diagnostic method, and can mutually identify with other swinery epidemic diseases such as classic swine fever, highly pathogenic PRRSs.
Ring mediated isothermal amplification method (Loop mediated isothermal amplification method, LAMP)
It is a kind of emerging nucleic acid amplification technologies, it realizes the nucleic acid rapid amplifying under isothermy using unique design of primers.?
Also occur in existing periodical literature article, such as Jiang Yanzeng of more LAMP detection African swine fever etc. 1., Yang Ji fly etc. 2., king
Rosy clouds etc. 3., Wang Hua etc. 4. establish African swine fever virus loop-mediated isothermal amplification fast detection method, however, in the market simultaneously
Do not occur relevant product.In order to which by the detection and product of the Technology application to ASFV, reply 2018 in China's discovery
African swine fever epidemic situation, the primer sets that this research uses African swine fever virus P72 gene to detect as LAMP, the primer group-specific
Encode African swine fever virus gene P72.ASFV can be effectively detected, new technological means is provided for African swine fever prevention and control, favorably
In strain detection and rapid screening of entering and leaving the border, and testing result accuracy is high, reproducible.
Summary of the invention:
In order to solve African swine fever detection common detection methods low efficiency, Bu Nengshi stronger for the dependence of instrument
The technical issues of existing on-site test, the present invention is intended to provide a kind of African swine fever virus LAMP detection primer group, kit and its
The quick detection of African swine fever virus may be implemented in detection method, the LAMP detection method, and testing result can be estimated directly,
It only needs heating that entire reaction can be realized, gets rid of dependence of traditional nucleic acid detection technique for PCR instrument, and the method is examined
Survey high sensitivity, high specificity, reproducible, detection speed is fast, can be as effective African swine fever on-site test means.
The purpose of the present invention is to provide African swine fever virus LAMP detection primer groups.The primer sets specific detection Africa
The encoding gene B646L gene of swine fever virus capsid protein p72.
Another object of the present invention is to provide African swine fever virus LAMP detection kits.The kit is mediated using ring
Isothermal amplification technique can be examined effectively by the detection of the encoding gene B646L gene to African swine fever virus capsid protein p72
ASFV out provides new technological means for African swine fever prevention and control, is conducive to the detection of different genotype strain and entry and exit quickly sieve
It looks into.
Third object of the present invention is to provide African swine fever virus LAMP detection method, which can quickly examine
ASF out, testing result accuracy is high, reproducible.The method can be used for the diagnosis of disease, such as the diagnosis of African swine fever；
It can be used for the diagnostic purpose of non-disease, for example, the confirmation of virus, Classification Identification of virus etc. in scientific research.
To achieve the goals above, the present invention provides African swine fever virus LAMP detection primer group, and the primer sets include:
A pair of of outer primer, a pair of of inner primer, a pair of of ring primer.Its nucleotide sequence difference is as follows:
Outer primer:
ASFV-2-F3:CCGTAACTGCTCATGGTA (SEQ ID No.1).
ASFV-2-B3:AGAAAGTTAATAGCAGATGCC (SEQ ID No.2).
Inner primer:
ASFV-2-FIP:TCATCGCACCCGGATCATGTTCTGCAGCTCTTACATAC (SEQ ID No.3).
ASFV-2-BIP:GGGAGGAATACCAACCCAGTTAATCCGTGTCCCAACTAAT (SEQ ID No.4).
Ring primer:
ASFV-2-LoopF:TTAATCGCATTGCCTCCG (SEQ ID No.5).
ASFV-2-LoopB:TAACGTATCCAGAGCAAGAGA (SEQ ID No.6).
The present invention separately provides a kind of African swine fever virus LAMP detection kit, and the kit includes above-described draws
Object group.
Preferably, the kit includes above-described primer sets, archaeal dna polymerase, LAMP reaction solution, glycine betaine, sun
Property control and negative control.
Preferably, outer primer, ring primer, inner primer molar ratio be 1:(4-6): (8-12).
Preferably, the archaeal dna polymerase is Bst archaeal dna polymerase.
Preferably, the LAMP reaction solution contains 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM
MgSO4Aqueous solution.
Preferably, the positive control is the Plasmid DNA containing target gene fragment, and negative control is sterile water, preferably
Using distilled water, tri-distilled water or DEPC water.
Preferably, the positive control is the Plasmid DNA containing p72 gene.
Preferably, the sequence of the p72 gene is as shown in SEQ ID No.7.
The present invention separately provides a kind of African swine fever virus LAMP detection method, includes the following steps:
(1) sample to be tested DNA is extracted；
(2) loop-mediated isothermal amplification: 25 μ L reaction systems of configuration, the reaction system contain above-described primer
Then group, archaeal dna polymerase, LAMP reaction solution and sample to be tested DNA are reacted with sterile water polishing to 25 μ L at 63-68 DEG C
30-60min；
(3) interpretation of result: observe by the naked eye whether solution in reaction tube becomes cloudy or by agarose gel electrophoresis point
Whether analysis there is scalariform band, as solution becomes cloudy or determines if there is scalariform band by agarose gel electrophoresis in reaction tube
It is otherwise feminine gender for the positive.
Preferably, color developing agent or fluorescent material can also be increased in loop-mediated isothermal amplification system, pass through face
Color change or fluorescence detection carry out the judgement of result.
The African swine fever virus LAMP detection method can be used for the diagnosis of disease, such as the diagnosis of African swine fever；?
It can be used for the diagnostic purpose of non-disease.
Based on above technical scheme, the invention has the advantages that and the utility model has the advantages that
First, the present invention chooses target gene of the conserved genetic sequences p72 of African swine fever virus as detection, Neng Goujian
The accuracy of testing result can be guaranteed by surveying Multi-genotype and different strains, avoid the appearance of missing inspection, for pig farm or
Culturing area, which carries out purification work, to have great importance.
Second, the present invention uses LAMP technology, and specificity is high, is only capable of the genome of specific amplification African swine fever virus
Sequence, and design of primers science, avoid the formation of primer dimer, ensure that going on smoothly for reaction.
Third, African swine fever virus LAMP detection method high sensitivity of the invention, the lowest detection limit are 25 copies sun
Property Plasmid DNA, higher than the LAMP detection method of prior art report, compared to the PCR detection method that OIE recommends, sensitivity mentioned
It is nearly 20 times high.
4th, it is easy to operate, complicated instrument is not needed, special reagent is not needed, it is only necessary to water bath with thermostatic control can react,
Reaction condition is mild；And it is glimmering to observe by the naked eye muddiness, agarose gel electrophoresis, addition chromogenic reagent or addition
Stimulative substance carries out the approach abundant such as fluorescence detection and determines testing result, is not only suitable for pig farm on-site test, is also suitble to scientific research list
Position is used and is promoted.
Detailed description of the invention:
Fig. 1: digestion verification figure: 1, pUC57-P72 plasmid；2, pass through the plasmid pUC57-P72 after SalI-XbaI digestion；
M, Marker.
Fig. 2: LAMP testing result figure: 1, plasmid pUC57-P72 is detected as template；2, detect ddH2O；3, with
The DNA that SPF pig lymph node extracts carries out LAMP detection as template.Note: solution is limpid after underscore indicates reaction in figure, for yin
Property.
Fig. 3: LAMP testing result electrophoretogram: M, Marker；1, plasmid pUC57-P72 is detected as template；2, inspection
Survey ddH2O；3, LAMP detection is carried out using the DNA that SPF pig lymph node extracts as template.
Fig. 4: LAMP sensitivity test result figure: 1, plasmid 1 × 107Copy/μ l；2, plasmid 1 × 106Copy/μ l；3, matter
Grain 1 × 105Copy/μ l；4, plasmid 1 × 104Copy/μ l；5, plasmid 1 × 103Copy/μ l；6, plasmid 1 × 102Copy/μ l；7,
Plasmid 1 × 101Copy/μ l；8,50 copies of plasmid/μ l；9,25 copies of plasmid/μ l；10,13 copies of plasmid/μ l；11, plasmid 6 is copied
Shellfish/μ l；12, negative control.
The repeated testing result figure of Fig. 5: LAMP test experience sample: 1, positive control；2, positive plasmid pUC57-
P72-1；3, positive plasmid pUC57-P72-2；4, positive plasmid pUC57-P72-3；5, positive plasmid pUC57-P72-4；6, sun
Property grain pUC57-P72-5.
Fig. 6: LAMP specific detection result electrophoretogram: 1, African swine fever positive plasmid pUC57-P72；2, pig Japanese B
Encephalitis viruses；3, swine fever virus；4, pig parvoviral；5, porcine pseudorabies virus；6, pig circular ring virus；7, transmissible gastroenteritis of swine
Virus.
Fig. 7: LAMP specific detection result figure: 1, African swine fever positive plasmid pUC57-P72；2, pig Japanese B encephalitis
Virus；3, swine fever virus；4, pig parvoviral；5, porcine pseudorabies virus；6, pig circular ring virus；7, transmissible gastroenteritis of swine disease
Poison.Note: solution is limpid after underscore indicates reaction in figure, for feminine gender.
Specific embodiment:
Below in conjunction with specific embodiment, the present invention is further illustrated, but not limited to this.
Embodiment 1: sample, design of primers and preparation
1.1 plasmids, sample source and experiment place
It is raw by raw work according to the African swine fever virus P72 protein gene (shown in SEQ ID No.7) provided on Genebank
Object engineering (Shanghai) limited liability company synthetic plasmid pUC57-P72, dissolved dilution to 1.0 × 107Copy/μ L.
Pig Japanese B encephalitis virus, swine fever virus, pig parvoviral, porcine pseudorabies virus, pig circular ring virus, pig are infected
Property marcy agent by Shaanxi Nowe Li Hua Biotechnology Co., Ltd provide.
SPF pig lymph node, SPF Swine serum are acquired from Shaanxi Nowe Li Hua Biotechnology Co., Ltd.
The identification of 1.2 positive plasmids
Digestion is carried out to the site SalI-XbaI of positive plasmid pUC57-P72, verifies the correctness of plasmid.To positive matter
The site SalI-XbaI of grain pUC57-P72 carries out digestion identification, the results showed that, plasmid form is normal, target gene size just
Really, carrier size is correct, electrophoretic band is clear, without genome, without miscellaneous band.As a result as shown in Figure 1.
The design of 1.3 ASFV LAMP primers and screening
According to the ASFV strain P72 gene order that Genebank is announced, set by http://PrimerExplorer.jp
5 sets of primers are counted, primer sequence is as shown in table 1.By Sangon Biotech (Shanghai) Co., Ltd..Specific primer sequence is as follows
Shown in table 1:
1 African swine fever virus LAMP detection primer group of table
Embodiment 2: the foundation of African swine fever virus LAMP detection kit
A kind of African swine fever virus LAMP detection kit, the kit include above-described primer sets.
Preferably, the kit includes primer sets described in above embodiments 1, Bst archaeal dna polymerase, LAMP reaction
Liquid, glycine betaine, positive control and negative control.
Preferably, outer primer, ring primer, inner primer molar ratio be 1:(4-6): (8-12).
Preferably, the LAMP reaction solution contains 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM
MgSO4Aqueous solution.
Preferably, the positive control is the Plasmid DNA containing target gene fragment, and negative control is sterile water, preferably
Using distilled water, tri-distilled water or DEPC water.
Preferably, the positive control is the Plasmid DNA containing p72 gene.
Preferably, the sequence of the p72 gene is as shown in SEQ ID No.7.
[points for attention]
1. in order to reduce cross contamination, ask division operation (the configuration operation of reagent and template preferably in the different areas into
Row).
2. each area's article be it is dedicated, can not cross-reference, prevent from polluting.
3. work clothes should be worn in experimentation, band gloves, mask.
Embodiment 3: the foundation of African swine fever LAMP detection method
The foundation of 3.1 LAMP reaction systems
According to the kit of embodiment 2,25 μ l reactants of African swine fever virus LAMP detection are determined using the above primer
The content and ratio of each component, place it in thermostatic container and are expanded in system.Observe by the naked eye white opacity and gel
Electrophoresis combines, and judges testing result.Wherein 25 μ L are shown in reaction system such as table 2.
2 25 μ L reaction system of table
3.2 LAMP reaction
3.2.1 the determination in LAMP reaction time
According to above-mentioned fixed LAMP reaction system, it is assumed that under conditions of 65 DEG C, by positive plasmid pUC57-P72 points
Not carry out 20min, 30min, 40min, 50min, 60min specific amplification, record as a result, determine optimum reacting time.
It is respectively 10 by concentration4The positive plasmid pUC57-P72 of copy at 65 DEG C, respectively 20min, 30min,
Specific amplification is carried out under the time of 40min, 50min, 60min, the results showed that, 40min and after, positive plasmid pUC57-
White opacity (see Fig. 2) is presented in P72LAMP reaction tube, through gel electrophoresis, it is seen that scalariform band (see Fig. 3) i.e. testing result is
It is positive.Therefore, optimum reacting time 40min.
3.2.2 the determination of LAMP reaction temperature
According to above-mentioned fixed reaction system and reaction time.It is respectively 10 by concentration4Copy positive plasmid pUC57-
P72 at 60-70 DEG C, setting 61 DEG C, 63 DEG C, 65 DEG C, 67 DEG C, 69 DEG C of five temperature reacted, observe test result, determine
Optimal reaction temperature.
The result shows that white opacity is presented in positive plasmid pUC57-P72LAMP reaction tube under conditions of 65 DEG C.Cause
This, optimal reaction temperature is 65 DEG C.
3.2.3 the sensitivity technique of African swine fever virus LAMP detection kit
Plasmid pUC57-P72 ddH is lyophilized in the African swine fever positive2O is by the abundant dissolved dilution of plasmid to 1 × 107A copy
Number, dissolved plasmid saves in -20 DEG C, spare.1 × 10 will be diluted to7Copy/μ l plasmid does 10-0~10-7Gradient is dilute
It releases, LAMP amplification is carried out to the positive plasmid sample of different copy numbers respectively.
African swine fever positive plasmid pUC57-P72 is from 10-1~10-6It is amplifiable in diluted plasmid again to arrive expected sun
Property, the plasmid number that can be measured is 100 copies/μ l (see Fig. 4).3 are shown in Table to the sensitivity results of above-mentioned sample detection.
The sensitivity Detection result of 3 LAMP test experience sample of table
For the sensitivity of further accurate validation African swine fever virus LAMP detection kit, concentration is copied for 100
Shellfish/μ l plasmid solution has carried out further dilution, has carried out further sensitivity technique using the above identical method, spirit
Sensitivity results are shown in Table 4 and Fig. 4.
The sensitivity Detection result of 4 LAMP test experience sample of table
As it can be seen that the sensitivity of African swine fever virus LAMP detection kit constructed by the present invention is 25 copies/μ l, it can
The minimum plasmid number measured is 25 copies/μ L.Compared to OIE recommend PCR detection method about 500 copy sensitivity (referring to ginseng
Examine document 1.), sensitivity of the invention improves about 20 times.
The sensitivity tests of 3.3 African swine fever virus LAMP detection kits
5 parts of sensibility quality-control samples: African swine fever positive plasmid pUC57-P72-1, African swine fever sun are detected with kit
It is property grain pUC57-P72-2, African swine fever positive plasmid pUC57-P72-3, African swine fever positive plasmid pUC57-P72-4, non-
Respectively plasmid ddH is lyophilized in 5 kind of 4 μ g African swine fever positive by continent swine fever positive plasmid pUC57-P72-52O is sufficiently molten by plasmid
Solution is diluted to 1 × 108A copy number repeats detection 3 times.
5 parts of sensibility quality-control samples are detected with the African swine fever virus LAMP detection kit that this research is developed, repeat to examine
It surveys 3 times, the sensibility quality-control sample of 5 parts of 3 detections is positive (see Fig. 5).Sensitivity and repetition to above-mentioned sample detection
Property the results are shown in Table 5.
The repeated testing result of 5 LAMP test experience sample of table
The specific test of 3.4 African swine fever virus LAMP detection kits
Using conventional method extract pig Japanese B encephalitis virus, swine fever virus, pig parvoviral, porcine pseudorabies virus,
Pig circular ring virus, transmissible gastro-enteritis virus nucleic acid as template, apply this research together with positive plasmid pUC57-P72
The African swine fever virus LAMP detection kit of development is detected respectively, observes its specificity.
The experimental results showed that detecting pig Japanese B encephalitis virus (JEV), swine fever virus (CSFV), pig with the kit
Parvovirus (PPV), porcine pseudorabies virus (PRV), pig circular ring virus (PCV), transmissible gastro-enteritis virus (TGEV), as a result
It is all negative.Known African swine fever positive plasmid pUC57-P72-2 is detected, result is the positive.It the results are shown in Table 6, Fig. 6-7.
The specific assay of 6 ASFV LAMP of table
Embodiment 4: the remolding sensitivity of African swine fever LAMP detection method compared with
In research process of the present invention, a large amount of Optimization Work is carried out for primer, has obtained multiple groups primer, and compare
The multiple groups primer of the prior art, primer sets sequence such as the following table 7 for specifically comparing:
The primer sets sequence (control group 1--5) of 7 prior art of table
Note: the sequence of the above control group 1-4 is primer sequence in the prior art, is specifically shown in the correlation of background technology part
Document, control group 5 (SEQ ID No:8-13) are to screen preferable one group of primer in our company's R&D process.
Note: the above sequence 1-5 is primer sequence in the prior art, is specifically shown in the pertinent literature of background technology part.
Using the method for " sensitivity technique of 3.2.3 African swine fever virus LAMP detection kit " in above embodiments 3
Determine the primer of the embodiment of the present invention 1 and the sensibility of the above control group 1-5, specific testing result such as following table 8-9.
The sensitivity Detection result (one) of 8 LAMP test experience sample of table
| Copy number | 108 | 107 | 106 | 105 | 104 | 103 | 102 | 101 | 100 |
| Embodiment 1 | + | + | + | + | + | + | + | - | - |
| Control 1 | + | + | + | + | + | + | + | - | - |
| Control 2 | + | + | + | + | + | + | + | - | - |
| Control 3 | + | + | + | + | + | + | - | - | - |
| Control 4 | + | + | + | + | + | + | - | - | - |
| Control 5 | + | + | + | + | + | + | - | - | - |
The sensitivity Detection result (two) of 9 LAMP test experience sample of table
| Copy number | 102 | 50 | 25 | 13 | 6 | 3 |
| Embodiment 1 | + | + | + | - | - | - |
| Control 1 | + | - | - | - | - | - |
| Control 2 | + | - | - | - | - | - |
| Control 3 | - | - | - | - | - | - |
| Control 4 | - | - | - | - | - | - |
| Control 5 | - | - | - | - | - | - |
As it can be seen that the sensitivity of African swine fever virus LAMP detection kit constructed by the present invention is 25 copies/μ l, it can
The minimum plasmid number measured is 25 copies/μ L.Primer sets 1-5 compared with prior art, the primer sets of the embodiment of the present invention 1 have
Higher sensitivity.There is more importantly meaning for African swine fever epidemic situation early stage or preclinical detection.
Above embodiments are a further detailed description of the invention, and provide embodiment only for illustrating the present invention, without
It is to limit the scope of the invention.
Bibliography:
1. Jiang Yanzeng, the foundation of African swine fever virus the loop-mediated isothermal amplification fast detection method, " Chinese Academy of Agricultural Sciences
Academic dissertation ".
2. Yang Ji flies etc., the foundation and application of African swine fever virus loop-mediated isothermal amplification Fast Detection Technique, " China is dynamic
Object infectious disease journal ", 2011,19 (4): 7-12.
3. Wang Caixia etc., loop-mediated isothermal amplification technology quickly detects African swine fever virus, " animal medicine progress ", 2010
The 2nd phase of volume 31 year: 15-19.
4. Wang Hua etc., the foundation of African swine fever virus ring mediated isothermal amplification diagnostic method, " Chinese veterinary science ",
2010,40 (09): 940-944).
Sequence table
<110>Shaanxi Nowe Li Hua Biotechnology Co., Ltd
<120>a kind of African swine fever virus ASFV-LAMP detection primer group and kit
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> ASFV-2-F3(ASFV)
<400> 1
ccgtaactgc tcatggta 18
<210> 2
<211> 21
<212> DNA
<213> ASFV-2-B3(ASFV)
<400> 2
agaaagttaa tagcagatgc c 21
<210> 3
<211> 38
<212> DNA
<213> ASFV-2-FIP(ASFV)
<400> 3
tcatcgcacc cggatcatgt tctgcagctc ttacatac 38
<210> 4
<211> 40
<212> DNA
<213> ASFV-2-BIP(ASFV)
<400> 4
gggaggaata ccaacccagt taatccgtgt cccaactaat 40
<210> 5
<211> 18
<212> DNA
<213> ASFV-2-LoopF(ASFV)
<400> 5
ttaatcgcat tgcctccg 18
<210> 6
<211> 21
<212> DNA
<213> ASFV-2-LoopB(ASFV)
<400> 6
taacgtatcc agagcaagag a 21
<210> 7
<211> 2366
<212> DNA
<213> P72(ASFV)
<400> 7
gtcacggacg ttgtaaaacg acggccagtg aattcgagct cggtacctcg cgaatgcatc 60
tagaatggca tcaggaggag ctttttgtct tattgctaac gatgggaagg ccgacaagat 120
tatattggcc caagacttgc tgaatagcag gatctctaac attaaaaatg tgaacaaaag 180
ttatgggaaa cccgatcccg aacccacttt gagtcaaatc gaagaaacac atttggtgca 240
ttttaatgcg cattttaagc cttatgttcc agtagggttt gaatacaata aagtacgccc 300
gcatacgggt acccccacct tgggaaacaa gcttaccttt ggtattcccc agtacggaga 360
ctttttccat gatatggtgg gccatcatat attgggtgca tgtcattcat cctggcagga 420
tgctccgatt cagggcacgt cccagatggg ggcccatggg cagcttcaaa cgtttcctcg 480
caacggatat gactgggaca accaaacacc cttagagggc gccgtttaca cgcttgtaga 540
tccttttgga agacccattg tacccggcac aaagaatgcg taccgaaact tggtttacta 600
ctgcgaatac cccggagaac gactttatga aaacgtaaga ttcgatgtaa atggaaattc 660
cctagacgaa tatagttcgg atgtcacaac gcttgtgcgc aaattttgca tcccagggga 720
taaaatgact ggatataagc acttggttgg ccaggaggta tcggtggagg gaaccagtgg 780
ccctctccta tgcaacattc atgatttgca caagccgcac caaagcaaac ctattcttac 840
cgatgaaaat gatacgcagc gaacgtgtag ccataccaac ccgaaatttc tttcacagca 900
ttttcccgag aactctcaca atatccaaac agcaggtaaa caagatatta ctcctatcac 960
ggacgcaacg tatctggaca taagacgtaa tgttcattac agctgtaatg gacctcaaac 1020
ccctaaatac tatcagcccc ctcttgcgct ctggattaag ttgcgctttt ggtttaatga 1080
gaacgtgaac cttgctattc cctcagtatc cattcccttc ggcgagcgct ttatcaccat 1140
aaagcttgca tcgcaaaagg atttggtgaa tgaatttcct ggactttttg tacgccagtc 1200
acgttttata gctggacgcc ccagtagacg caatatacgc tttaaaccat ggtttatccc 1260
aggagtcatt aatgaaatct cgctcacgaa taatgaactt tacatcaata acctgtttgt 1320
aacccctgaa atacacaacc tttttgtaaa acgcgttcgc ttttcgctga tacgtgtcca 1380
taaaacgcag gtgacccaca ccaacaataa ccaccacgat gaaaaactaa tgtctgctct 1440
taaatggccc attgaatata tgtttatagg attaaaacct acctggaaca tctccgatca 1500
aaatcctcat caacaccgag attggcacaa gttcggacat gttgttaacg ccattatgca 1560
gcccactcac cacgcagaga taagctttca ggatagagat acagctcttc cagacgcatg 1620
ttcatctata tctgatatta gccccgttac gtatccgatc acattaccta ttattaaaaa 1680
catttccgta actgctcatg gtatcaatct tatcgataaa tttccatcaa agttctgcag 1740
ctcttacata cccttccact acggaggcaa tgcgattaaa acccccgatg atccgggtgc 1800
gatgatgatt acctttgctt tgaagccacg ggaggaatac caacccagtg gtcatattaa 1860
cgtatccaga gcaagagaat tttatattag ttgggacacg gattacgtgg ggtctatcac 1920
tacggctgat cttgtggtat cggcatctgc tattaacttt cttcttcttc agaacggttc 1980
agctgtgctg cgttacagta cctaagtcga ctgcagaggc ctgcatgcaa gcttggcgta 2040
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 2100
acgagccgga agcataaagt gtaaagcctg ggggtgccta atgagtgagc taactcacat 2160
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 2220
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tggggcgctc ttccgcttcc 2280
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 2340
aaggcggtaa tacggtatcc acagaa 2366
<210> 8
<211> 16
<212> DNA
<213> 1F3(ASFV)
<400> 8
actgctcatg gtatca 16
<210> 9
<211> 16
<212> DNA
<213> 1B3(ASFV)
<400> 9
atagcagatg ccgata 16
<210> 10
<211> 34
<212> DNA
<213> 1FIP(ASFV)
<400> 10
ggtaatcatc atcgcaccat acccttccac tacg 34
<210> 11
<211> 36
<212> DNA
<213> 1BIP(ASFV)
<400> 11
ttaacgtatc cagagcaaga agccgtagtg atagac 36
<210> 12
<211> 16
<212> DNA
<213> 1LoopF(ASFV)
<400> 12
ttaatcgcat tgcctc 16
<210> 13
<211> 19
<212> DNA
<213> 1LoopB(ASFV)
<400> 13
ttatattagt tgggacacg 19
Claims (10)
1. a kind of African swine fever virus LAMP detection primer group, the primer sets specific detection African swine fever virus capsid protein
The encoding gene B646L gene of p72, the primer sets include: a pair of of outer primer, a pair of of inner primer, a pair of of ring primer, nucleosides
Acid sequence difference is as follows:
Outer primer:
ASFV-2-F3:CCGTAACTGCTCATGGTA(SEQ ID No.1)
ASFV-2-B3:AGAAAGTTAATAGCAGATGCC(SEQ ID No.2)
Inner primer:
ASFV-2-FIP:TCATCGCACCCGGATCATGTTCTGCAGCTCTTACATAC (SEQ ID No.3)
ASFV-2-BIP:GGGAGGAATACCAACCCAGTTAATCCGTGTCCCAACTAAT(SEQ ID No.4)
Ring primer:
ASFV-2-LoopF:TTAATCGCATTGCCTCCG(SEQ ID No.5)
ASFV-2-LoopB:TAACGTATCCAGAGCAAGAGA(SEQ ID No.6).
2. a kind of African swine fever virus LAMP detection kit, the kit includes primer sets described in claim 1.
3. African swine fever virus LAMP detection kit according to claim 2, which is characterized in that the kit includes
Primer sets, archaeal dna polymerase, LAMP reaction solution, glycine betaine, positive control and negative control described in claim 1.
4. African swine fever virus LAMP detection kit according to claim 2 or 3, which is characterized in that outer primer, ring draw
Object, inner primer molar ratio be 1:(4-6): (8-12).
5. African swine fever virus LAMP detection kit according to claim 3, which is characterized in that the archaeal dna polymerase
For Bst archaeal dna polymerase.
6. African swine fever virus LAMP detection kit according to claim 3, which is characterized in that the LAMP reaction solution
Contain 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM MgSO4Aqueous solution.
7. African swine fever virus LAMP detection kit according to claim 3, which is characterized in that the positive control is
Plasmid DNA containing target gene fragment, negative control are sterile water, it is preferred to use distilled water, tri-distilled water or DEPC water.
8. African swine fever virus LAMP detection kit according to claim 3, which is characterized in that the positive control is
Plasmid DNA containing p72 gene, the sequence of the p72 gene is as shown in SEQ ID No.7.
9. a kind of African swine fever virus LAMP detection method, includes the following steps:
(1) sample to be tested DNA is extracted；
(2) loop-mediated isothermal amplification: 25 μ L reaction systems of configuration, the reaction system contain as claimed in claim 2 draw
Then object group, archaeal dna polymerase, LAMP reaction solution and sample to be tested DNA are reacted with sterile water polishing to 25 μ L at 63-68 DEG C
30-60min；
(3) interpretation of result: observe by the naked eye whether solution in reaction tube becomes cloudy or be by agarose gel electrophoresis analysis
It is no scalariform band occur, as solution becomes cloudy or is determined as sun if there is scalariform band by agarose gel electrophoresis in reaction tube
Property, it is otherwise feminine gender.
10. according to application of the described in any item kits of claim 2-8 in food inspection, which is characterized in that will be described
Kit is used for the detection of live fresh pork, Frozen Pork or pork product, judges live fresh pork, Frozen Pork or meat products
Raw meat is polluted with the presence or absence of African swine fever virus.
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Cited By (13)
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| CN110791592A (en) * | 2019-12-04 | 2020-02-14 | 宁波爱基因科技有限公司 | Primer and kit for rapidly detecting African swine fever virus |
| CN110791591A (en) * | 2019-11-18 | 2020-02-14 | 华南农业大学 | LAMP (loop-mediated isothermal amplification) detection primer and kit for distinguishing African swine fever virus wild strain and double-gene deletion vaccine strain |
| 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 |
| CN110885905A (en) * | 2019-11-12 | 2020-03-17 | 华南农业大学 | LAMP (loop-mediated isothermal amplification) detection primer and kit for distinguishing African swine fever virus wild strains and gene deletion strains |
| CN111226805A (en) * | 2019-11-20 | 2020-06-05 | 南京农业大学 | Method and system for live breed conservation of germplasm resources of swine species in China under epidemic situation of African swine fever |
| CN112094948A (en) * | 2020-09-27 | 2020-12-18 | 上海真测生物科技有限公司 | Application of target gene combination in African swine fever virus detection and kit |
| CN112442550A (en) * | 2019-08-27 | 2021-03-05 | 洛阳普泰生物技术有限公司 | PCR amplification primer pair for identifying and detecting African swine fever virus and kit prepared by same |
| CN112646926A (en) * | 2020-10-16 | 2021-04-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | African swine fever virus LAMP-LFD visual detection method, kit and use method thereof |
| CN113025751A (en) * | 2021-03-22 | 2021-06-25 | 福建傲农生物科技集团股份有限公司 | Primer combination and kit of African swine fever virus and application of primer combination and kit |
| CN113073148A (en) * | 2021-04-08 | 2021-07-06 | 青岛农业大学 | On-site differential diagnosis kit for African swine fever virus and application thereof |
| CN113215311A (en) * | 2021-04-23 | 2021-08-06 | 华南农业大学 | Primer combination and kit for identifying African swine fever virus gene deletion strain and African swine fever epidemic strain by centrifugal microfluidic chip |
| CN113234862A (en) * | 2021-06-16 | 2021-08-10 | 龙岩学院 | African swine fever virus LAMP detection primer group and kit |
| CN116875744A (en) * | 2023-09-08 | 2023-10-13 | 江西农业大学 | Kit for detecting African swine fever virus |
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| CN112646926A (en) * | 2020-10-16 | 2021-04-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | African swine fever virus LAMP-LFD visual detection method, kit and use method thereof |
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| CN113073148A (en) * | 2021-04-08 | 2021-07-06 | 青岛农业大学 | On-site differential diagnosis kit for African swine fever virus and application thereof |
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| CN116875744A (en) * | 2023-09-08 | 2023-10-13 | 江西农业大学 | Kit for detecting African swine fever virus |
| CN116875744B (en) * | 2023-09-08 | 2023-12-29 | 江西农业大学 | Kit for detecting African swine fever virus |
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