CN105463135A - Method for fast detecting loop-mediated isothermal amplification of African swine fever viruses - Google Patents
Method for fast detecting loop-mediated isothermal amplification of African swine fever viruses Download PDFInfo
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Abstract
The invention discloses a method for fast detecting loop-mediated isothermal amplification of African swine fever viruses. An LAMP optimal reaction system is prepared from 25 microliters of matter which comprises l microliter of 8U Bst DNA polymerase, 2.5 microliters of a 10*ThermoPol buffer solution, 6 microliters of 2.5 mM each dNTPs, 4 microliters of 25 mM MgCl2, 1 microliter of outer primers F3 and B3 (5 micrometers), 1 microliter of inner primers FIP and BIP (50 micrometers), 1 microliter of a DNA template, 2.5 microliters of 10M bataine, and the balance ddH2O. An LAMP reaction is performed in a constant-temperature water bath kettle, reaction temperature is 63 DEG C, and reaction time is 45 min. The method is extremely high in specificity and sensitivity, fast, efficient, low in cost, easy and convenient to operate, capable of being effectively applied to fast diagnosis of African swine fever viruses, and applicable to field operation of an animal quarantine institution and a livestock farm.
Description
Technical field
The invention belongs to African swine fever virus detection technique field, particularly relate to a kind of method of rapid detection African swine fever virus ring mediated isothermal amplification.
Background technology
African swine fever (Africanswinefever, ASF) is caused by African swine fever virus (Africanswinefevervirus, ASFV) that the one of pig is acute, high degree in contact sexually transmitted disease.Nineteen twenty-one, this disease was at world's large-scale outbreak subsequently since Kenya's Late Cambrian African swine fever, caused huge financial loss.The clinical symptom of African swine fever is similar to classic swine fever with pathological change, feature be high heat, skin cyanosis and lymphoglandula, kidney, gastrointestinal mucosa obviously hemorrhage, virulent strain infects family's pig mortality ratio can up to 100%, therefore be classified as category-A epidemic disease by OIE (OIE), China is classified as a class animal epidemic.China there is no the generation of African swine fever epidemic disease at present, and due to the fast development of world commerce, the factors such as personnel's dealing easily, make China be faced with huge challenge when prevention and control poisoning intrusion.And at present effective measure also be there is no for the treatment of African swine fever and the prevention and control of vaccine, be existingly still quarantine to this sick prevention and control measure is topmost, once detect that this disease exists, without exception animal strictly slaughtered and take harmless treatment.Therefore the detection quarantine method setting up this virus seems particularly important.K205R guards special gene as African swine fever virus, and can, in this characteristic of the early expression of Virus entry body, make K205R gene receive increasing concern gradually both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of method of rapid detection African swine fever virus ring mediated isothermal amplification, be intended to the basis based on African swine fever virus K205R gene builds a kind of method for quick, timely monitoring African swine fever epidemic disease, for importing into of China's this disease of prevention and control provides reference.
The present invention is achieved in that a kind of method of rapid detection African swine fever virus ring mediated isothermal amplification, and the method for described rapid detection African swine fever virus ring mediated isothermal amplification comprises:
Step one, synthetic GenBank African swine fever virus K205R gene order, after synthesis is loaded with the coli strain pure culture of goal gene plasmid, utilizes plasmid extraction kit to extract goal gene plasmid DNA as template, order-checking qualification ,-80 DEG C save backup;
Step 2, utilizes Viral RNA genome to extract test kit, and save backup in-80 DEG C according to the DNA of step extraction pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus), extract Pestivirus suis RNA, reverse transcription is that cDNA saves backup in-80 DEG C;
Step 3, adopting PrimerExplorerV4, for the design of African swine fever K205R gene order and screening 2 pairs of LAMP primer, is inner primer FIPSEQIDNO:1 and BIPSEQIDNO:2, outer primer F3SEQIDNO:3 and B3SEQIDNO:4 respectively, carry out primer dilution ,-20 DEG C save backup;
Step 4, LAMP reaction system is 25 μ L:1 μ L8UBstDNA polysaccharases, 2.5 μ L10 × ThermoPolbuffer damping fluids, 6 μ L2.5mMdNTP, 4 μ L25mMMgCl
2, each 1 μ L of outer primer F3, B3 (5 μMs), each 1 μ L of inner primer FIP, BIP (50 μMs), plasmid DNA template 1 μ L, 2.5 μ L10M trimethyl-glycines, add ddH
2o mends to 25 μ L; Carry out LAMP reaction in thermostat water bath, temperature of reaction is 63 DEG C, and the reaction times is 45min; ThermoPolbuffer damping fluid is 20mMTris – HCl, 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%w/vTritonX-100;
Step 5,100 times of rear SYBRGreenI dyestuffs of dilution are added in LAMP reaction tubes, under ultraviolet lamp, positive reaction has green fluorescence to occur, otherwise negative reaction there will not be, get the LAMP reaction product of 5 μ L in 1.5% agarose gel electrophoresis, analyzed by ultraviolet gel imaging system instrument, the visible stepped band of positive reaction pipe;
Step 6,
ultramicrospectrophotometermeasure African swine fever plasmid DNA concentration, and it being for subsequent use to carry out 10 times of gradient dilutions successively, is 6 × 10 to final concentration respectively
4the DNA of copies/ μ L ~ 6copies/ μ L tests, and explores detection sensitivity.
Further, in LAMP reaction tubes, add 100 times of rear SYBRGreenI dyestuffs of dilution, fully after mixing, under ultraviolet lamp, positive reaction has green fluorescence to occur, otherwise negative reaction there will not be.The LAMP reaction product of getting 5 μ L is in 1.5% agarose gel electrophoresis, and analyzed by ultraviolet gel imaging system instrument, positive reaction pipe is stepped band.
Another object of the present invention is to provide a kind of African swine fever virus comprising the method for described rapid detection African swine fever virus ring mediated isothermal amplification to detect.
Another object of the present invention is to provide a kind of Pestivirus suis comprising the method for described rapid detection African swine fever virus ring mediated isothermal amplification to detect.
Another object of the present invention is to provide a kind of pig parvoviral comprising the method for described rapid detection African swine fever virus ring mediated isothermal amplification to detect.
Another object of the present invention is to provide a kind of porcine circovirus 2 type comprising the method for described rapid detection African swine fever virus ring mediated isothermal amplification to detect.
Another object of the present invention is to provide a kind of PRV (Pseudorabies virus) comprising the method for described rapid detection African swine fever virus ring mediated isothermal amplification to detect.
The method of rapid detection African swine fever virus ring mediated isothermal amplification provided by the invention, compared with prior art, has following advantage:
1, specificity is high, and 4 primers, respectively for 6 different zones, not mating of any primer, all can cause reaction to carry out.
2, sensitivity is high, compares have higher susceptibility with conventional additive method.
3, rapidly and efficiently, do not need complicated temperature match curing conditions and set different response procedures, usual proliferation time can not more than 1 hour, and conventional PCR method generally at least needs 2 hours.
4, with low cost, easy and simple to handle, do not need special plant and instrument, can complete reaction in thermostat water bath, naked eyes can judged result, is a kind of detection technique being applicable to field condition and basic unit.
5, the African swine fever virus LAMP detection method set up, do not needing can complete under specific apparatus and complex reaction condition, fast easy, and specificity is good, 63 DEG C of isothermal reaction 45min can complete, and the African swine fever virus DNA of 6 copies/μ L can be detected, reaction result judges simple, under the condition not having specific apparatus, under uv irradiating, after adding fluorescence dye, there is the appearance of green fluorescence, the Visual retrieval of virus can be carried out.
The African swine fever LAMP detection method that the present invention sets up, can effectively be applied in the quick diagnosis of African swine fever, and be applicable to Animal Quarantine mechanism of China and plant's execute-in-place, for China provides reference frame for the monitoring of African swine fever epidemic disease, lay a good foundation for preventing this disease from entering China simultaneously.African swine fever plasmid DNA is carried out 10 times of doubling dilutions successively, measures DNA concentration conversion and become copy number to be 6 × 10
4---6 × 10
0copy/μ L, detects by the above LAMP method that describes, confirms that the method can detect the African swine fever virus DNA of 6 copies/μ L, have very high sensitivity; Detect African swine fever virus, Pestivirus suis, pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus) simultaneously, amplified production is identified through 1.5% agarose gel electrophoresis, only has African swine fever virus generation specific amplification, and Pestivirus suis, pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus) are without amplified band, show that the method has good specificity to African swine fever virus.
Accompanying drawing explanation
Fig. 1 is the method flow diagram of the rapid detection African swine fever virus ring mediated isothermal amplification that the embodiment of the present invention provides.
Fig. 2 is the different recognition site schematic diagram of the LAMP primer that provides of the embodiment of the present invention in gene order.
Fig. 3 be the African swine fever virus LAMP product that provides of the embodiment of the present invention 1.5% agarose gel electrophoresis result schematic diagram.
Fig. 4 is that the African swine fever virus LAMP that the embodiment of the present invention provides detects product visualization result schematic diagram;
In figure: SYBRGreen I detection of fluorescent dyes; M:DNA relative molecular mass standard DL2000; 1:LAMP reaction product; 2: negative control product.
Fig. 5 is the African swine fever virus LAMP detection sensitivity schematic diagram that the embodiment of the present invention provides;
In figure: M:DNA relative molecular mass standard DL2000; 1: negative control; 2:6 × 10
4copy; 3:6 × 10
3copy; 4:6 × 10
2copy; 5:6 × 10
1copy; 6:6 × 10
0copy.
Fig. 6 is the African swine fever virus LAMP detection specificity test schematic diagram that the embodiment of the present invention provides;
In figure: DNA relative molecular mass standard DL2000; 1: African swine fever virus; 2: Pestivirus suis; 3: pig parvoviral; 4: porcine circovirus 2 type; 5: PRV (Pseudorabies virus).
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The present invention sets up African swine fever virus (ASFV) ring mediated isothermal amplification (loop-mediatedisothermalamplification, LAMP) method for quick, specificity LAMP primer is designed and synthesized for African swine fever virus K205R gene order, Optimal reaction conditions, analyze its sensitivity and specificity, result shows that set up African swine fever LAMP detection method can detect the DNA of 6copies/ μ L, highly sensitive and specificity and having good stability.This method is simple, fast, thermostat water bath 63 DEG C reaction 45min can complete.Reaction product, except agarose gel electrophoresis analysis, can carry out Visual retrieval, and add fluorescence dye has green fluorescence under ultraviolet lamp shines, and effectively can detect African swine fever virus fast.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, the method for the rapid detection African swine fever virus ring mediated isothermal amplification of the embodiment of the present invention comprises the following steps:
S101: synthetic GenBank announced African swine fever virus K205R gene order (accession number: NC_001659.1), after synthesis is loaded with the coli strain pure culture of goal gene plasmid, plasmid extraction kit is utilized to extract goal gene plasmid DNA as template, order-checking qualification ,-80 DEG C save backup;
S102: utilize Viral RNA genome to extract test kit, save backup in-80 DEG C according to the DNA of step extraction pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus), extract Pestivirus suis RNA, reverse transcription is that cDNA saves backup in-80 DEG C;
S103: adopt the online software of PrimerExplorerV4 (http://primerexplorer.jp/e/), for the design of African swine fever K205R gene order and screening 2 pairs of LAMP primer, inner primer FIPSEQIDNO:1 and BIPSEQIDNO:2, outer primer F3SEQIDNO:3 and B3SEQIDNO:4 respectively, 6 different zones of the identification target gene that they are special; FIP is made up of F2 and F1c (complementary with F1); BIP is made up of B2 and B1c (complementary with B1), and carry out primer dilution ,-20 DEG C save backup;
S104:LAMP optimal reaction system is 25 μ L:1 μ L8UBstDNA polysaccharases, 2.5 μ L10 × ThermoPolbuffer damping fluid (20mMTris – HCl, 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%w/vTritonX-100), 6 μ L2.5mMeachdNTPs, 4 μ L25mMMgCl
2, each 1 μ L of outer primer F3, B3 (5 μMs), each 1 μ L of inner primer FIP, BIP (50 μMs), 1 μ LDNA template, 2.5 μ L10M trimethyl-glycines, add ddH
2o mends to 25 μ L; In thermostat water bath, carry out LAMP reaction, temperature of reaction is 63 DEG C, and the reaction times is 45min;
S105: add 100 times of rear SYBRGreenI dyestuffs of dilution in LAMP reaction tubes, under ultraviolet lamp, positive reaction has green fluorescence to occur, otherwise negative reaction there will not be, get the LAMP reaction product of 5 μ L in 1.5% agarose gel electrophoresis, analyzed by ultraviolet gel imaging system instrument, the visible stepped band of positive reaction pipe;
S106:
ultramicrospectrophotometer(
nanodrop2000) measure African swine fever plasmid DNA concentration, and it is for subsequent use to carry out 10 times of gradient dilutions successively.By the LAMP detection method after optimizing as mentioned above, be 6 × 10 to final concentration respectively
4the DNA of copies/ μ L ~ 6copies/ μ L tests, and explores detection sensitivity.
Below in conjunction with test, effect of the present invention is further described.
1 materials and methods
1.1 instruments and reagent
Supercentrifuge Sigma3-18K is purchased from Sigma company; Thermostat water bath is purchased from Yuyao telecommunication instrument industrial corporation; Full automatic gel Image analysis system is purchased from Bio-Rad company; Voltage stabilization and current stabilization type electrophoresis apparatus DYY-III, nucleic acid electrophoresis system DYY-2C are purchased from Beijing Liuyi Instrument Factory.
Magnesium ion, dNTPs, trimethyl-glycine, purchased from Beijing Tian Gen biochemical technology company limited; BstDNA polysaccharase is purchased from NewEnglandBiolabs; DNAMarkerDL2000, plasmid extraction kit, agarose all give birth to engineering (Dalian) company limited purchased from treasured; Other reagent are all purchased from domestic analytical pure product.
1.2 plasmids and virus
Synthetic GenBank announced African swine fever virus K205R gene order (accession number: NC_001659.1), by Shanghai, handsome company synthesizes.After synthesis is loaded with the coli strain pure culture of goal gene plasmid, utilize plasmid extraction kit to extract goal gene plasmid DNA as template, after delivering to the order-checking qualification of Hua Da company ,-80 DEG C save backup.
Viral RNA genome is utilized to extract test kit, the DNA of step extraction pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus) saves backup in-80 DEG C to specifications, extract Pestivirus suis RNA, reverse transcription is that cDNA saves backup in-80 DEG C, and positive is all provided by Animal Quarantine research department of Sichuan Agricultural University.
The Design and synthesis of 1.3 primers
The key factor of LAMP technology stability is 2 pairs of Auele Specific Primers, according to LAMP primer principle of design, this test adopts the online software of PrimerExplorerV4 (http://primerexplorer.jp/e/), for the design of African swine fever K205R gene order and screening 2 pairs of LAMP primer, inner primer FIPSEQIDNO:1 and BIPSEQIDNO:2, outer primer F3SEQIDNO:3 and B3SEQIDNO:4 respectively, 6 different zones (Fig. 2) of the identification target gene that they are special.FIP is made up of F2 and F1c (complementary with F1); BIP is made up of (table 1) B2 and B1c (complementary with B1).Primer is synthesized by Shanghai Sheng Gong biotechnology limited-liability company, and carry out primer dilution by synthesis specification sheets ,-20 DEG C save backup.
Table 1. African swine fever virus LAMP detection method primer information table
agene order reference by location ASFV bacterial strain BA71V (GenBank accession number: NC_001659.1).
The optimization of 1.4LAMP reaction conditions
LAMP reaction is carried out in 25 μ L systems, and reaction condition optimization is as follows: dNTP final concentration is 0.1mM ~ 0.8mM; Mgcl
2the optimization of final concentration is from 2mM ~ 10mM; The final concentration ratio of outer primer and inner primer is optimized by 1:1,1:2,1:4,1:6,1:8,1:10,1:12, and the optimization of trimethyl-glycine final concentration is respectively 0M, 0.5M, 1.0M, 1.5M.LAMP temperature of reaction is optimized for 60 DEG C ~ 65 DEG C, and the reaction times is optimized by 15min, 30min, 45min, 60min, after repeatedly repetition test, determine optimum reaction condition.
1.5LAMP product detects
In LAMP reaction tubes, add 100 times of rear SYBRGreenI dyestuffs of dilution, under ultraviolet lamp, positive reaction has green fluorescence to occur, otherwise negative reaction there will not be.The LAMP reaction product of getting 5 μ L, in 1.5% agarose gel electrophoresis, is analyzed by ultraviolet gel imaging system instrument, the visible stepped band of positive reaction pipe.
1.6LAMP sensitivity technique
ultramicrospectrophotometer(
nanodrop2000) measure African swine fever plasmid DNA concentration, and it is for subsequent use to carry out 10 times of gradient dilutions successively.By the LAMP detection method after optimizing as mentioned above, be 6 × 10 to final concentration respectively
4the DNA of copies/ μ L ~ 6copies/ μ L tests, and explores detection sensitivity.
1.7LAMP specific test
African swine fever virus is detected by the LAMP method set up, Pestivirus suis, pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus) are detected simultaneously, above virus purification, in the sick pig with typical clinical symptom performance, assesses the specificity of the method with this.
2. results and analysis
The establishment of 2.1LAMP reaction conditions
Through the optimization of repeatedly reaction conditions, establishing LAMP optimal reaction system is 25 μ L (table 2): BstDNA polysaccharase (8U) 1 μ L, ThermoPolbuffer (10 ×) 2.5 μ L, dNTP (2.5mM) 6 μ L, MgCl
2(25mM) 4 μ L, each 1 μ L of outer primer F3, B3 (5 μMs), each 1 μ L of inner primer FIP, BIP (50 μMs), plasmid DNA template 1 μ L, trimethyl-glycine (10M) 2.5 μ L, adds ddH
2o mends to 25 μ L.Optimal reaction temperature is 63 DEG C, optimum reacting time is 45min, when reaction product is after 1.5% agarose gel electrophoresis, there is stepped band clearly, do not add the sample of African swine fever virus DNA as negative control (Fig. 3), confirm the successful of the LAMP method built.
Table 2. African swine fever LAMP detection system information
2.2 African swine fever virus LAMP visual detect
Carry out the detection of African swine fever virus LAMP visual.Add SYBRGreen I dyestuff after 100 times of dilutions in African swine fever virus LAMP reaction tubes after, fully mix, under ultraviolet lamp shines, positive reaction pipe has green fluorescence, and (Fig. 4) appears in negative reaction pipe unstressed configuration.
2.3LAMP susceptibility is tested
Sensitivity test is carried out with the African swine fever virus LAMP set up.Result shows that LAMP method can detect the African swine fever virus DNA (Fig. 5) of 6copies/ μ L, shows that set up African swine fever virus LAMP detection method has good susceptibility.
2.4 African swine fever virus LAMP specific tests
In the test of African swine fever specific detection, by the LAMP method set up, African swine fever virus, Pestivirus suis, pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus) are detected respectively.Can see that African swine fever LAMP positive products occurs scalariform band after 1.5% agarose gel electrophoresis clearly, and Pestivirus suis, pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus) there is no the appearance (Fig. 6) of band.Confirm that the method has good specificity to African swine fever virus.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a method for rapid detection African swine fever virus ring mediated isothermal amplification, is characterized in that, the method for described rapid detection African swine fever virus ring mediated isothermal amplification comprises:
Step one, synthetic GenBank African swine fever virus K205R gene order, after synthesis is loaded with the coli strain pure culture of goal gene plasmid, utilizes plasmid extraction kit to extract goal gene plasmid DNA as template, order-checking qualification ,-80 DEG C save backup;
Step 2, utilizes Viral RNA genome to extract test kit, and save backup in-80 DEG C according to the DNA of step extraction pig parvoviral, porcine circovirus 2 type, PRV (Pseudorabies virus), extract Pestivirus suis RNA, reverse transcription is that cDNA saves backup in-80 DEG C;
Step 3, adopting PrimerExplorerV4, for the design of African swine fever K205R gene order and screening 2 pairs of LAMP primer, is inner primer FIPSEQIDNO:1 and BIPSEQIDNO:2, outer primer F3SEQIDNO:3 and B3SEQIDNO:4 respectively, carry out primer dilution ,-20 DEG C save backup;
Step 4, LAMP reaction system is 25 μ L:1 μ L8UBstDNA polysaccharases, 2.5 μ L10 × ThermoPolbuffer damping fluids, 6 μ L2.5mMdNTP, 4 μ L25mMMgCl
2, each 1 μ L of outer primer F3, B3 (5 μMs), each 1 μ L of inner primer FIP, BIP (50 μMs), plasmid DNA template 1 μ L, 2.5 μ L10M trimethyl-glycines, add ddH
2o mends to 25 μ L; Carry out LAMP reaction in thermostat water bath, temperature of reaction is 63 DEG C, and the reaction times is 45min; ThermoPolbuffer damping fluid is 20mMTris – HCl, 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%w/vTritonX-100;
Step 5,100 times of rear SYBRGreenI dyestuffs of dilution are added in LAMP reaction tubes, under ultraviolet lamp, positive reaction has green fluorescence to occur, otherwise negative reaction there will not be, get the LAMP reaction product of 5 μ L in 1.5% agarose gel electrophoresis, analyzed by ultraviolet gel imaging system instrument, the visible stepped band of positive reaction pipe;
Step 6, ultramicrospectrophotometer measures African swine fever plasmid DNA concentration, and it is for subsequent use to carry out 10 times of gradient dilutions successively, is 6 × 10 respectively to final concentration
4the DNA of copies/ μ L ~ 6copies/ μ L tests, and explores detection sensitivity.
2. the method for rapid detection African swine fever virus ring mediated isothermal amplification as claimed in claim 1, it is characterized in that, get 5 μ LLAMP reaction product and identify through 1.5% agarose gel electrophoresis, after 80V electrophoresis 30min, sepharose is analyzed by gel imaging system instrument, result of determination.
3. the method for rapid detection African swine fever virus ring mediated isothermal amplification as claimed in claim 1, it is characterized in that, in LAMP reaction tubes, add the SYBRGreenI dyestuff of 100 times of dilutions, fully after mixing, reaction tubes is put into ultraviolet gel imaging system instrument, result of determination.
4. the method for rapid detection African swine fever virus ring mediated isothermal amplification as claimed in claim 1, it is characterized in that, African swine fever virus LAMP method primer sequence is as follows:
FIP:GCATAGCCTCCTTAATCGTTGTTAGATTCTGATGATAAATGGCACT
BIP:CCTACCTTCATCAAACACGAACAGGATTTTTTTTAGGTGTTTCACTTG
F3：GCCATTATCGCCCAACTT
B3：CGTGAAGAACATTGCATTCG。
5. the method for rapid detection African swine fever virus ring mediated isothermal amplification as claimed in claim 1, it is characterized in that, positive reaction product is identified through 1.5% agarose gel electrophoresis, the visible stepped band of gel imaging system instrument analysis; The SYBRGreenI dyestuff of 100 times of dilutions is added in positive reaction pipe, fully after mixing, visible green fluorescence under ultraviolet lamp shines.
6. the African swine fever virus comprising the method for rapid detection African swine fever virus ring mediated isothermal amplification described in claim 1-5 any one detects.
7. the Pestivirus suis comprising the method for rapid detection African swine fever virus ring mediated isothermal amplification described in claim 1-5 any one detects.
8. the pig parvoviral comprising the method for rapid detection African swine fever virus ring mediated isothermal amplification described in claim 1-5 any one detects.
9. the porcine circovirus 2 type comprising the method for rapid detection African swine fever virus ring mediated isothermal amplification described in claim 1-5 any one detects.
10. the PRV (Pseudorabies virus) comprising the method for rapid detection African swine fever virus ring mediated isothermal amplification described in claim 1-5 any one detects.
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| CN106947838A (en) * | 2017-05-31 | 2017-07-14 | 广东出入境检验检疫局检验检疫技术中心 | African swine fever virus nonstructural gene real-time fluorescence LAMP detection primer group, kit and detection method |
| WO2017212904A1 (en) * | 2016-06-06 | 2017-12-14 | 国立大学法人 宮崎大学 | Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined |
| CN110229932A (en) * | 2019-06-10 | 2019-09-13 | 北京森康生物技术开发有限公司 | African swine fever virus nucleic acid is hands-free to take fluorescence isothermal amplification detection kit |
| CN110656202A (en) * | 2019-05-28 | 2020-01-07 | 陕西诺威利华生物科技有限公司 | African swine fever virus LAMP detection primer group and application thereof |
| CN110724762A (en) * | 2019-10-21 | 2020-01-24 | 华中农业大学 | LAMP detection primer and detection method for African swine fever virus |
| CN110760619A (en) * | 2019-12-06 | 2020-02-07 | 四川大学 | Primer group, probe, kit and detection method for rapidly detecting African swine fever virus based on RPA method |
| CN111500790A (en) * | 2020-06-10 | 2020-08-07 | 四川农业大学 | Primer probe set for detecting African swine fever by fluorescent quantitative PCR and application thereof |
| WO2020253537A1 (en) * | 2019-06-21 | 2020-12-24 | 苏州克睿基因生物科技有限公司 | Method and kit for detecting african swine fever virus |
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| CN106947838A (en) * | 2017-05-31 | 2017-07-14 | 广东出入境检验检疫局检验检疫技术中心 | African swine fever virus nonstructural gene real-time fluorescence LAMP detection primer group, kit and detection method |
| CN106947838B (en) * | 2017-05-31 | 2020-12-01 | 广州海关技术中心 | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method |
| CN110656202A (en) * | 2019-05-28 | 2020-01-07 | 陕西诺威利华生物科技有限公司 | African swine fever virus LAMP detection primer group and application thereof |
| CN110229932A (en) * | 2019-06-10 | 2019-09-13 | 北京森康生物技术开发有限公司 | African swine fever virus nucleic acid is hands-free to take fluorescence isothermal amplification detection kit |
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| WO2020253537A1 (en) * | 2019-06-21 | 2020-12-24 | 苏州克睿基因生物科技有限公司 | Method and kit for detecting african swine fever virus |
| CN114391046A (en) * | 2019-06-21 | 2022-04-22 | 苏州晶睿生物科技有限公司 | Method and kit for detecting African swine fever virus |
| CN110724762A (en) * | 2019-10-21 | 2020-01-24 | 华中农业大学 | LAMP detection primer and detection method for African swine fever virus |
| CN110760619A (en) * | 2019-12-06 | 2020-02-07 | 四川大学 | Primer group, probe, kit and detection method for rapidly detecting African swine fever virus based on RPA method |
| CN111500790A (en) * | 2020-06-10 | 2020-08-07 | 四川农业大学 | Primer probe set for detecting African swine fever by fluorescent quantitative PCR and application thereof |
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