CN104388577A - Loop-mediated isothermal amplification primer for detecting erwinia amylovory and kit - Google Patents
Loop-mediated isothermal amplification primer for detecting erwinia amylovory and kit Download PDFInfo
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Abstract
The invention relates to a loop-mediated isothermal amplification primer for detecting erwinia amylovory and a kit. The primer comprises a pair of outer primers and a pair of inner primers, and nucleotide sequences are respectively SEQ ID NO:1-4. Primer mixed liquor, loop-mediated isothermal amplification mixed liquor, isothermal amplification reaction liquid I and erwinia amylovory positive control DNA (deoxyribonucleic acid) are contained in the kit. The method for detecting erwinia amylovory comprises extraction of total nucleic acid of samples to be detected, LAMP isothermal amplification, and judgment of reaction results of amplified nucleic acid SYBR Green I through fluorescent staining method. The kit designs the primer according to a conserved gene sequence on strain pathogenic plasmid pEA29 of erwinia amylovory, and guarantees the specificity of the detection method.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of the loop-mediated isothermal amplification primer and the test kit that detect erwinia amylovora.
Background technology
Erwinia amylovora (Eriwinia amylovora (Burrill) Winslow et al) is international important plant quarantine object, " People's Republic of China (PRC) enter the territory the dangerous venereal disease of Plant Quarantine, worm, weed species " that newly puts into effect in China is listed in quarantine harmful organisms, is the destructive disease of cash crop apple, pears, hawthorn etc. by this germ fire blight of pear.Fire blight of pear is found as far back as North America, nearly decades.Along with this disease of development of world commerce is with seedling, fruit and wrapping material bamboo telegraph, almost throughout whole Continent of Europe.Various countries are all very responsive to fire blight of pear, and majority state is all classified as quarantine harmful organisms.In recent years, in order to meet the demand of domestic market, China introduces excellent pears, apple nursery stock and fruit in a large number from the ground such as Korea S, Japan in succession.Therefore, fire blight of pear forms potential threat to China's Fruits production and fruit trade of passing in and out.Erwinia amylovora can infect 220 various plants that the Rosaceae more than 40 belongs to, and brings significant damage to local diversity of organism and agricultural, causes barrier also to fruit trade.Extensively there is the Suitable sites of fire blight of pear and susceptible host in China, its wide temperate climate area is also the normal region of this disease, here Chinese main apple producing region has been concentrated, along with the susceptible Apple Culture kind of height as loud, high-pitched sound, the plantation of Qiao Najin promotes, the intrusion risk of this disease increases further.Erwinia amylovora generally invades from the floral organ of host, then causes deadwood and the death of whole plant, and most typical symptom is that flower, fruit and blade are by after the infringement of fire blast bacterium, very fast blackening brown is withered, just as burning, but still hang not fall in the tree, so gain the name.Fire blight of pear long-distance communications are susceptible host's reproductive material mainly, comprises seedling, scion, stock, fruit, contaminated transportation means, migratory bird and air-flow.Wherein the most dangerous route of transmission is the biography band by sick scion, nursery stock, fruit.The domestic generation also not having fire blight of pear at present, but the artificial and natural propagation of fire blight of pear constitutes potential threat to China, and there has been the distribution of fire blight of pear in especially adjacent Japan, and accurately sensitive detection technique is the effective means stoping this disease to be imported into.
At present, the domestic quarantine to fire blight of pear there is no examination criteria.Its method of inspection is also a lot, wait until that advanced Protocols in Molecular Biology comprises the application etc. of DNA probe, the application of round pcr and fatty acid analysis, monoclonal antibody from the separation and Culture of classics, Pathogenicity, observation of symptoms, but in quarantine, adopt the more method made a definite diagnosis in conjunction with Pathogenicity for immunofluorescence fluorescent dye, sense cycle is oversize, can not adapt to the actual demand of import-export commodity.In recent years, many new Molecular biology research methods are also applied in the quarantine of erwinia amylovora.Although these method specificitys are high, sense cycle is short, all needs PCR aftertreatment, usually needs just can complete for one to several days, and another problem is that PCR aftertreatment usually occurs polluting and the accuracy of impact test and reliability
Summary of the invention
The first object of the present invention is to provide the loop-mediated isothermal amplification primer detecting erwinia amylovora, second object there is provided a kind of test kit utilizing this primer to detect erwinia amylovora, thus can realize detecting quickly and accurately erwinia amylovora, make up the deficiencies in the prior art.
One aspect of the invention relates to the loop-mediated isothermal amplification primer detecting erwinia amylovora, and be pair of outside primers F 3, B3 and pair of inside primers F IP, BIP, its nucleotide sequence is respectively SEQ ID NO:1-4.
Another aspect of the present invention provides a kind of loop-mediated isothermal amplification quick detection kit of erwinia amylovora, includes following component:
1) primer mixed solution: 80 μm of ol/L primers F IP 2 μ L, 80 μm of ol/L primer BIP 2 μ L, 10 μm of ol/L primers F 32 μ L, 10 μm of ol/L primer B32 μ L;
2) ring mediated isothermal amplification mixed solution: 8U/ μ L Bst DNA PoLymerase 2 μ L, 2.5 μm of ol/LdNTP 4 μ L, 1 × ThermoL Buffer 2.5uL, ddH
2o 31.5 μ L; Wherein 1 × Thermol Buffer composition is: 20mmol/LTris-HCl, 10mmol/LKCl, 10mmol/L (NH4)
2sO4,2mmol/LMgSO4,0.01%Triton X-100;
3) erwinia amylovora positive control dna 2 μ L.
4) fluorescent color-developing agent: composition is 100 × fluorescence dye SYBR GREEN I.
The working method of loop-mediated isothermal amplification (LAMP) detection kit of erwinia amylovora of the present invention is as follows:
1) extraction of DNA profiling in testing sample: by commercially available DNA extraction kit, step extracts DNA in testing sample to specifications.The purity ratio OD260/OD280 of the sample DNA wherein extracted is within the scope of 1.6-2.0, and nucleic acid concentration is within the scope of 40-100ng/ μ L.
2) LAMP constant-temperature amplification: add primer mixed solution 8 μ L, ring mediated isothermal amplification mixed solution 40 μ L, template DNA 2 μ L in PCR pipe, mixing.By the PCR pipe isothermal reaction 60min in 61 DEG C of water-baths prepared, then 80 DEG C of 10min termination reactions, are cooled to 4 DEG C.
3) SYBR Green I fluorescence colour judges reaction result: add 3 μ l fluorescent color-developing agents in the reaction product and mix gently, and standing 5min carries out colour developing to be observed.If shows green, containing erwinia amylovora in testing sample; Manifest in orange then testing sample not containing erwinia amylovora.If be not inconsistent with above-mentioned condition, then this detected result is invalid, should again detect.
Test kit of the present invention is for detecting the erwinia amylovora in plant prod.
Compared with prior art, primer specificity of the present invention is strong, and sensitivity is good; Without the need to the aftertreatment of PCR, greatly reduce the possibility of pollution, detection time is short, and efficiency is high; Detecting instrument is simple, only needs common water-bath, reduces equipment investment, is applicable to Check and Examination of Port quarantine mechanism and laboratories popularization.
Accompanying drawing explanation
Fig. 1: LAMP primer specific amplification experimental result, in Fig. 1, A is Gel electrophoresis results figure, B is SYBR Green I detection figure;
Wherein: M:DL 2000DNA Marker; 1: erwinia amylovora (Erwiniaamylovora (Burrill) Winslow et al.) reference culture (ATCC strain number: 51853); 2: erwinia amylovora reference culture (ATCC strain number: 51853); 3: Asia erwinia amylovora (Erwiniapyrifoliae) bacterial strain (CGMCC strain number: 1.7277); 4: lady's slipper Erwinia (Erwiniacypripedii) reference culture (ATCC strain number: 29268); 5: chrysanthemum Phyllostachys pubescens (Erwiniachrysanthemi) bacterial strain (ACCC strain number: 04160); 6: addicted to vascular bundle Erwinia (Erwinia tracheiphila) reference culture (ATCC strain number: 27004); 7: piscidia Erwinia (Erwinia psidii) reference culture (ATCC strain number: 49406); 8: rhubarb horsetails Erwinia sp (Erwinia rhapontici) (CGMCC strain number: 1.6978) 9: negative control; 10: blank.Using sterilized water as blank, replace the reaction soln of template as negative control using the deionized water of sterilizing.The object of this experiment is the specificity in order to verify LAMP primer in this invention.
Fig. 2: the erwinia amylovora LAMP amplification experimental result of Pear leaves sample to be checked;
Wherein: 1: negative control; 2-3: erwinia amylovora reference culture (ATCC 51853); 4: blank; 5:DL 2000DNA Marker (be respectively from top to bottom 2000,1000,750,500,250 and 100bp); 6-8: the Pear leaves A infected by erwinia amylovora, B, C; 9: not by the Pear leaves D that erwinia amylovora infects.Using sterilized water as blank, replace the reaction soln of template as negative control using the deionized water of sterilizing.The object of this experiment is to detect primer of the present invention and can test kit be implemented accurately to detect to Pear leaves sample to be measured.
Fig. 3: LAMP primer susceptibility amplification experimental result;
Wherein, M:DL2000DNA marker; Erwinia amylovora (Erwiniaamylovora) reference culture (the ATCC strain number: 51853) concentration is respectively: 10 of 2 ~ 8
-6, 10
-5, 10
-4, 10
-3, 10
-2, 10
-1, 10
0; 1: negative control; B: blank.Using sterilized water as blank, replace the reaction soln of template as negative control using the deionized water of sterilizing.
Embodiment
Isothermal amplification technology is applied to the rapid detection of erwinia amylovora by the present invention's application loop-mediated isothermal amplification (LAMP) primer and test kit, compare with the PCR detection method of this bacterium, there is following unique advantage: (1) amplification is increased at one temperature, do not need expensive PCR amplification instrument, be convenient to basic unit and promote the use of.6 different zones of (2) 4 Auele Specific Primers and 2 common recognition sequences of ring primer, therefore specificity is high.(3) amplification efficiency is high, therefore highly sensitive based on the detection method of this technology.(4) generally completed at about 60 minutes, more consuming time than Standard PCR short.(5) need not PCR aftertreatment, greatly reduce possibility of pollution.Test kit of the present invention according to conserved genetic sequences design primer on fire blight of pear bacteria strain, thus ensure that the specificity of detection method.Detection pears fire epidemic disease bacterium pEA29 plasmid DNA is the reason for two aspects: 1, plasmid DNA is more much smaller than Fluorescent stained chromosomes molecular dna, and copy number can have more several times to hundred times, catches than being easier to.2, to so far, research finds that all nature erwinia amylovoras are all unique in this plasmid, and other sibling specieses (comprising Asia erwinia amylovora) are not all containing this plasmid.Research also finds that the virulence losing the artificial mutation bacterial strain of plasmid declines, and therefore pEA29 plasmid is most important for the virulence of erwinia amylovora.
Embodiment 1: the LAMP primer specific amplification experiment of erwinia amylovora
1, the Design and synthesis of primer
The design of LAMP primer is mainly for four of target gene different regions, and 4 primers are designed in 4 different sites that are that hold based on target gene 3 ' and 5 ' end.Utilize the different primer of design and rely on high reactivity strand displacement archaeal dna polymerase, strand displacement DNA is synthesized in ceaselessly oneself's circulation.The present invention selects fire blight of pear bacteria strain to cause a disease plasmid pEA29 the preceding paragraph conserved genetic sequences to design LAMP primer.All nature pears fire epidemic disease bacteriums are all unique in this pathogenic plasmid, and other sibling specieses (comprising Asia erwinia amylovora) are not all containing this plasmid, and research finds that the virulence losing the artificial mutation bacterial strain of plasmid declines, therefore pEA29 plasmid is most important for the virulence of erwinia amylovora.The fire blight of pear bacteria strain that the present invention selects this section of conservative gene caused a disease on plasmid pEA29 is one of distinguished sequence of erwinia amylovora, and the accession number of this sequence on GenBank is FN434114.1.Adopt LAMP primer design software Primer Explorer V4.0 to design LAMP primer, filter out one group of good LAMP primer of specificity by specific test.Primer is synthesized by Shanghai Sheng Gong company, and its sequence is respectively:
F35’-GTGATTTCCGCTGTGGCG-3’(SEQ ID NO：1)
B35’-GCCGTGTTGTCTTGAACTCA-3’(SEQ ID NO：2)
FIP
5’-CCGAAGCGTTTTCCTGACGCTAATTTGAGCGCGATCTGCT-3’(SEQID NO：3)
BIP5’-CGGCCTCCTACCCTGAATGATGTCATCCCTGTACTCAGACGA-3’(SEQ ID NO：4)
2, for the extraction of examination material DNA profiling
The extraction that test kit specification sheets carries out each pathogenic bacteria STb gene is extracted according to Tiangen bacteria total DNA.The purity ratio OD of the sample DNA wherein extracted
260/ OD
280within the scope of 1.6-2.0, nucleic acid concentration is within the scope of 40-100ng/ μ L.
Wherein comprise for examination material: M:DL 2000DNA Marker; 1: erwinia amylovora (Erwiniaamylovora (Burrill) Winslow et al.) reference culture (ATCC strain number: 51853); 2: erwinia amylovora reference culture (ATCC strain number: 51853); 3: Asia erwinia amylovora (Erwiniapyrifoliae) bacterial strain (CGMCC strain number: 1.7277); 4: lady's slipper Erwinia (Erwiniacypripedii) reference culture (ATCC strain number: 29268); 5: chrysanthemum Phyllostachys pubescens (Erwiniachrysanthemi) bacterial strain (ACCC strain number: 04160); 6: addicted to vascular bundle Erwinia (Erwinia tracheiphila) reference culture (ATCC strain number: 27004); 7: piscidia Erwinia (Erwinia psidii) reference culture (ATCC strain number: 49406); 8: rhubarb horsetails Erwinia sp (Erwinia rhapontici) (CGMCC strain number: 1.6978); 9: negative control; 10: blank.Using sterilized water as blank, replace the reaction soln of template as negative control using the deionized water of sterilizing.Wherein erwinia amylovora reference culture, lady's slipper Erwinia reference culture, all buy from ATCC addicted to vascular bundle Erwinia reference culture, piscidia Erwinia reference culture, Asia erwinia amylovora bacterial strain, chrysanthemum Phyllostachys pubescens bacterial strain are all purchased from Chinese agriculture Culture Collection (ACCC).
3, LAMP reaction system
Reaction system is 50 μ l, wherein comprises: (1) primer mixed solution: 80 μm of ol/L primers F IP2 μ L, 80 μm of ol/L primer BIP 2 μ L, 10 μm of ol/L primers F 32 μ L, 10 μm of ol/L primer B32 μ L;
2) ring mediated isothermal amplification mixed solution: 8U/ μ L Bst DNA PoLymerase 2 μ L, 2.5 μm of ol/LdNTP 4 μ L, 1 × ThermoL Buffer 2.5uL, ddH
2o 31.5 μ L; Wherein 1 × Thermol Buffer composition is: 20mmol/LTris-HCl, 10mmol/LKCl, 10mmol/L (NH4)
2sO4,2mmol/LMgSO4,0.01%Triton X-100;
(3) template DNA 2 μ L.
4, LAMP amplification
Following component is added: primer mixed solution 8 μ L, ring mediated isothermal amplification mixed solution 40 μ L, template DNA 2 μ L in PCR pipe.By the PCR pipe isothermal reaction 60min in 61 DEG C of water-baths prepared, then 80 DEG C of 10min termination reactions, are cooled to 4 DEG C.
5, result judges
Test kit recommend method of the present invention (SYBR Green I fluorescence colour): add 3 μ l fluorescent color-developing agents in the reaction product and mix gently, standing 5min carries out colour developing to be observed.If shows green, containing erwinia amylovora in testing sample; Manifest in orange then testing sample not containing erwinia amylovora.If be not inconsistent with above-mentioned condition, then this detected result is invalid, should again detect.Result is see Fig. 1.The erwinia amylovora reference culture fluorescent dye shows green of 1 and 2; The contrast nearly edge bacterium fluorescent dye of 3-8 manifests orange.Result shows the high specificity of primer pair erwinia amylovora of the present invention, can precise Identification erwinia amylovora.
Gel electrophoresis (in order to prove the exactness of SYBR Green I fluorescence colour): after nucleic acid amplification completes, 6 × Loading buffer is added in reaction tubes, getting product 5 μ L adds in the sample well of gel slab, with 1% agarose gel electrophoresis 40 minutes containing ethidium bromide (EB), electrophoretic image system is taken pictures.Result is see Figure 1A.Wherein 1,2 is erwinia amylovora reference culture, all can occur scalariform specific band.The nearly edge bacterium of contrast of 3-8 does not all have scalariform specific band to occur.SYBR Green I fluorescent dye is test kit using method, and Gel electrophoresis results figure is the exactness in order to prove SYBR Green I fluorescent staining method.Gel electrophoresis method: the product reacted by LAMP, with 1.5% agarose gel electrophoresis containing ethidium bromide (EB).Nucleic acid belt is observed under ultraviolet transilluminator.After positive amplification product gel electrophoresis, as seen from the conditions of streaking of loading wells and the scalariform band of a lot of different amplification length.
The result display of Figure 1A and Figure 1B, the amplified production of A with B obtains consistent result, and the difference between the positive and feminine gender is fairly obvious.Wherein, in figure A, after the amplified production gel electrophoresis of the erwinia amylovora reference culture of 1 and 2, as seen from the conditions of streaking of loading wells and the scalariform band of a lot of different amplification length, and in 3-8 all there is not this phenomenon in the nearly edge bacterium of other contrast.In Figure 1B, the erwinia amylovora reference culture fluorescent dye shows green of 1 and 2; The contrast bacterium fluorescent dye of 3-8 manifests orange.Therefore the SYBR Green I detection method reliable results that test kit is used.
Embodiment 2: the erwinia amylovora LAMP of Pear leaves sample to be checked increases and tests
1, the Design and synthesis (with embodiment example 1) of primer
2, testing sample total nucleic acid is extracted
By commercially available DNA extraction kit, step extracts DNA in testing sample to specifications.The purity ratio OD260/OD280 of the sample DNA wherein extracted is within the scope of 1.6-2.0, and nucleic acid concentration is within the scope of 40-100ng/ μ L.
This experiment comprises for examination material: 1: negative control; 2-3: erwinia amylovora reference culture (ATCC51853); 4: blank; 5:DL 2000DNA Marker; 6-8: the Pear leaves A infected by erwinia amylovora, B, C; 9: not by the Pear leaves D that erwinia amylovora infects.Using sterilized water as blank, replace the reaction soln of template as negative control using the deionized water of sterilizing.
3, LAMP reaction system (with embodiment example 1)
4, LAMP amplification (with embodiment example 1)
5, result judges
Test kit recommend method of the present invention (SYBR Green I fluorescence colour): add 3 μ l fluorescent color-developing agents in the reaction product and mix gently, standing 5min carries out colour developing to be observed.Result is see Fig. 2 B.By the Pear leaves A that erwinia amylovora infects, B, C and the equal shows green of erwinia amylovora reference culture (ATCC 51853) fluorescent dye, the Pear leaves D do not infected by erwinia amylovora and negative control, blank fluorescent dye all manifest orange.Result shows that primer of the present invention can erwinia amylovora in precise Identification Pear leaves sample to be checked.
Gel electrophoresis (in order to prove the exactness of SYBR Green I fluorescence colour): after nucleic acid amplification completes, 6 × Loading buffer is added in reaction tubes, getting product 5 μ L adds in the sample well of gel slab, with 1% agarose gel electrophoresis 40 minutes containing ethidium bromide (EB), electrophoretic image system is taken pictures.Result is see Fig. 2 A.Wherein by the Pear leaves A that erwinia amylovora infects, B, after the amplified production gel electrophoresis of C and erwinia amylovora reference culture (ATCC 51853), the scalariform band of the visible conditions of streaking from loading wells and a lot of different amplification length, all there is not this phenomenon in the Pear leaves D do not infected by erwinia amylovora and negative control, blank.Gel electrophoresis achieves consistent result with the SYBR Green I fluorescence colour that test kit of the present invention is recommended.
SYBR Green I fluorescent dye is test kit using method, and Gel electrophoresis results figure is the exactness in order to prove SYBR Green I fluorescent staining method.The result display of Fig. 2 A, by the Pear leaves A that erwinia amylovora infects, B, after the amplified production gel electrophoresis of C and erwinia amylovora reference culture (ATCC 51853), the scalariform band of the visible conditions of streaking from loading wells and a lot of different amplification length, all there is not this phenomenon in the Pear leaves D do not infected by erwinia amylovora and negative control, blank.The result display of Fig. 2 B, by the Pear leaves A that erwinia amylovora infects, B, C and the equal shows green of erwinia amylovora reference culture (ATCC 51853) fluorescent dye, the Pear leaves D do not infected by erwinia amylovora and negative control, blank fluorescent dye all manifest orange.Fig. 2 A obtains consistent result with the amplified production of Fig. 2 B, and the difference between the positive and feminine gender is fairly obvious.
Embodiment 3: the LAMP primer susceptibility amplification experiment of erwinia amylovora
1, the Design and synthesis (with embodiment example 1) of primer
2, the extraction of fire blight of pear bacteria strain DNA profiling
The extraction that test kit specification sheets carries out each pathogenic bacteria STb gene is extracted according to Tiangen bacteria total DNA.Eppendorf nucleic acid-protein instrument is utilized by the DNA obtained to measure concentration, concentration 100ng/ μ L.Then 10 times of serial dilutions are carried out to the DNA obtained, do template with different extent of dilution and carry out LAMP amplification, to detect its sensitivity.
3, LAMP reaction system (with embodiment example 1)
4, LAMP amplification (with embodiment example 1)
5, result judges
After nucleic acid amplification completes, gel electrophoresis is adopted to carry out sensitivity analysis.Result is see Fig. 3.Along with the reduction of template concentrations, scalariform specific band is thin out gradually, to extent of dilution 10
-6time there is no amplified production.Result shows that the greatest dilution of primer of the present invention and test kit detection erwinia amylovora is 10
-5.Concentration according to DNA calculates, and the detection of LAMP method of the present invention is limited to 2pgDNA.
Claims (6)
1. detect a loop-mediated isothermal amplification primer for erwinia amylovora, it is characterized in that, described primer is F3, B3, FIP and BIP, and its nucleotide sequence is respectively SEQ ID NO:1-4.
2. primer according to claim 1 detects the application in erwinia amylovora goods in preparation.
3. a loop-mediated isothermal amplification detection kit for erwinia amylovora, is characterized in that, described test kit comprises following component:
1) primer mixed solution: 80 μm of ol/L primers F IP 2 μ L, 80 μm of ol/L primer BIP 2 μ L, 10 μm of ol/L primers F 32 μ L, 10 μm of ol/L primer B32 μ L;
2) ring mediated isothermal amplification mixed solution: 8U/ μ L Bst DNA PoLymerase 2 μ L, 2.5 μm of ol/LdNTP 4 μ L, 1 × ThermoL Buffer 2.5uL, ddH
2o 31.5 μ L; Wherein 1 × Thermol Buffer composition is: 20mmol/LTris-HCl, 10mmol/LKCl, 10mmol/L (NH4)
2sO4,2mmol/LMgSO4,0.01%Triton X-100;
(3) erwinia amylovora positive control dna 2 μ L.
4. the loop-mediated isothermal amplification detection kit of erwinia amylovora according to claim 3 is in the application detecting erwinia amylovora in plant prod.
5. detect a method for erwinia amylovora in plant prod, it is characterized in that, described method comprises following step:
1) extraction of DNA profiling in testing sample: extract DNA in testing sample by DNA extraction kit;
2) LAMP constant-temperature amplification: add primer mixed solution 8 μ L, ring mediated isothermal amplification mixed solution 40 μ L, template DNA 2 μ L in PCR pipe, mixing; By the PCR pipe isothermal reaction 60min in 61 DEG C of water-baths prepared, then 80 DEG C of 10min termination reactions, are cooled to 4 DEG C;
3) SYBR Green I fluorescence colour judges reaction result: add 3 μ l fluorescent color-developing agents in the reaction product and mix gently, and standing 5min carries out colour developing to be observed: if shows green, containing erwinia amylovora in testing sample; Manifest in orange then testing sample not containing erwinia amylovora.
6. method as claimed in claim 5, is characterized in that, the purity ratio OD of the sample DNA of described extraction
260/ OD
280within the scope of 1.6-2.0, nucleic acid concentration is within the scope of 40-100ng/ μ L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101954392B1 (en) | 2017-10-31 | 2019-03-05 | 연세대학교산학협력단 | Molecular marker for detecting Erwinia amylovora and uses thereof |
| CN113136442A (en) * | 2021-04-27 | 2021-07-20 | 中国检验检疫科学研究院 | Method and kit for detecting erwinia amylovora based on LFD-RPA technology and application of method and kit |
| KR102318379B1 (en) * | 2021-08-02 | 2021-10-28 | 지노타입 주식회사 | Marker composition for rapid and simultaneous detecting Erwinia amylovora and Erwinia pyrifoliae |
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| CN113136442B (en) * | 2021-04-27 | 2022-08-23 | 中国检验检疫科学研究院 | Method and kit for detecting erwinia amylovora based on LFD-RPA technology and application of method and kit |
| KR102318379B1 (en) * | 2021-08-02 | 2021-10-28 | 지노타입 주식회사 | Marker composition for rapid and simultaneous detecting Erwinia amylovora and Erwinia pyrifoliae |
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