CN104762409A - Method for detecting pseudomonas syringaepv altinidia through recombinase-mediated isothermal amplification technology - Google Patents
Method for detecting pseudomonas syringaepv altinidia through recombinase-mediated isothermal amplification technology Download PDFInfo
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Abstract
The invention discloses a method for detecting pseudomonas syringaepv altinidia through the recombinase-mediated isothermal amplification technology and belongs to the technical field of phytobacteriology diagbosis. The method mainly includes the steps that general DNA of the pseudomonas syringaepv altinidia is extracted, an RPA reaction and 2.5% agarose gel electrophoresis detection are performed, and fast detection of the pseudomonas syringaepv altinidia is performed in the aspect of the molecular level. The method has the advantages of being fast, sensitive, convenient to use and specific. Compared with a conventional PCR method, primers obtained through the method in a screened mode have high specificity and stability on amplification of target fragments, amplification time is short, annealing is not needed during the reaction, the high activity of the RPA reaction can be kept when required temperature can be maintained under any condition of a constant-temperature incubator, a water bath kettle and human body temperature, and no expensive instruments are needed. Due to the method, a detection system of the pseudomonas syringaepv altinidia is successively established, the practicability of the system is verified through sample detection, and the method for detecting the pseudomonas syringaepv altinidia is fast and effective.
Description
Technical field
The invention belongs to phytobacteriology diagnostic techniques field, particularly adopt isothermal amplification technique to detect phytopathogen, be specially a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium.
Background technology
China is the major production areas of Kiwifruit, first of its cultivated area and the output Jun Ju world.Prospect on Kiwifruit Bacterial Canker is a kind of destructive disease, and have harm in various degree to each large producing region of China's Kiwifruit, serious threat, to the Sustainable development of China's Kiwifruit industry, is listed in Forest Plant Quarantine controlling object.This disease breaks with tremendous force, and directly can have influence on output and the fruit quality of Kiwifruit during morbidity, causes serious financial loss to China's Kiwifruit industry.
Prospect on Kiwifruit Bacterial Canker harm is serious and control is difficult, is that Kiwifruit is produced and a theoretic major issue always.Be difficult at present find a kind of effective means to prevent and treat.Its pathogenic bacteria is Pseudomonas syringae pv.actinidiae (Pseudomonas syringae pv.actinidiae; PSA); this bacterium has longer latent period; virulence is strong; once infect; to cause after 2-3 and ruin garden on a large scale, therefore, accurately detecting it is in early days the important prerequisite that kiwi berry bacterial canker is prevented and treated.Therefore, set up efficiently, fast, convenient, detection method is particularly important accurately, this contributes to the early diagnosis of nursery stock and prevents and treats in time.
Traditional Physiology and biochemistry method qualification Prospect on Kiwifruit Bacterial Canker bacterium is a kind of loaded down with trivial details, time-consuming method, and does not often reach expected effect, can not realize the early detection to germ.In recent years, the qualification developing into phytopathogen of molecular detection technology provides more strong instrument, and technique directly can determine pathogenic bacteria from DNA aspect, makes the qualification of cause of disease, and the diagnosis of disease becomes more quick and precisely.Polymerase chain reaction (Polymerase Chain Reaction, PCR) be a kind of conventional germ molecular detection technology, by sex change,--annealing--extension three primitive reaction steps are formed: the 1. sex change of template DNA: template DNA is after being heated to about 94 DEG C certain hours, make template DNA double-strand or dissociate through the double-stranded DNA that pcr amplification is formed, make it to become strand, so that it is combined with primer, for lower whorl reaction is prepared; 2. the annealing (renaturation) of template DNA and primer: template DNA becomes after strand through heat denatured, and temperature is down to about 55 DEG C, and the complementary sequence of primer and template DNA strand matches and combines; 3. the extension of primer: DNA profiling--primer binding substances is at 72 DEG C, under the effect of archaeal dna polymerase (as Taq DNA polymerase), take dNTP as reaction raw materials, target sequence is template, by base pair complementarity and semiconservative replication principle, synthesize a semiconservative replication chain that the is new and complementation of template DNA chain, recirculation sex change--annealing--extends three processes and just can obtain more " semiconservative replication chain ", and this new chain can become again the template of circulation next time.Round pcr needs certain temperature condition and suitable sex change, annealing and extension time to meet reaction requirement, and process control is bad, easily occurs that false positive maybe cannot increase target stripe, consuming time long.
Recombinase-mediated isothermal duplication (Recombinase Polymerase Amplification, RPA) technology is considered to the nucleic acid detection technique that can replace PCR.RPA technological essence is exactly the reaction process of a constant temperature, its most suitable temperature between 37 DEG C-42 DEG C, without the need to sex change, alternating temperature, the processes such as annealing, react at normal temperatures, reaction process is simple, within general about 15 minutes, can obtain the amplified production that can detect.Current this technology is applied to coronavirus, HIV virus, the detection of the aspects such as cancer, but in phytopathy mycology field, particularly in the application that the early detection of Prospect on Kiwifruit Bacterial Canker bacterium is but not relevant.
Summary of the invention
For solving the problems of the technologies described above, recombinase-mediated isothermal amplification technique is applied to diagnosis and the prevention and control field of Prospect on Kiwifruit Bacterial Canker bacterium by the present invention, sets up a kind of method of early diagnosis.
The invention provides a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium.The method mainly comprises the extraction of Prospect on Kiwifruit Bacterial Canker bacterium STb gene, and RPA reaction and 2.5% agarose gel electrophoresis detect, and carry out rapid detection from molecular level to Prospect on Kiwifruit Bacterial Canker bacterium.
A kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium of the present invention, mainly comprises the following steps:
1) design of recombinase-mediated isothermal duplication (RPA) primer;
2) cultivation of Prospect on Kiwifruit Bacterial Canker bacterium;
3) extraction of Prospect on Kiwifruit Bacterial Canker bacterium STb gene;
4) RPA reaction;
5) 2.5% agarose gel electrophoresis detects.
The design of primers of RPA is longer than PCR primer, generally needs to reach 30-35bp bases longs, and primer is too short can reduce recombination fraction, affects amplification rate and detection sensitivity.Preferred as one, described design of primers is:
RPA Psa1-F:5’-CCTGATAAGGGTGAGGTCGGCAGTTCGAATC-3’
RPA Psa1-R:5’-TCACGCACCCTTCAATCAGGATGGAATGCTC-3’
Preferred as one, the cultivation of described Prospect on Kiwifruit Bacterial Canker bacterium is: Prospect on Kiwifruit Bacterial Canker bacterium is put into LB liquid medium, cultivates 12 hours under 22 DEG C of conditions.
Preferred as one, being extracted as of described Prospect on Kiwifruit Bacterial Canker bacterium STb gene: collect ulcer bacteria by after above-mentioned medium centrifugal, extract STb gene from germ.
Preferred as one, described RPA reaction is: with the DNA of Prospect on Kiwifruit Bacterial Canker bacterium for template, RPA reaction tubes is placed on 37 DEG C of water bath with thermostatic control 15-30 minute, obtains the target product increased.
RPA reaction depends on three kinds of enzymes: recombinase protein, single-stranded DNA binding protein and archaeal dna polymerase.Recombinase protein can form DNA nucleoprotein microfilament in conjunction with single stranded DNA (primer) in the presence of ATP, and nucleoprotein microfilament two ends can extend, and the method that can be shortened by front end elongation rear end be moved to double chain DNA molecule.Microfilament can catch hold of the DNA molecular of surrounding, the oligonucleotide sequence of strand and DNA sequence dna is compared, and when finding the sequence of coupling, two molecules will be closely adhered to guarantees that restructuring is ensued together.To search, the helical conformation of DNA and the length of nucleoprotein microfilament identify that the efficiency of homologous sequence plays an important role, under the help of single strand binding protein, template DNA starts to unwind, primer invasion double-stranded DNA starting match formation copy needed for 3' hydroxyl terminal freely, carry out copying extension under the effect of archaeal dna polymerase, to form new DNA complementary strand reaction product be also increase with exponential, whole reaction process is carried out quickly, within general tens minutes, can obtain the amplified production of detectable Prospect on Kiwifruit Bacterial Canker bacterium.
As a kind of optimal way in the specific embodiment of the invention, described RPA reaction can be realized by following manner: in the 0.2ml TwistAmp Basic reaction tubes containing lyophozyme powder, add rehydration damping fluid 29.5 μ L, deionized water 12.2 μ L, then be divided in the reaction tubes of two 0.2ml, primer 1 μ L (10 μm of ol/L) is respectively added in two pipes, bacterial genomes DNA2 μ L (50ng/ μ L), finally add magnesium acetate solution 1.25 μ L (280mmol/L), in 37 DEG C of waters bath with thermostatic control, 15-30 minute is reacted after abundant mixing, obtain amplified production.
Several enzymes containing RPA reaction in lyophilized powder, comprise recombinase, single strand binding protein and polysaccharase etc., and these enzymes are dissolved in the effect of rehydration damping fluid, and provide stable buffer system in RPA reaction, and magnesium acetate has the effect starting RPA reaction.
Preferred as one, described RPA reaction carries out in 37 DEG C of waters bath with thermostatic control.
Preferred as one, the time of described RPA reaction is 15-30 minute.
Preferred as one, the optimum reacting time of described RPA reaction is 20 minutes.
Preferred as one, described 2.5% agarose gel electrophoresis is detected as: detecting carrying out the amplified production that RPA is obtained by reacting, determining whether Kiwifruit infects described ulcer bacteria.
Beneficial effect of the present invention: the present invention utilizes RPA to react to carry out the target product that obtains in water bath with thermostatic control, has the features such as quick, sensitive, convenient, specificity.RPA reaction is not high to conditional request, can maintain temperature required any condition RPA reaction can be kept to have higher activity, without the need to the plant and instrument of costliness in constant incubator, water-bath, human temperature etc.Compared with conventional PCR method, the amplification that the present invention screens the primer pair target fragment of acquisition has higher specificity and stability, and reaction without primer dimer, and copies mechanism in parody, and reaction does not need annealing process, and whole reaction is simple and quick.The inventive method has been successfully applied to the detection of Prospect on Kiwifruit Bacterial Canker fungus diseases, and to demonstrate RPA be a kind of effective means detecting Prospect on Kiwifruit Bacterial Canker fungus diseases.
Accompanying drawing explanation
Fig. 1 is the RPA primer screening schematic diagram of the embodiment of the present invention 1, and wherein M is Marker 2000,1 is primer RPA-Psa1F and R, 2 be primer RPA-Psa2F and R.
Fig. 2 be the embodiment of the present invention 2 the different RPA reaction times on detect impact, wherein M is Marker2000; 1 be 5min, 2 be 10min, 3 be 20min, 4 be 30min, 5 be 40min, 6 be 60min, 7 for 120min.
Fig. 3 is RPA and the PCR sensitivity technique figure of the embodiment of the present invention 3, and wherein A is that RPA reacts, B is PCR reaction; 1 represents that DNA concentration is that 50ng/ μ l, 2 represents that DNA concentration is that 1ng/ μ l, 3 represents that DNA concentration is that 0.5ng/ μ l, 4 represents that DNA concentration is that 0.25ng/ μ l, 5 represents that DNA concentration is that 0.15ng/ μ l, 6 represents that DNA concentration is 0.05ng/ μ l.
Fig. 4 be each PCR of RPA of the embodiment of the present invention 4 to field sample specific detection figure, wherein M is Marker 2000; 1-2 is PCR, 3-4 is RPA; 1 and 3 is Kiwifruit morbidity limb, 2 and 4 Chinese goosebeery healthy limbs.
Embodiment
In order to more understand the present invention, below in conjunction with specific embodiment, the method that the described employing recombinase-mediated isothermal amplification technique of invention detects Prospect on Kiwifruit Bacterial Canker bacterium is described in detail, but described embodiment does not limit the present invention.
Material and reagent
Material:
Chinese goosebeery healthy branch, infects the branch of ulcer bacteria.Pick up from Sichuan Province China Cangxi, Yaan, Dujiang weir, gather branch and be stored in 4 DEG C of refrigerators.
Main agents:
TwistAmp Basic test kit is purchased from TwistDx company of Britain;
DNA of bacteria extracts test kit purchased from the biological company limited of sky root.
Embodiment 1:
1. design of primers:
This experiment is many groups RPA primer according to Prospect on Kiwifruit Bacterial Canker bacterium 16S-23S rDNA sequences Design, and then optimize wherein two groups of primers and carry out RPA amplified reaction, these two groups of primer sequences are:
| Primer | Sequence 5 '-3 ' |
| RPA Psa1-F | CCTGATAAGGGTGAGGTCGGCAGTTCGAATC |
| RPA Psa1-R | TCACGCACCCTTCAATCAGGATGGAATGCTC |
| RPA PSA2-F | AGAAGCAGCTTTTGCTTTGCACACCCGATTTT |
| RPA PSA2-R | AATCAACATTCACGCACCCTTCAATCAGGATG |
2. the cultivation of Prospect on Kiwifruit Bacterial Canker bacterium:
Prospect on Kiwifruit Bacterial Canker bacterium is put into LB liquid medium, cultivates 12 hours under 22 DEG C of conditions.
3. ulcer bacteria total DNA extraction:
Get 1ml above-mentioned cultivation bacterium liquid, collected by centrifugation ulcer bacteria, extract the specification sheets of test kit according to DNA of bacteria, extract DNA, the concentration of spectrophotometric measurement DNA, is diluted for 50ng/ μ L, is stored in-20 DEG C.
4.RPA reacts:
RPA reaction is carried out to the STb gene of Prospect on Kiwifruit Bacterial Canker bacterium, rehydration damping fluid (Rehydration Buffer) 29.5 μ L is added in the 0.2ml TwistAmp Basic reaction tubes containing lyophozyme powder, deionized water 12.2 μ L, then be divided in the reaction tubes of two 0.2ml, get 1 pipe, add each 1 μ L of primer (10 μm of ol/L), bacterial genomes DNA 2 μ L (50ng/ μ L), finally add magnesium acetate solution 1.25 μ L (280mmol/L), reaction tubes is placed on 37 DEG C of waters bath with thermostatic control 20 minutes after abundant mixing, obtains amplified production.
5. 2.5% agarose gel electrophoresis detects:
Detect carrying out the amplified production that RPA is obtained by reacting, result display utilizes RPA Psa1F and R, and this can detect target sizes fragment to primer, RPA Psa2F and R fails to amplify target fragment, determines a pair special primer detected for Prospect on Kiwifruit Bacterial Canker bacterium thus.Experimental result as shown in Figure 1.
The rapidity analysis of embodiment 2:RPA
According to the primer of above-described embodiment 1 Screening and Identification, design several reaction times gradient, i.e. 5min, 10min, 20min, 30min, 40min, 60min, 120min, analyze the RPA reaction times to the impact detected.Found that reaction 5min and 10min, can't detect target stripe, target fragment can be detected after 20min, but with the prolongation in reaction times, amplified fragments does not increase, illustrate that RPA has higher enzymic activity, can increase within the relatively short time to DNA fragmentation, this experiment determines that the optimum reacting time that RPA reacts is 20min.Experimental result as shown in Figure 2.
The sensitivity analysis that embodiment 3:PCR and RPA detects
The STb gene of Prospect on Kiwifruit Bacterial Canker bacterium is diluted to 50ng/ μ L, then dilutes by the extension rate of 50 times, 100 times, 200 times, 300 times, 600 times, detect for PCR and RPA.PCR detects the primer with reference to Rees-George (Rees-George, J., Vanneste, J.L., Cornish, D.A., Pushparajah, I.P.S., Yu, J., Templeton, M.D., and Everett, K.R.2010.Detection of Pseudomonas syringae pv.actinidiae using polymerase chain reaction (PCR) primers based on the 16S-23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions.Plant Pathol.59:453-464) publish an article in primer, wherein upstream primer is 5 '-TTTTGCTTTGCACACCCGATTTT-3 ', downstream primer is 5 '-CACGCACCCTTCAATCAGGATG-3 '.Experimental result display PCR and RPA all can see target fragment when the DNA that concentration is 50ng/ μ L is diluted to 600 times, proves that RPA with PCR is the same and has higher detection spirit lightness.Experimental result as shown in Figure 3.
Embodiment 4:PCR and RPA detects field sample
In order to more determine the accuracy of RPA technology for detection Prospect on Kiwifruit Bacterial Canker bacterium, with PCR reaction for contrast experiment, compare two kinds of technology to the detectivity of field diseased plant sample.Major experimental step is as follows:
1) Kiwifruit limb total DNA extraction: the Kiwifruit limb be stored in 4 DEG C of refrigerators is extracted STb gene.
2) RPA and PCR reaction: with the Kiwifruit limb STb gene extracted for template, carries out RPA and PCR reaction respectively;
3) RPA and pcr amplification product detect: utilize 2.5% sepharose to carry out electrophoresis detection, found that two kinds of methods all can detect target fragment.Experimental result as shown in Figure 4.
Have detected field diseased plant sample by the RPA method described in the present invention and PCR method, result illustrates that RPA method can reach the technique effect of PCR method simultaneously, can be special amplify target fragment.But the time that RPA detects but is less than reaction times of PCR greatly, and RPA detection method does not need expensive plant and instrument, and schedule of operation is simple, more conveniently applies.
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 (9)
1. adopt recombinase-mediated isothermal amplification technique to detect a upstream primer for Prospect on Kiwifruit Bacterial Canker bacterium, it is characterized in that, nucleotides sequence is classified as: CCTGATAAGGGTGAGGTCGGCAGTTCGAATC.
2. adopt recombinase-mediated isothermal amplification technique to detect a downstream primer for Prospect on Kiwifruit Bacterial Canker bacterium, it is characterized in that, nucleotides sequence is classified as: TCACGCACCCTTCAATCAGGATGGAATGCTC.
3. the primer pair adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium, it is characterized in that, upstream primer nucleotides sequence is classified as CCTGATAAGGGTGAGGTCGGCAGTTCGAATC, and downstream primer nucleotides sequence is classified as: TCACGCACCCTTCAATCAGGATGGAATGCTC.
4. adopt primer described in claim 1-3 to carry out a method for recombinase-mediated isothermal amplification technique detection Prospect on Kiwifruit Bacterial Canker bacterium, it is characterized in that, mainly comprise the following steps:
1) extraction of Prospect on Kiwifruit Bacterial Canker bacterium STb gene;
2) recombinase-mediated isothermal duplication (RPA) reaction: with Prospect on Kiwifruit Bacterial Canker bacterium STb gene for template, carry out RPA reaction, obtain amplified production;
3) 2.5% agarose gel electrophoresis detects.
5. a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium as claimed in claim 4, is characterized in that, also comprise the design that RPA reacts primer.
6. a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium as claimed in claim 4, it is characterized in that, described RPA reaction carries out in 37 DEG C of waters bath with thermostatic control.
7. a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium as claimed in claim 4, it is characterized in that, the time of described RPA reaction is 15-30 minute.
8. a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium as claimed in claim 4, it is characterized in that, the optimum reacting time of described RPA reaction is 20 minutes.
9. a kind of method adopting recombinase-mediated isothermal amplification technique to detect Prospect on Kiwifruit Bacterial Canker bacterium as claimed in claim 4, it is characterized in that, described RPA reaction is: in the 0.2ml TwistAmp Basic reaction tubes containing lyophozyme powder, add rehydration damping fluid 29.5 μ L, deionized water 12.2 μ L, then be divided in the reaction tubes of two 0.2ml, get 1 pipe, add each 1 μ L of primer (10 μm of ol/L), germ genomic dna 2 μ L (50ng/ μ L), finally add magnesium acetate solution 1.25 μ L (280mmol/L), in 37 DEG C of waters bath with thermostatic control, 15-30 minute is reacted after abundant mixing, obtain amplified production.
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Cited By (12)
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| CN104975098A (en) * | 2015-07-17 | 2015-10-14 | 浙江泰晶生物科技有限公司 | Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature |
| CN105018466A (en) * | 2015-07-17 | 2015-11-04 | 浙江泰晶生物科技有限公司 | Normal-temperature and constant-temperature nucleic acid amplification method using fluorescence probe |
| CN105296393A (en) * | 2015-11-03 | 2016-02-03 | 中国科学院武汉植物园 | Method for rapidly identifying kiwi fruit canker susceptible sample |
| CN105400884A (en) * | 2015-12-17 | 2016-03-16 | 中国检验检疫科学研究院 | RPA detection-based quarantine solanum weed solanum elaeagnifolium detection method |
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| CN111363853A (en) * | 2020-04-23 | 2020-07-03 | 四川农业大学 | Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV |
| CN111690759A (en) * | 2020-08-04 | 2020-09-22 | 西南大学 | Specific primer, kit and method for detecting RPA of citrus canker pathogen |
| CN113186319A (en) * | 2021-06-22 | 2021-07-30 | 广东海洋大学 | Primer, kit and method for detecting Nocardia seriolae |
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| CN115896314A (en) * | 2022-07-14 | 2023-04-04 | 四川大学 | Equipment-free visual detection method for bacterial canker of kiwi fruit based on LF-RPA |
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| CN105018466A (en) * | 2015-07-17 | 2015-11-04 | 浙江泰晶生物科技有限公司 | Normal-temperature and constant-temperature nucleic acid amplification method using fluorescence probe |
| CN104975098A (en) * | 2015-07-17 | 2015-10-14 | 浙江泰晶生物科技有限公司 | Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature |
| CN105296393A (en) * | 2015-11-03 | 2016-02-03 | 中国科学院武汉植物园 | Method for rapidly identifying kiwi fruit canker susceptible sample |
| CN105400884A (en) * | 2015-12-17 | 2016-03-16 | 中国检验检疫科学研究院 | RPA detection-based quarantine solanum weed solanum elaeagnifolium detection method |
| CN108192987A (en) * | 2018-02-10 | 2018-06-22 | 绵阳市农业科学研究院 | The primer and its methods and applications of quantitative PCR detection Prospect on Kiwifruit Bacterial Canker opportunistic pathogen |
| CN108315448A (en) * | 2018-02-10 | 2018-07-24 | 绵阳市农业科学研究院 | The primer and its detection method of detection Prospect on Kiwifruit Bacterial Canker opportunistic pathogen and application |
| CN111363853A (en) * | 2020-04-23 | 2020-07-03 | 四川农业大学 | Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV |
| CN111363853B (en) * | 2020-04-23 | 2022-07-29 | 四川农业大学 | Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV |
| CN113832255B (en) * | 2020-06-08 | 2023-07-14 | 西北农林科技大学 | Primer group and kit for RT-PCR detection of 4 novel kiwi fruit viruses |
| CN113832255A (en) * | 2020-06-08 | 2021-12-24 | 西北农林科技大学 | Primer group and kit for RT-PCR detection of 4 kinds of new kiwi fruit viruses |
| CN111690759A (en) * | 2020-08-04 | 2020-09-22 | 西南大学 | Specific primer, kit and method for detecting RPA of citrus canker pathogen |
| CN111690759B (en) * | 2020-08-04 | 2023-10-17 | 西南大学 | Specific primer, kit and method for detecting RPA of citrus canker pathogen |
| CN113186319A (en) * | 2021-06-22 | 2021-07-30 | 广东海洋大学 | Primer, kit and method for detecting Nocardia seriolae |
| CN115896314A (en) * | 2022-07-14 | 2023-04-04 | 四川大学 | Equipment-free visual detection method for bacterial canker of kiwi fruit based on LF-RPA |
| CN115948407A (en) * | 2022-10-10 | 2023-04-11 | 四川省农业科学院植物保护研究所 | Pseudomonas syringae kiwi fruit pathogenic variety aptamer, screening method and application |
| CN115948407B (en) * | 2022-10-10 | 2023-12-19 | 四川省农业科学院植物保护研究所 | Pseudomonas syringae kiwi fruit pathogenic variant aptamer, screening method and application |
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