CN108424866A - A kind of sturgeon source Aeromonas media AMth-1 and PCR detection primer and application - Google Patents
A kind of sturgeon source Aeromonas media AMth-1 and PCR detection primer and application Download PDFInfo
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Abstract
The invention belongs to Bacteria Detection field, specifically discloses a kind of sturgeon source Aeromonas media AMth 1 and PCR detection primers and application, inventor have isolated a kind of Aeromonas media from mandarin sturgeon for the first time, which is Aeromonas media（Aeromonas media）AMth 1, for the bacterial sequences design primer, F1：5'GTTGATACCTAATACCTGYCAGC 3', R1：5'‑GGACTACCAGGGTATCTAATC‑3'；F2：5'CCAGCCCTGCGCCGACAA 3', R2：5'CACCAGGGTCGCCAGCGCTTCA 3' are detected sturgeon source Aeromonas media, specific good, the high sensitivity of the primer.
Description
Technical field
The invention belongs to bacteria molecule detection technique fields, and in particular to a kind of sturgeon source Aeromonas media AMth-1 and
PCR detection primers and application.
Technical background
Aeromonas is the gram-Negative bacillus of a kind of amphimicrobian.Early in 1986, Colwell etc. was by Aeromonas
Belong to by being separated in vibrionaceae, is under the jurisdiction of newly-built aeromonas section.So far, Aeromonas includes：Aeromonas hydrophila, globefish
14 phenotype kinds such as mouse Aeromonas, Aeromonas sobria, Aeromonas veronii and Aeromonas media and 16 gene kinds.Gas
Monad is widely distributed in the environment, exists with mesophile in aquatic ecosystem.Aeromonas media (Aeromonas m
Edia), be one kind of Aeromonas, most found in river water earlier than nineteen eighty-three, then mud, waste water, animal body with
And the presence of the bacterium is all detected in human body.Aeromonas media is a kind of newfound conditioned pathogen, can cause the mankind
Cardinal symptom with the infection of animal, the mankind is diarrhea.The report that Aeromonas media causes a disease to aquatic animal is relatively fewer, only
Wang Gaoxue etc. (2007) reports Aeromonas media and causes stichopus japonicus putrid skin disease；Yellow civilization etc. (2013) report intermediate gas unit cell
Bacterium causes mullet to fall ill；Yang Yibin etc. (2016) passes through the experiments such as pathogen separation Purification and artificial challenge, it was demonstrated that intermediate
Aeromonas can cause channel catfish hemorrhagic dead.
In the application, applicant has found Aeromonas media infection, infection symptoms in the mandarin sturgeon of artificial breeding for the first time
The predominantly extensive bleeding of bleeding of skin and internal organs.Mandarin sturgeon and acipenser dabryanus are existing the most ancient in China Yangtze river basin
Fish, belong to country-level focused protection wild animal, the species conservation of the discovery of Aeromonas media to both sturgeons
Constitute potential threat.Therefore, further investigation is carried out to sturgeon source Aeromonas media to be of great significance.
Aeromonas hydrophila (Aeromonashydrophila) is concentrated mainly on to the research of Aeromonas at present, and
It is very few to the research of Aeromonas media.There has been no the open reports of sturgeon source Aeromonas media, and also there are no effective detection sides
Method.
Invention content
It is described the object of the present invention is to provide sturgeon source Aeromonas media (Aeromonas media) AMth-1
Aeromonas media is isolated from mandarin sturgeon pathological tissues, which send on April 9th, 2018 to China typical culture collection
Center carries out preservation, Classification And Nomenclature：Intermediate Aerononas punctata (Aeromonas media) AMth-1, deposit number：CCTC C
NO:M2018185, address：Wuhan, China Wuhan University.
It is another object of the present invention to provide the specific sequence 16SrRNA of sturgeon source Aeromonas media AMth-1
It is classified as shown in SEQ ID NO.1 with the nucleotides sequence of c pn60,16SrRNA, the nucleotides sequence of cpn60 is classified as SEQ ID NO. 2
It is shown.
It is also an object of the present invention to provide the primers of detection sturgeon source Aeromonas media AMth-1, and described draws
Object is F1：5'-GTTGATACCTAATACCTGYCAGC-3', R1：5'-GGACTACCAGGGTATCTAATC-3'；F 2：5'-
CCAGCCCTGCGCCGACAA-3', R2：5'-CACCAGGGTCGCCAGCGCTTCA-3'.
It is also an object of the present invention to provide the application of sturgeon source Aeromonas media AMth-1, the application packet
It includes and is prepared into Aeromonas media bacterial vaccine using the bacterium, or be prepared into the laboratory control of sturgeon source Aeromonas media
Product, or it is used to prepare the model of screening treatment sturgeon source Aeromonas media bacterium medicine.
Final object of the present invention is to provide a kind of answering for sturgeon source Aeromonas media AMth-1PCR detection primers
With being prepared into sturgeon source gas monad detection kit including the use of the primer.
In order to achieve the above object, the present invention takes following technical measures：
A kind of Aeromonas media AMth-1, the bacterium are isolated from mandarin sturgeon pathologic liver tissue, the bacterium in
It send on April 9th, 2018 to China typical culture collection center and carries out preservation, Classification And Nomenclature：Intermediate Aerononas punctata
(Aeromon as media) AMth-1, deposit number：CCTCC NO:M2018185, address：Wuhan, China Wuhan University.
Aeromonas media AMth-1 (present invention or be AMth-1) is inoculated into BHI (BD, BectoTM Brain
Heart Infusion) culture is enlarged on solid agar medium, after being cultivated for 24 hours in 28 DEG C of incubators, picking single bacterium colony
Secondary lineation culture is carried out in BHI solid agar mediums under the same conditions, is preserved in -80 DEG C of glycerine.
The morphological feature of Aeromonas media AMth-1：Size be (1~4) μ m (0.1~1)/μm, thalline both ends are blunt
Circle, monopole flagellum move torpescence.Without brood cell, there is narrow pod membrane, belongs to Grain-negative brevibacterium.
The application of Aeromonas media AMth-1, the application are prepared into Aeromonas media epidemic disease including the use of the bacterium
Seedling, or it is prepared into the laboratory reference substance of sturgeon source Aeromonas media, or it is used to prepare gas list among screening treatment sturgeon source
The model of born of the same parents' bacterium bacterium medicine.
The specific sequence 16SrRNA and cpn60 of sturgeon source Aeromonas media AMth-1, the nucleotide sequence of 16SrRNA
Shown in SEQ ID NO.1, the nucleotides sequence of cpn60 is classified as shown in SEQ ID NO.2.
The application of the specific sequence of sturgeon source Aeromonas media AMth-1, including the use of 16SrRNA and/or cpn60 sequences
Arrange the Aeromonas media AMth-1 for detecting or distinguishing Xun Yuan.
Also belong to for the primer of specific sequence 16SrRNA and/or the cpn60 design of sturgeon source Aeromonas media AMth-1
In protection scope of the present invention, the primer includes but not limited to be based on the diversity sequence, utilizes all of prior art design
Such as fluorescent quantitation primer, LAMP primer, digital pcr primer etc..
A kind of sturgeon source Aeromonas media AMth-1PCR detection primers, including F1：5'-GTTGATACCTAATACCT
GYCAGC-3', R1：5'-GGACTACCAGGGTATCTAATC-3'；F2：5'-CCAGCCCTGCGCCGAC AA-3', R2：5'-
CACCAGGGTCGCCAGCGCTTCA-3'。
A kind of application of sturgeon source Aeromonas media AMth-1PCR detection primers, including the use of for gas list among sturgeon source
The primer of specific sequence 16SrRNA and/or the cpn60 design of born of the same parents bacterium AMth-1 is prepared into for detecting Aeromonas media inspection
Test agent box.
Preferably, when being prepared into kit, the nondiagnostic detection method of the kit includes：
1) DNA of sample to be tested is extracted；
2) PCR system
Using 25 μ L reaction systems：A concentration of 10 μM of two pairs of primers Fs 1, R1 or F2, R2 respectively take 0.5 μ L to be added two respectively
PCR reaction tubes are separately added into 2 μ L, 25mM MgCl of 2.5mMdNTPs in two PCR reaction tubes again21.5 μ L, 5U Taq
2.5 μ L of 0.5 μ L of polymerase, template cDNA1 μ L, 10 × ThermoPol Reaction Buffer, with sterilizing distilled water by two
The equal polishing of tube reaction system is to 25 μ L.Reaction condition, 94 DEG C of pre-degeneration 3min；94 DEG C of 30s, 56 DEG C of 15s, 72 DEG C of 30s, 30
Cycle；72 DEG C of extension 10min, 4 DEG C of preservations.
3) testing result judges
The final reacting product of PCR amplification takes 4 μ L, is added and extremely contains 0.5 μ g/mL ethidium bromides (EB)
In 2% Ago-Gel of dyestuff, the electrophoresis 30min under 100V voltages, in gel at imaging amplification production on phase system
The collection of illustrative plates of object.Electrophoresis picture shows 352bp and 385bp signature bands, then result is the positive；Such as without any band, then result
For feminine gender.
Compared with prior art, the present invention has the following advantages：
1. the present invention has isolated Aeromonas media from mandarin sturgeon body for the first time, have for the research of sturgeon source gas monad
Standby ground-breaking meaning.
1. the method for the present invention is fast and convenient：The PCR method that the present invention is built only needs a few hours, the conventional bacteria that compares point
From method time a couple of days, detection efficiency is greatly improved.
2. the method high specificity of the present invention：The identification in the distinguished sequence area of two pairs of primer pair target sequences, ensure that the side PCR
The high degree of specificity of method amplification.
Description of the drawings
Fig. 1 is the experimental result schematic diagram of the sensitivity of two pairs of primers provided by the invention；
Wherein, swimming lane 1：Primary template, swimming lane 2：10 times of dilutions, swimming lanes 3：100 times of dilutions, swimming lanes 4：1000 times of dilutions,
Swimming lane 5：10000 times of dilutions, swimming lanes 6：100000 times of dilutions, swimming lanes 7：1000000 times of dilutions.
Fig. 2 is the sturgeon tissue progress that sturgeon source Aeromonas media AMth-1 is infected using primer detection provided by the invention
The result schematic diagram of detection；
Wherein swimming lane P：Positive control；N：Negative control (template is sterile water) M：2000bp marker (from top to bottom according to
Secondary is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp)
Swimming lane 1：Sample to be tested 1；
Swimming lane 2：Sample to be tested 2；
Swimming lane 3：Sample to be tested 3；
Swimming lane 4：Sample to be tested 4.
Specific implementation mode
Technical solution of the present invention is if not otherwise specified the conventional scheme of this field, agents useful for same or raw material,
If not otherwise specified, it is purchased from commercial channel or discloses.
Embodiment 1：
A kind of sturgeon source Aeromonas media AMth-1, separation process are as follows：
1) after the tissues such as the mandarin sturgeon liver of Carried bacteria, spleen being taken out and are milled, 100 times of sterile PBS dilutions, 20
It takes supernatant to filter after 00rpm centrifugations, is inoculated into BHI (BD, BectoTM Brain Heart Infusion) solid agar culture
It is separately cultured on base, after being cultivated for 24 hours in 28 DEG C of incubators, occurs the bacterium colony of white translucent in culture dish.
2) picking single bacterium colony, Gram's staining observe ne ar.
3) bacterium is collected, 16SrRNA and cpn60 amplifications and sequencing are carried out to it, sequence is respectively SEQ ID NO.1 institutes
Show with shown in SEQ ID NO.2.
The morphological feature of mandarin sturgeon Aeromonas media：Belong to Grain-negative brevibacterium.Size is (1~4) μ m (0.1
~1)/μm, thalline both ends blunt circle, monopole flagellum moves torpescence.Without brood cell, there is narrow pod membrane.The bacterium is in 2018
It sent April 9 to China typical culture collection center and carries out preservation, Classification And Nomenclature：Intermediate Aerononas punctata (Aeromon as
Media) AMth-1, deposit number：CCTCC NO:M2018185, address：Wuhan, China Wuhan University.
Embodiment 2：
16SrDNA (SEQ ID NO.1) based on sturgeon source Aeromonas media AMth-1 and cpn60 (SEQ ID NO.2)
The primer designed with the difference of the corresponding sequence of other bacteriums：
The present invention is illustrated by taking regular-PCR as an example, and based on other primers of diversity sequence design, fluorescent quantitation draws
Object, LAMP primer, digital pcr primer etc. can also complete the detection of sturgeon source Aeromonas media AMth-1.
A kind of sturgeon source Aeromonas media AMth-1PCR detection primers, including F1：5'-GTTGATACCTAATACCT
GYCAGC-3', R1：5'-GGACTACCAGGGTATCTAATC-3'；F2：5'-CCAGCCCTGCGCCGAC AA-3', R2：5'-
CACCAGGGTCGCCAGCGCTTCA-3'。
Embodiment 3：
Detection using Aeromonas media AMth-1PCR detection primers to sturgeon source Aeromonas media AMth-1, method
Including：
1. the extraction of mandarin sturgeon tissue DNA：The total DNA of sample to be tested is extracted using commercialization genome extracts kit,
- 20 DEG C of DNA profiling will be obtained to save backup.
2. PCR amplification：
Using 25 μ L reaction systems：A concentration of 10 μM of two pairs of primers Fs 1, R1 or F2, R2 respectively take 0.5 μ L to add respectively respectively
Enter two PCR reaction tubes, then is separately added into 2 μ L, 25mM MgCl of 2.5mM dNTPs21.5 μ L, 5U Taq polymerase, 0.5 μ L,
1 μ L, 10 × ThermoPol Reaction Buffer of template cDNA, 2.5 μ L, it is with sterilizing distilled water that two tube reaction systems are equal
Polishing is to 25 μ L.Reaction condition, 94 DEG C of pre-degeneration 3min；94 DEG C of 30s, 56 DEG C of 15s, 72 DEG C of 30s, 30 cycles；72 DEG C of extensions
10min, 4 DEG C of preservations.
F1：5'-GTTGATACCTAATACCTGYCAGC-3', R1：5'-GGACTACCAGGGTATCTAATC-3'；
F2：5'-CCAGCCCTGCGCCGACAA-3', R2：5'-CACCAGGGTCGCCAGCGCTTCA-3'
3. PCR amplification product analysis：
The 4 μ L of final reacting product for taking PCR amplification to expand are added and extremely contain 0.5 μ g/mL ethidium bromides
(EB) in 2% Ago-Gel of dyestuff, the electrophoresis 30min under 100V voltages expands in gel at imaging on phase system
Increase production the collection of illustrative plates of object.If F1 and R1 can amplify 352bp and F2 and R2 can amplify 385bp signature bands, which is
It is positive；Then it is feminine gender such as without any band.
Embodiment 4：
Sturgeon source Aeromonas media AMth-1PCR detection primer specific tests
Using method described in embodiment 3, the Aeromonas media AMth-1 samples in sturgeon source to be checked in table 1 are examined
It surveys, while negative control group (distilled water) and positive controls (AMth-1) is set, bacteria samples to be checked are specially：Mandarin sturgeon is thermophilic
Hydrophila (AHth-1) (immune response and protecting effect after Amur Sturgeon is immunized in aeromonas hydrophila inactivated vaccine,
Zhou Yong), mandarin sturgeon Aeromonas veronii (AVth-1), mandarin sturgeon meninx sepsis Elizabethan bacterium (EM), Aeromonas sobria
(ASth-1)。
Aerononas punctata AMth-1 PCR detection kit specific tests among 1 sturgeon source of table
The results are shown in Table 1, and only positive controls (AMth-1) can detect 352bp and 385bp signature bands, remaining
Group illustrates that Aerononas punctata AMth-1 detection primers have very strong specificity among sturgeon source without characteristic bands.
Embodiment 5：
The sensitivity of sturgeon source Aeromonas media AMth-1PCR detection primers
By 10 times of gradient dilutions of AMth-1DNA templates of a concentration of 2000ng/ μ L, the method for embodiment 3, detection profit are utilized
It is limited with the detection of primer PCR provided by the invention, the results are shown in Figure 1, it is seen that 105Dilution also can detect purpose band again,
The cDNA templates of namely minimum detectable 20pg/ μ L.
Embodiment 6：
Sturgeon source Aeromonas media AMth-1PCR detection primers are in preparing sturgeon source Aeromonas media detection kit
Using：
1. mandarin sturgeon organizes the extraction of total DNA：The total of sample to be tested is extracted using commercialization genome extracts kit
DNA will obtain -20 DEG C of DNA profiling and save backup.
2. PCR amplification：
Using 25 μ L reaction systems：A concentration of 10 μM of two pairs of primers Fs 1, R1 or F2, R2 respectively take 0.5 μ L to be added two respectively
PCR reaction tubes, then it is separately added into 2 μ L, 25mM MgCl of 2.5mM dNTPs21.5 μ L, 5U Taq polymerase, 0.5 μ L, template
1 2.5 μ L of μ L, 10 × Buffer of cDNA, with sterilizing distilled water by the two equal polishings of tube reaction system to 25 μ L.Reaction condition, 94
DEG C pre-degeneration 3min；94 DEG C of 30s, 56 DEG C of 15s, 72 DEG C of 30s, 30 cycles；72 DEG C of extension 10min, 4 DEG C of preservations. F1：5'-
GTTGATACCTAATACCTGYCAGC-3', R1：5'-GGACTACCAGGGTATCTAATC-3'； F2：5'-
CCAGCCCTGCGCCGACAA-3', R2：5'-CACCAGGGTCGCCAGCGCTTCA-3'
3. PCR amplification product analysis：
The 4 μ L of final reacting product of PCR amplification are taken, is added and extremely contains 0.5 μ g/mL ethidium bromides (EB)
In 2% Ago-Gel of dyestuff, the electrophoresis 30min under 100V voltages, in gel at imaging amplification production on phase system
The collection of illustrative plates of object.Electrophoresis picture shows 352bp and 385bp signature bands, then result is the positive；Such as without any band, then result
For feminine gender.
The results are shown in Figure 2, and positive control (Aerononas punctata AMth-1 among mandarin sturgeon) display 385bp and 352bp is special
Sign property band, negative (water) control is without apparent band, No. 1-4 sturgeon group for having infected Aerononas punctata AMth-1 among mandarin sturgeon
Tissue samples show 352bp and 385bp signature bands, illustrate that sample 1-4 is that Aerononas punctata is positive among mandarin sturgeon, with reality
Situation is consistent.
Embodiment 7：
Applications of the Aeromonas media AMth-1 in the laboratory reference substance for preparing sturgeon source Aeromonas media：
By sturgeon source Aeromonas media AMth-1 provided by the invention product as a contrast, by other bacteriums to be confirmed and this
The bacterium of invention carries out the comparison on Physiology and biochemistry, and by sample to be tested with sequence shown in SEQ ID NO.1 and SEQ ID NO.2
Row are compared.
Embodiment 8：
Aeromonas media AMth-1 is in preparing the model for screening treatment sturgeon source Aeromonas media bacterium medicine
Using：
1. picking Aeromonas media AMth-1 single bacterium colonies, which are inoculated on BHI fluid nutrient mediums, is enlarged culture, and 28
In DEG C shaking table after culture 15h, 4 DEG C of 4000rpm centrifugations 15min collect bacteriums；
2. the Aeromonas media AMth-1 bacteriums being collected by centrifugation are resuspended in PBS buffer solution, adjustment bacterial suspension is dense
It spends to 1.9 × 109CFU/mL；
3. (body is long for the second filial acipenser dabryanus of selection 30 tails health：3.0±0.8cm；Weight：25.1 ± 3.1g), it is divided into 2
Group, experimental group and each 15 tail of control group；
4. 200 μ L (3.8 × 10 are injected intraperitoneally per tail fish in experimental group8CFU) bacterial suspension, control group is per tail fish intraperitoneal injection
20 0 μ L PBS buffer solution；
5. counting the death rate of the experimental group and control group fish in 7 days, experimental group infectious age is 100% (15 tail),
Control group is without death.
Sequence table
<110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120>A kind of sturgeon source Aeromonas media AMth-1 and PCR detection primer and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1346
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gcgagcggcg gacgggtgag taatgcctgg gaaattgccc agtcgagggg gataacagtt 60
ggaaacgact gctaataccg catacgccct acgggggaaa gcaggggacc ttcgggcctt 120
gcgcgattgg atatgcccag gtgggattag cttgttggtg aggtaatggc tcaccaaggc 180
gacgatccct agctggtctg agaggatgat cagccacact ggaactgaga cacggtccag 240
actcctacgg gaggcagcag tggggaatat tgcacaatgg gggaaaccct gatgcagcca 300
tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact ttcagcgagg aggaaaggtt 360
gatacctaat acctgycagc tgtgacgtta ctcgcagaag aagcaccggc taactccgtg 420
ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg 480
cacgcaggcg gttggataag ttagatgtga aagccccggg ctcaacctgg gaattgcatt 540
taaaactgtc cagctagagt cttgtagagg ggggtagaat tccaggtgta gcggtgaaat 600
gcgtagagat ctggaggaat accggtggcg aaggcggccc cctggacaaa gactgacgct 660
caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 720
gatgtcgatt tggaggctgt gtccttgaga cgtggcttcc ggagctaacg cgttaaatcg 780
accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca 840
agcggtggag catgtggttt aattcgatgc aacgcgaaga accttacctg gccttgacat 900
gtctggaatc ctgtagagat rcgggagtgc cttcgggaat cagaacacag gtgctgcatg 960
gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccctg 1020
tcctttgttg ccagcacgta atggtgggaa ctcaagggag actgccggtg ataaaccgga 1080
ggaaggtggg gatgacgtca agtcatcatg gcccttacgg ccagggctac acacgtgcta 1140
caatggcgcg tacagagggc tgcaagctag cgatagtgag cgaatcccaa aaagcgcgtc 1200
gtagtccgga tcggagtctg caactcgact ccgtgaagtc ggaatcgcta gtaatcgcaa 1260
atcagaatgt tgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1320
gagtgggttg caccagaagt agatag 1346
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acgacgccgc cggtgacggt accaccactg ccaccgtact ggcgcaagcc atcgtcaacg 60
aaggcctgaa ggccgttgcc gccggcatga accccatgga tctgaagcgc ggtatcgaca 120
aggccgtcgt ggcagccgtc gccgagctgc aggccctgtc ccagccctgc gccgacaaca 180
acgccatcgc tcaggtcggt accatctctg ccaactccga cgagaaagtg ggtgcactga 240
tcgccgaagc catggacaag gtcggccgtg acggcgtgat caccgtagaa gacggtcagg 300
gtctggaaga cgagctggct gtcgtcgaag gcatgcagtt cgaccgtggc tacctctccc 360
cctacttcat caacaagccg gaaaccggct ccgtcgagct ggacgacccc ttcatcctgc 420
tggtggacaa gaagatctcc aacatccgcg aaatgctgcc ggtgctggaa ggcgtggcca 480
aggcgggcaa accgctgatc atcgtggccg aagacgtgga aggtgaagcg ctggcgaccc 540
tggtggtcaa caccatgcgc ggcatcgtga aagtcgccgc cgtcaaggcg ccgggcttcg 600
gcgaccgtcg caaggccatg ctgcaggata tc 632
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gttgatacct aatacctgyc agc 23
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggactaccag ggtatctaat c 21
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ccagccctgc gccgacaa 18
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
caccagggtc gccagcgctt ca 22
Claims (10)
1. a kind of Aeromonas media of separation, the Aeromonas media is Aeromonas media（Aeromonas media）AMth-1, deposit number are：CCTCC NO:M2018185.
Sequence shown in 2.SEQ ID NO.1 in the detection between application in Aeromonas, the guarantor of the Aeromonas media
Hiding number is：CCTCC NO:M2018185.
Sequence shown in 3.SEQ ID NO.2 in the detection between application in Aeromonas, the guarantor of the Aeromonas media
Hiding number is：CCTCC NO:M2018185.
4. for the primer of sequence design shown in SEQ ID NO.1 or SEQ ID NO.2.
5. the primer described in claim 4 is preparing the application in detecting Aeromonas media kit, the intermediate gas list
The deposit number of born of the same parents bacterium is：CCTCC NO:M2018185.
6. Aeromonas media detection primer described in claim 1, the primer is F1：5'-
GTTGATACCTAATACCTGYCAGC-3', R1：5'-GGACTACCAGGGTATCTAATC-3'；F2：5'-
CCAGCCCTGCGCCGACAA-3', R2：5'-CACCAGGGTCGCCAGCGCTTCA-3'.
7. application of the primer in preparing Aeromonas media detection kit described in claim 6, the intermediate gas list
The deposit number of born of the same parents bacterium is：CCTCC NO:M2018185.
8. application of the Aeromonas media described in claim 1 in preparing the anti-Aeromonas media vaccine of mandarin sturgeon.
9. application of the Aeromonas media described in claim 1 in preparing sturgeon source Aeromonas media reference substance.
10. Aeromonas media described in claim 1 is preparing screening treatment sturgeon source Aeromonas media bacterium medicine
Application in model.
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| CN114317315A (en) * | 2020-09-30 | 2022-04-12 | 中国科学院分子植物科学卓越创新中心 | Aeromonas intermedia (Aeromonas media) and application thereof |
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