CN108424866B - Acipenser sinensis intermediate aeromonas AMth-1, PCR detection primer and application - Google Patents
Acipenser sinensis intermediate aeromonas AMth-1, PCR detection primer and application Download PDFInfo
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Abstract
The invention belongs to the field of bacteria detection, and particularly discloses sturgeon-derived Aeromonas intermedia AMth-1, a PCR (polymerase chain reaction) detection primer and applicationAeromonas media) AMth-1, primers designed against this bacterial sequence, F1: 5'-GTTGATACCTAATACCTGYCAGC-3', R1: 5'-GGACTACCAGGGTATCTAATC-3', respectively; f2: 5'-CCAGCCCTGCGCCGACAA-3', R2: 5'-CACCAGGGTCGCCAGCGCTTCA-3', detecting the sturgeon source aeromonas intermedia, and the primer has good specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of bacterial molecule detection, and particularly relates to sturgeon-derived Aeromonas intermedia AMth-1, a PCR detection primer and application.
Technical Field
Aeromonas is a group of facultative anaerobic gram-negative bacilli. As early as 1986, Colwell et al have classified the genus Aeromonas from the family Vibrionaceae, and belonged to the newly established family Aeromonas. To date, aeromonas includes: 14 phenotypic species and 16 genetic species such as aeromonas hydrophila, aeromonas caviae, aeromonas sobria, aeromonas veronii and aeromonas intermedia. Aeromonas is widely distributed in the environment and exists as mesophilic bacteria in an aquatic ecosystem. Aeromonas intermedia (Aeromonas m edia), a species of Aeromonas, was first discovered in 1983 in river water, and the presence of the bacterium was subsequently detected in sludge, wastewater, animals, and humans. Aeromonas intermedia is a newly discovered conditioned pathogen that causes infections in humans and animals, the primary symptom of which is diarrhea. The reports of the aeromonas intermedia on the pathogenicity of aquatic animals are relatively few, and only the wangao university and the like (2007) report that the aeromonas intermedia causes the skin rot of the stichopus japonicus; xantho-civilian et al (2013) reported that Aeromonas intermedia causes myxocyprinus pathogenesis; yang-bin et al (2016) verified that Aeromonas intermedia can cause hemorrhagic death of Ictalurus punctatus through pathogen separation, purification, identification, artificial infection and other experiments.
In the application, the applicant finds the aeromonas intermedia infection in the artificially cultured Chinese sturgeon for the first time, and the infection symptoms are mainly superficial hemorrhage and extensive hemorrhage of internal organs. Chinese sturgeons and acipenser dabryanus are the oldest fishes existing in Yangtze river basin in China, belong to the national first-level important protection of wild animals, and the discovery of intermediate aeromonas species poses a potential threat to the species protection of the two sturgeons. Therefore, the sturgeon-derived aeromonas intermedia is of great significance for further research.
Currently, research on the genus aeromonas is mainly focused on aeromonas hydrophila (aeromonas hydrophila), while research on intermediate aeromonas is very little. There is no public report of sturgeon-derived aeromonas intermedia, and no effective detection method exists.
Disclosure of Invention
The invention aims to provide sturgeon-derived Aeromonas intermedia (Aeromonas media) AMth-1, which is isolated from pathological change tissues of Chinese sturgeons, is delivered to a Chinese type culture collection for preservation in 2018, 4 and 9 months, and is classified and named as follows: aeromonas intermedia (Aeromonas media) AMth-1 with a deposit number: CCTC NO: M2018185, address: wuhan university in Wuhan, China.
The other purpose of the invention is to provide the specific sequences 16SrRNA and c pn60 of the sturgeon-derived Aeromonas intermedia AMth-1, wherein the nucleotide sequence of the 16SrRNA is shown as SEQ ID NO.1, and the nucleotide sequence of cpn60 is shown as SEQ ID number 2.
The invention also aims to provide a primer for detecting sturgeon-derived aeromonas intermedia AMth-1, wherein the primer is F1: 5'-GTTGATACCTAATACCTGYCAGC-3', R1: 5'-GGACTACCAGGGTATCTAATC-3', respectively; f2: 5'-CCAGCCCTGCGCCGACAA-3', R2: 5'-CACCAGGGTCGCCAGCGCTTCA-3' are provided.
The invention also aims to provide application of the sturgeon-derived intermediate aeromonas AMth-1, which comprises preparing an intermediate aeromonas bacterium vaccine by using the bacterium, or preparing a laboratory reference substance of the sturgeon-derived intermediate aeromonas bacterium, or preparing a model for screening a medicament for treating the sturgeon-derived intermediate aeromonas bacterium.
The last purpose of the invention is to provide the application of the PCR detection primer of the sturgeon-derived Aeromonas intermedia AMth-1, which comprises preparing a sturgeon-derived Aeromonas detection kit by using the primer.
In order to achieve the purpose, the invention adopts the following technical measures:
an aeromonas intermedia AMth-1, wherein the bacteria are separated from pathological liver tissues of Acipenser sinensis, and the bacteria are delivered to a China center for type culture collection for storage at 2018, 4, 9 and are classified and named: aeromonas intermedia (Aeromonas media) AMth-1 with a deposit number: CCTCC NO, M2018185, address: wuhan university in Wuhan, China.
The Aeromonas intermedia AMth-1 (the invention or called AMth-1) is inoculated to BHI (BD, Becto Brain Heart Infusion) solid agar culture medium for amplification culture, after 24 hours of culture in an incubator at 28 ℃, single colonies are selected to be subjected to secondary streak culture on the BHI solid agar culture medium under the same conditions, and are preserved in glycerol at-80 ℃.
Morphological characteristics of Aeromonas intermedia AMth-1: the size of the cells is (1-4) mum x (0.1-1)/mum, the two ends of the cells are rounded, and the cells are unipolar flagella and inactive to movement. No spore, narrow capsule, gram negative bacillus brevis.
The application of the intermediate aeromonas AMth-1 comprises the step of preparing intermediate aeromonas vaccine by using the bacterium, or preparing sturgeon-derived intermediate aeromonas laboratory reference substance, or preparing a model for screening sturgeon-derived intermediate aeromonas bacterial drugs.
The sturgeon-derived intermediate aeromonas AMth-1 has the specific sequences of 16SrRNA and cpn60, the nucleotide sequence of 16SrRNA is shown as SEQ ID NO.1, and the nucleotide sequence of cpn60 is shown as SEQ ID NO. 2.
The application of the specific sequence of the sturgeon-derived intermediate aeromonas AMth-1 comprises the step of utilizing a 16SrRNA and/or cpn60 sequence to detect or distinguish the sturgeon-derived intermediate aeromonas AMth-1.
Primers designed aiming at the specific sequence 16SrRNA and/or cpn60 of the Acipenser sinensis AMth-1 also belong to the protection scope of the invention, and the primers include but are not limited to fluorescent quantitative primers, LAMP primers, digital PCR primers and the like designed by utilizing the prior art based on the difference sequence.
A PCR detection primer for Acipenser sinensis intermediate Aeromonas AMth-1 comprises F1: 5'-GTTGATACCTAATACCT GYCAGC-3', R1: 5'-GGACTACCAGGGTATCTAATC-3', respectively; f2: 5'-CCAGCCCTGCGCCGAC AA-3', R2: 5'-CACCAGGGTCGCCAGCGCTTCA-3' are provided.
An application of PCR detection primers for sturgeon-derived Aeromonas intermedia AMth-1 comprises preparing a detection kit for detecting Aeromonas intermedia by using primers designed aiming at a specific sequence 16SrRNA and/or cpn60 of the Acipenser-derived Aeromonas intermedia AMth-1.
Preferably, when prepared as a kit, the non-diagnostic detection method of the kit comprises:
1) extracting DNA of a sample to be detected;
2) PCR system
A 25 μ L reaction was used: two pairs of primers F1 and R1 or F2 and R2 with the concentration of 10 mu M are respectively added into two PCR reaction tubes by 0.5 mu L, and 2 mu L of 2.5mM dNTPs and 25mM MgCl are respectively added into the two PCR reaction tubes2mu.L of 1.5. mu.L, 0.5. mu.L of 5U Taq polymerase, 1. mu.L of template cDNA, 2.5. mu.L of 10 × ThermoPol Reaction Buffer, and both Reaction tubes were filled to 25. mu.L with sterilized double distilled water. Reaction conditions, pre-denaturation at 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 15s and 72 ℃ for 30 s; extending for 10min at 72 ℃, and storing at 4 ℃.
3) Determination of detection result
mu.L of the final reaction product amplified by the polymerase chain reaction is added into 2% agarose gel containing 0.5. mu.g/mL Ethidium Bromide (EB) dye, electrophoresis is carried out for 30min under the voltage of 100V, and the pattern of the amplified product is observed by imaging on a gel phase forming system. Displaying 352bp and 385bp characteristic strips by an electrophoresis picture, and determining that the result is positive; if there is no band, the result is negative.
Compared with the prior art, the invention has the following advantages:
1. the invention separates the intermediate aeromonas from the Chinese sturgeon body for the first time, and has pioneering significance for the research of sturgeon source aeromonas.
1. The method of the invention is rapid, simple and convenient: the PCR method established by the invention only needs hours, and greatly improves the detection efficiency compared with the traditional bacteria separation method for days.
2. The method of the invention has strong specificity: the recognition of the specific sequence region of the target sequence by the two pairs of primers ensures the high specificity of the PCR amplification.
Drawings
FIG. 1 is a diagram showing the results of the sensitivity test of two pairs of primers provided by the present invention;
wherein, lane 1: original template, lane 2: 10-fold dilution, lane 3: 100-fold dilution, lane 4: 1000-fold dilution, lane 5: 10000-fold dilution, lane 6: 100000-fold dilution, lane 7: 1000000-fold dilution.
FIG. 2 is a schematic diagram showing the result of detecting sturgeon tissues infected with Acipenser intermedius AMth-1 by using the primers provided by the invention;
wherein lane P: a positive control; n: negative control (template sterile water) M: 2000bp marker (2000 bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom)
Lane 1: a sample 1 to be tested;
lane 2: a sample to be tested 2;
lane 3: a sample to be tested 3;
lane 4: sample 4 to be tested.
Detailed Description
The technical scheme of the invention is a conventional scheme in the field if not specifically stated, and the used reagents or raw materials are purchased from commercial sources or published if not specifically stated.
Example 1:
a sturgeon-derived aeromonas intermedia AMth-1 is isolated as follows:
1) taking out and grinding tissues such as Chinese sturgeon liver, spleen and the like carrying bacteria, diluting by 100 times with sterile PBS, centrifuging at 2000 rpm, taking supernatant, filtering, inoculating to a BHI (Becto Brain Heart Infusion) solid agar culture medium for isolated culture, and culturing in an incubator at 28 ℃ for 24 hours to obtain white semitransparent colonies in a culture dish.
2) Single colonies were picked and gram stained for bacterial morphology.
3) The bacteria were collected and subjected to 16S rRNA and cpn60 amplification and sequencing, with the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
The morphological characteristics of Chinese sturgeon Aeromonas intermedia are as follows: belongs to gram negative brevibacterium. The size is (1-4) mum multiplied by (0.1-1)/mum, the two ends of the thallus are round, the single-pole flagellum is inactive to movement. No spores, narrow capsule. The bacteria are sent to China center for type culture Collection for preservation in 2018, 4, 9 and are classified and named: aeromonas intermedia (Aeromonas media) AMth-1 with a deposit number: CCTCC NO, M2018185, address: wuhan university in Wuhan, China.
Example 2:
primers were designed based on differences between 16SrDNA (SEQ ID NO.1) and cpn60(SEQ ID NO.2) of Acipenser sinensis-derived Aeromonas intermedia AMth-1 and corresponding sequences of other bacteria:
the invention is explained by taking common PCR as an example, and other primers, fluorescent quantitative primers, LAMP primers, digital PCR primers and the like designed based on the difference sequence can also complete the detection of the sturgeon-derived Aeromonas intermedia AMth-1.
A PCR detection primer for Acipenser sinensis intermediate Aeromonas AMth-1 comprises F1: 5'-GTTGATACCTAATACCT GYCAGC-3', R1: 5'-GGACTACCAGGGTATCTAATC-3', respectively; f2: 5'-CCAGCCCTGCGCCGAC AA-3', R2: 5'-CACCAGGGTCGCCAGCGCTTCA-3' are provided.
Example 3:
the detection of the sturgeon-derived intermediate aeromonas AMth-1 by using the intermediate aeromonas AMth-1PCR detection primer comprises the following steps:
1. extracting Chinese sturgeon tissue DNA: and extracting the total DNA of the sample to be detected by adopting a commercial genome extraction kit, and storing the obtained DNA template at-20 ℃ for later use.
2. Polymerase chain reaction amplification:
a 25 μ L reaction was used: two pairs of primers F1, R1 or F2, R2 with a concentration of 10. mu.M, respectively, 0.5. mu.L of each primer was added to two PCR reaction tubes, 2. mu.L of 2.5mM dNTPs and 25mM MgCl were added to the tubes2mu.L of 1.5. mu.L, 0.5. mu.L of 5U Taq polymerase, 1. mu.L of template cDNA, 2.5. mu.L of 10 XThermoPol Reaction Buffer, and both Reaction systems were filled to 25. mu.L with sterilized double distilled water. Reaction conditions, pre-denaturation at 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 15s and 72 ℃ for 30 s; extending for 10min at 72 ℃, and storing at 4 ℃.
F1：5'-GTTGATACCTAATACCTGYCAGC-3'，R1：5'-GGACTACCAGGGTATCTAATC-3'；
F2：5'-CCAGCCCTGCGCCGACAA-3'，R2：5'-CACCAGGGTCGCCAGCGCTTCA-3'
3. Analysis of polymerase chain reaction amplification products:
taking 4 mu L of final reaction product amplified by the polymerase chain reaction, adding the final reaction product into 2% agarose gel containing 0.5 mu g/mL Ethidium Bromide (EB) dye, carrying out electrophoresis for 30min under the voltage of 100V, and imaging and observing the spectrum of the amplified product on a gel phase forming system. If the F1 and the R1 can amplify 352bp and the F2 and the R2 can amplify 385bp characteristic bands, the sample is positive; if there is no band, it is negative.
Example 4:
acipenser sinensis intermediate aeromonas AMth-1PCR detection primer specificity test
By using the method described in embodiment 3, a sturgeon source aeromonas intermedia AMth-1 sample to be detected in table 1 is detected, and a negative control group (double distilled water) and a positive control group (AMth-1) are simultaneously set, wherein the bacterial sample to be detected specifically comprises: aeromonas hydrophila (AHth-1) (immune response reaction and protection effect after the inactivated vaccine of aeromonas hydrophila is used for immunizing sturgeon, pericourage), aeromonas acipensis sinensis (AVth-1), Elispot (EM) in Chinese sturgeon meninges, and aeromonas sobria (ASth-1).
TABLE 1 sturgeon-derived Aeromonas intermedia AMth-1PCR detection kit specificity test
The results are shown in Table 1, only the positive control group (AMth-1) can detect characteristic bands of 352bp and 385bp, and the rest groups have no characteristic band, which indicates that the detection primer of the sturgeon-derived Aeromonas intermedia AMth-1 has strong specificity.
Example 5:
sensitivity of PCR detection primer for Acipenser sinensis intermediate aeromonas AMth-1
The AMth-1DNA template with the concentration of 2000 ng/. mu.L is diluted by 10 times of gradient, the detection limit of PCR using the primer provided by the invention is detected by the method of example 3, and the result is shown in FIG. 1, which shows 105The double dilution also detected the band of interest, i.e., the lowest detectable 20 pg/. mu.L cDNA template.
Example 6:
the application of the PCR detection primer of the sturgeon-derived intermediate aeromonas AMth-1 in the preparation of the sturgeon-derived intermediate aeromonas detection kit comprises the following steps:
1. extracting total DNA of Chinese sturgeon tissues: and extracting the total DNA of the sample to be detected by adopting a commercial genome extraction kit, and storing the obtained DNA template at-20 ℃ for later use.
2. Polymerase chain reaction amplification:
a 25 μ L reaction was used: two pairs of primers F1, R1 or F2, R2 with a concentration of 10. mu.M, respectively, were added to two PCR reaction tubes in an amount of 0.5. mu.L, respectively, and 2. mu.L of 2.5mM dNTPs, respectively, and 25mM MgCl were added thereto in an amount of 2. mu.L2mu.L of 1.5. mu.L, 0.5. mu.L of 5U Taq polymerase, 1. mu.L of template cDNA, 2.5. mu.L of 10 XBuffer, sterilizedThe reaction system of both tubes was filled to 25. mu.L with double distilled water. Reaction conditions, pre-denaturation at 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 15s and 72 ℃ for 30 s; extending for 10min at 72 ℃, and storing at 4 ℃. F1: 5'-GTTGATACCTAATACCTGYCAGC-3', R1: 5'-GGACTACCAGGGTATCTAATC-3', respectively; f2: 5'-CCAGCCCTGCGCCGACAA-3', R2: 5'-CACCAGGGTCGCCAGCGCTTCA-3'
3. Analysis of polymerase chain reaction amplification products:
mu.L of the final reaction product amplified by the polymerase chain reaction is taken and added into 2 percent agarose gel containing 0.5 mu g/mL Ethidium Bromide (EB) dye, electrophoresis is carried out for 30min under the voltage of 100V, and the spectrum of the amplified product is observed by imaging on a gel phase forming system. Displaying 352bp and 385bp characteristic strips by an electrophoresis picture, and determining that the result is positive; if there is no band, the result is negative.
The results are shown in figure 2, positive control (Acipenser sinensis intermediate aeromonas AMth-1) shows characteristic bands of 385bp and 352bp, negative (water) control has no obvious band, and sturgeon tissue samples infected with Acipenser sinensis intermediate aeromonas AMth-1 in No. 1-4 show characteristic bands of 352bp and 385bp, which indicates that the samples 1-4 are positive for Acipenser sinensis intermediate aeromonas and are consistent with the actual situation.
Example 7:
the application of the aeromonas intermedia AMth-1 in preparing sturgeon-derived aeromonas intermedia laboratory reference substances comprises the following steps:
taking the sturgeon-derived Aeromonas intermedia AMth-1 provided by the invention as a reference substance, performing physiological and biochemical comparison on other bacteria to be confirmed and the bacteria of the invention, and comparing a sample to be detected with sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.
Example 8:
the application of the intermediate aeromonas AMth-1 in preparing a model for screening a medicament for treating sturgeon-derived intermediate aeromonas bacteria is as follows:
1. selecting single colony of Aeromonas intermedia AMth-1 to inoculate to BHI liquid culture medium for amplification culture, culturing in a shaking table at 28 ℃ for 15h, and centrifuging at 4 ℃ and 4000rpm for 15min to collect bacteria;
2. resuspending the centrifugally collected Aeromonas intermedia AMth-1 bacteriaThe bacterial suspension concentration was adjusted to 1.9X 10 in PBS buffer9CFU/mL；
3. Selecting 30 healthy second-generation Acipenser dabryanus (body length: 3.0 +/-0.8 cm; body weight: 25.1 +/-3.1 g) and dividing into 2 groups, wherein 15 pieces of Acipenser dabryanus are respectively selected from an experimental group and a control group;
4. experiment group Each fish intraperitoneal injection is 200 mu L (3.8X 10)8CFU) bacterial suspension, and 200 mu L PBS buffer solution is injected into the abdominal cavity of each fish in the control group;
5. the mortality rate of the fish in the experimental group and the fish in the control group within 7 days is counted, the infection mortality rate of the experimental group is 100 percent (15 tails), and the fish in the control group does not die.
Sequence listing
<110> Changjiang aquatic products institute of aquatic science and research in China
<120> sturgeon-derived Aeromonas intermedia AMth-1, PCR detection primer and application
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gcgcgattgg atatgcccag gtgggattag cttgttggtg aggtaatggc tcaccaaggc 180
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actcctacgg gaggcagcag tggggaatat tgcacaatgg gggaaaccct gatgcagcca 300
tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact ttcagcgagg aggaaaggtt 360
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agcggtggag catgtggttt aattcgatgc aacgcgaaga accttacctg gccttgacat 900
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cctacttcat caacaagccg gaaaccggct ccgtcgagct ggacgacccc ttcatcctgc 420
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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 (5)
1. An isolated Aeromonas intermedia (Aeromonas media) The preservation number of the aeromonas intermedia is as follows: CCTCC NO: M2018185.
2. The application of the primer combination in preparing the kit for detecting the Aeromonas intermedia is disclosed, wherein the preservation number of the Aeromonas intermedia is as follows: CCTCC NO of M2018185; the primer combination is as follows: f1: 5'-GTTGATACCTAATACCTGYCAGC-3', R1: 5'-GGACTACCAGGGTATCTAATC-3', respectively; and F2: 5'-CCAGCCCTGCGCCGACAA-3', R2: 5'-CACCAGGGTCGCCAGCGCTTCA-3' are provided.
3. Use of Aeromonas intermedia of claim 1 in the preparation of an Acipenser sinensis anti-Aeromonas intermedia vaccine.
4. Use of Aeromonas intermedia of claim 1 in the preparation of a sturgeon-derived control of Aeromonas intermedia.
5. Use of Aeromonas intermedia according to claim 1 for the preparation of a model for screening a medicament for the treatment of sturgeon-derived Aeromonas intermedia bacteriosis.
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| CN111926094B (en) * | 2020-07-17 | 2022-07-22 | 电子科技大学中山学院 | Bar code identification primer, identification method and kit for different species in aeromonas |
| CN114317315B (en) * | 2020-09-30 | 2023-12-22 | 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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