CN102382881A - Kit and method for detecting fish pathogenic bacteria - Google Patents
Kit and method for detecting fish pathogenic bacteria Download PDFInfo
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- CN102382881A CN102382881A CN2011103438264A CN201110343826A CN102382881A CN 102382881 A CN102382881 A CN 102382881A CN 2011103438264 A CN2011103438264 A CN 2011103438264A CN 201110343826 A CN201110343826 A CN 201110343826A CN 102382881 A CN102382881 A CN 102382881A
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- 241000949274 Edwardsiella ictaluri Species 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 11
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
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- 208000013223 septicemia Diseases 0.000 abstract 1
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Abstract
The invention provides a kit for detecting fish pathogenic bacteria; the kit comprises a primer with a nucleotide sequence as shown in SEQ ID NO. 1-2, and the primer is used to amplify genes of edwardsiella ictaluri. The invention also provides a method for detecting fish pathogenic bacteria, and an application of the primer shown in SEQ ID NO. 1-2. The kit provided by the invention can rapidly and accurately detect a pathogenic bacterium of ictalurus punctatus - edwardsiella ictaluri, and thus realizes pertinent control and detection of ictalurus punctatus enteric septicemia, and the invention has wide application prospects.
Description
Technical field
The present invention relates to a kind of test kit that detects fish bacterial pathogens, belong to biological technical field.
Background technology
Channel catfish Edward coli (Edwardsiella? Ictaluri) in 1979 for the first time as channel catfish (Ictalurus? Punctatus) aquaculture is a new pathogen has been reported in recent years from diagnosis of diseased fish catfish isolated bacteria 85% of channel catfish Edward's bacteria.
Edwardsiella ictaluri detected traditional way of conventional isolation and identification of bacteria, it takes a longer time, cumbersome procedures, the accuracy is low.In recent years, the method that detects the disease of diagnosis with PCR is risen.Liangwan Wen, "catfish sepsis PCR diagnostic methods to establish a", Dalian Fisheries University, August 2007 Volume 22, No. 4 discloses a PCR method catfish sepsis methods, but the method of detection target is a kind to be validated genes (Edwardsiella? ictaluri? putative? protein, eip), whether it is pathogenic genes are also to be further explored, not representative, and the objects to be detected literature Guangxi Research Institute of fish from the channel catfish Isolation and saved, not the standard strains, can not explain the literature method can accurately detect channel catfish Edward coli.
Urgent need to find an accurate detection method of channel catfish Edward coli.
Summary of the invention
In order to address the above problem, the invention provides a kind of new test kit.
The invention provides a kit for detecting fish pathogens, said kit comprises the nucleotide sequence shown in SEQ? ID? NO.1 ~ 2 shows primers for amplification of Edwardsiella ictaluri genes.
The present invention also provides a kind of method that detects fish bacterial pathogens, and it comprises the steps:
(1) genomic dna of extraction sample to be checked;
(2) be template with aforementioned genomic dna, with the primer amplification shown in SEQ ID NO.1～2;
(3) amplification is detected.
Sample to be checked is fish body or aquaculture water in the said step (1).
Said fish body is muscle, liver, the gill or spleen.
The invention finally provides a SEQ? ID? NO.1 ~ 2 shows the nucleotide sequence of the amplification or detection of Edwardsiella ictaluri from the use of a gene.
The present invention provides a test kit and test method specificity, high sensitivity, you can quickly and accurately determine whether fish or water contaminated by channel catfish Edward coli for catfish farming to provide protection.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Figure 1 Edwardsiella ictaluri conventional PCR primers verify the results.1:DNA; 2: single bacterium colony; 3: blank; M:DNA maker I, this maker have 6 bands, and molecular weight is followed successively by 600bp, 500bp, 400bp, 300bp, 200bp and 100bp from top to bottom.
Figure 2 catfish Edward coli-specific PCR assay system test results.1: control; 2: Pseudomonas fluorescens; 3: river Vibrio; 4: Aeromonas hydrophila; 5: guinea pig Aeromonas; 6: Mild Aeromonas; 7: Flavobacterium; 8: channel catfish Edward coli; M: DNA? marker? I.
Figure 3 channel catfish Edward coli PCR detection system sensitivity test results.1-8: the dna profiling of gradient dilution, concentration are followed successively by 25ng/ μ L-2.5fg/ μ L; 9: blank; M:DNA maker I.
Figure 4 catfish Edward coli PCR assay system sensitivity test results.1-8: the bacterium liquid of gradient dilution, concentration is followed successively by 0.28 * 10
8Cfu/mL-0.28 * 10
1Cfu/mL; 9: blank; M:DNAmaker I.
Fig. 5 channel catfish cause of disease detected result.M:DNA maker I; 1: muscle tissue; 2: muscle tissue; 3: hepatic tissue; 4: muscle tissue; 5: muscle tissue; 6: muscle tissue; 7: muscle tissue; 8: muscle tissue; 9: muscle tissue; 10: blank; 11: the fish gill; 12: the fish gill; 13: spleen; 14: blood; 15: positive control.
Embodiment
Experiment material:
Experimental strains: Flavobacterium, mild Aeromonas, Vibrio river, Pseudomonas fluorescens, guinea pigs, Aeromonas, Aeromonas hydrophila, were purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, channel catfish Edward coli ATCC33202, were purchased from ATCC.
Reagent: 5U/ μ L Taq DNA polymerase (Promega contains 10 * reaction buffer and 25mM Mg2+), 10mM dNTPs (Promega), DNA marker I (Tiangen); Sterile Millipore H2O (providing for oneself); Bacterial genomes DNA extraction test kit (Tiangen).
The experiment of embodiment 1 design of primers
1, design of primers
The conservative gene sequence of searching tarda phosphoserine transaminase from GenBank is according to these sequences Design primers.Which, according to Edwardsiella ictaluri serC gene coding sequence (gene sequence No. AF110153.1, as the causative gene), the design of a pair of primers is:
Ei1F(5-CATGATAATACCCGGTGTTGG-3)
Ei1R(5-GTATTGCTGGGGAACAACTC-3)，
Shown in SEQ ID NO.1 and SEQ ID NO.2, the size of its expection amplified fragments is 124bp.Primer is given birth to the biological Services Co., Ltd of worker by Shanghai and is synthesized.
2, primer checking
(1) template preparation: Preparation of activation and channel catfish Edward coli ATCC? 33202 bacterial suspension, and extracted genomic DNA.DNA adopts the bacterial genomes DNA extraction test kit of Tiangen to extract.
(2) amplification: on increasing appearance, increase in Bio-Rad Mycycler gradient with above-mentioned designed primer;
The PCR reaction system (final concentration) of 20 μ L:
The PCR program is: 95 ℃ of preparatory sex change 4min; 94 ℃ of sex change 15s, 56 ℃ of annealing 10s, 72 ℃ are extended 15s, 38 circulations; 72 ℃ the insulation 5min after under 4 ℃ of conditions termination reaction.
(3) detect: 2% agarose gel electrophoresis detects the PCR product.
3, result
The results shown in Figure 1, lanes 1 and 2 were amplified gene fragment of the expected, but only vague and blank bands of smaller molecular weight, which is a primer-dimer bands, without the expected gene fragment The present invention will be described primers designed to amplify channel catfish Edward coli genome gene fragment is valid.
The specificity of embodiment 2PCR detection architecture and sensitivity detect
1, experimental technique
(1) template preparation: The Flavobacterium, mild Aeromonas, Vibrio river, Pseudomonas fluorescens, guinea pigs, Aeromonas, Aeromonas hydrophila, channel catfish Edward coli genomic DNA as template determination specificity of the method; dilutions of channel catfish Edward coli bacteria (concentration range of 0.28 × 10
8 cfu/mL-0.28 × 10
1 cfu / mL), and the concentration gradient of genomic DNA (concentrations ranged from 25ng/μL ~ 2.5fg/μL) Determination of the sensitivity of the template.The bacterial genomes DNA extraction test kit that DNA adopts Tiangen to provide extracts.
(2) amplification: on increasing appearance, increase in Bio-Rad Mycycler gradient with embodiment 1 designed primer;
The PCR reaction system (final concentration) of 20 μ L:
The PCR program is: 95 ℃ of preparatory sex change 4min; 94 ℃ of sex change 15s, 56 ℃ of annealing 10s, 72 ℃ are extended 15s, 38 circulations; 72 ℃ the insulation 5min after under 4 ℃ of conditions termination reaction.
(3) detect: 2% agarose gel electrophoresis detects pcr amplification product.
2, experimental result
Specificity:
The results shown in Figure 2, Flavobacterium, mild Aeromonas, Vibrio river, Pseudomonas fluorescens, guinea pigs, Aeromonas, Aeromonas hydrophila bacteria several common cause non-target fish There was no target bacteria amplified bands, only vague and smaller molecular weight band, which is a primer-dimer bands, the test results were negative, channel catfish Edward coli have targets amplified bands, positive test results , the present invention provides a primer with good specificity.
Susceptibility:
The results shown in Figure 3-4, the established PCR system for Edwardsiella ictaluri genomic DNA detection limit of 25pg/μL, i.e. the bacteria Edwardsiella ictaluri the detection limit of 0.28 × 10
5 cfu / mL, the sensitivity is excellent.
Experiments show that, under Edwardsiella ictaluri serC coding gene sequence such as SEQ? ID? NO.1 and SEQ? ID? NO.2 primers shown were good specificity and sensitivity, for bacteria Edwardsiella ictaluri testing.
The composition and the method for use of embodiment 3 test kits
1, the composition of test kit
(1), PCR reaction solution (10 μ L/ time): damping fluid, the primer of nucleotide sequence shown in SEQ ID NO:1～2, Mg
2+, dNTPs;
(2), Taq enzyme (0.25 μ L/ time);
(3), 6%chelex-100 (DNA extraction liquid, 200 μ L/ time);
(4), sterilization ultrapure water.
2, method of use
(1) genomic dna of extraction sample to be checked;
(2) be template with the genomic dna that extracts sample to be checked, increase with the aforementioned agents box:
The PCR reaction system (final concentration) of 20 μ L:
The PCR program is: 95 ℃ of preparatory sex change 4min; 94 ℃ of sex change 15s, 56 ℃ of annealing 10s, 72 ℃ are extended 15s, 38 circulations; 72 ℃ the insulation 5min after under 4 ℃ of conditions termination reaction.
(3) 2% agarose gel electrophoresis detect pcr amplification product.
The detection of the sick fish of embodiment 4 channel catfish
1, the DNA of the ill fish of extraction body tissue
From the sick fish sample of the ill channel catfish of cultivation fish pond collection in worksite (Ietalurus Punetaus), the opening aphtha appears in the head of sick fish, the base portion of fin and afterbody, and eyes are outstanding; Branchial plate is scarlet; Belly swelling has flaxen liquid in the body cavity, liver is greyish white.Respectively get a fritter tissue block, add 200 μ l Millipore H respectively
2O mashes, and takes out residual tissue block, and the residue turbid solution is in 12, and the centrifugal 1min of 000r/min abandons supernatant, adds 200 μ l Millipore H
2O is resuspended.Fully get 50 μ l behind the mixing and join among the chelex-100 of 200 μ l 6%, mixing, 56 ℃ of insulation 20min, behind the concuss in 100 ℃ of insulation 8min, concuss, and in 12, the centrifugal 3min of 000r/min gets the template of supernatant as pcr amplification.
2, detect
As template, the test kit and the detection method that provide with embodiment 3 detect with the sample genomic dna that extracts.
3, result
In summary, the experiments show that the present invention provides a kit can quickly and accurately detect channel catfish Edward coli, for targeted treatment and effective prevention of fish diseases provides a reliable diagnostic results for the security of supply of aquatic products to provide a guarantee.
Claims (5)
1 A kit for detecting fish pathogens, characterized in that: said kit comprises the nucleotide sequence shown in SEQ? ID? NO.1 ~ 2 shows primers for amplification of Edwardsiella ictaluri bacterial genes.
2. a method that detects fish bacterial pathogens is characterized in that: comprise the steps:
(1) genomic dna of extraction sample to be checked;
(2) be template with the genomic dna in the step (1), increase with the primer shown in SEQ ID NO.1～2;
(3) amplification is detected.
3. method according to claim 2 is characterized in that: sample to be checked is fish body or aquaculture water in the said step (1).
4. according to the method for claim 3, it is characterized in that: said fish body is muscle, liver, the gill or spleen.
5.SEQ? ID? NO.1 ~ 2 shows the nucleotide sequence of the amplification or detection of Edwardsiella ictaluri from the use of a gene.
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| CN 201110343826 CN102382881B (en) | 2011-11-03 | 2011-11-03 | Kit and method for detecting fish pathogenic bacteria |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132411A (en) * | 2015-06-23 | 2015-12-09 | 通威股份有限公司 | Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri |
| CN115011711A (en) * | 2022-06-02 | 2022-09-06 | 中国科学院水生生物研究所 | Kit and method for visually detecting Edwardsiella ictaluri of zebra fish source through closed tube |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6951726B2 (en) * | 2002-09-24 | 2005-10-04 | Mississippi State University | Real-time PCR assay of the bacterium Edwardsiella ictaluri in channel catfish |
| CN102140513A (en) * | 2010-12-24 | 2011-08-03 | 中国科学院海洋研究所 | Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof |
-
2011
- 2011-11-03 CN CN 201110343826 patent/CN102382881B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6951726B2 (en) * | 2002-09-24 | 2005-10-04 | Mississippi State University | Real-time PCR assay of the bacterium Edwardsiella ictaluri in channel catfish |
| CN102140513A (en) * | 2010-12-24 | 2011-08-03 | 中国科学院海洋研究所 | Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JULIA W. PRIDGEON ET AL.,: "Identification of in vitro upregulated genes in a modified live vaccine strain of Edwardsiella ictaluri compared to a virulent parent strain", 《COMPARATIVE IMMUNOLOGY, MICROBIOLOGY AND INFECTIOUS DISEASE》 * |
| 梁万文 等: "斑点叉尾鮰肠败血症PCR诊断方法的建立", 《大连水产学院学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132411A (en) * | 2015-06-23 | 2015-12-09 | 通威股份有限公司 | Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri |
| CN115011711A (en) * | 2022-06-02 | 2022-09-06 | 中国科学院水生生物研究所 | Kit and method for visually detecting Edwardsiella ictaluri of zebra fish source through closed tube |
| CN115011711B (en) * | 2022-06-02 | 2023-05-26 | 中国科学院水生生物研究所 | Kit and method for visually detecting Edwardsiella ictaluri of zebra fish source |
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| CN102382881B (en) | 2013-07-03 |
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Effective date of registration: 20231111 Address after: No. 588, Middle Tianfu Avenue, Chengdu Hi tech Zone, China (Sichuan) Pilot Free Trade Zone, 610000, Sichuan Patentee after: Tongwei Agricultural Development Co.,Ltd. Address before: 610041, No. 2, pioneering Road, hi tech Zone, Sichuan, Chengdu Patentee before: TONGWEI Co.,Ltd. |