CN112048575A - Primer, probe and detection method for identifying grass carp hemorrhagic fever virus - Google Patents
Primer, probe and detection method for identifying grass carp hemorrhagic fever virus Download PDFInfo
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- CN112048575A CN112048575A CN202011153888.4A CN202011153888A CN112048575A CN 112048575 A CN112048575 A CN 112048575A CN 202011153888 A CN202011153888 A CN 202011153888A CN 112048575 A CN112048575 A CN 112048575A
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- 239000000523 sample Substances 0.000 title claims abstract description 31
- 241000700605 Viruses Species 0.000 title claims abstract description 26
- 206010061192 Haemorrhagic fever Diseases 0.000 title claims abstract description 22
- 241000252230 Ctenopharyngodon idella Species 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 238000001179 sorption measurement Methods 0.000 claims description 36
- 239000007788 liquid Substances 0.000 claims description 12
- 239000002699 waste material Substances 0.000 claims description 9
- 230000009089 cytolysis Effects 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 241000252228 Ctenopharyngodon Species 0.000 claims 2
- 238000003556 assay Methods 0.000 claims 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000702247 Reoviridae Species 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention provides a primer, a probe and a detection method for identifying grass carp hemorrhagic fever viruses.
Description
Technical Field
The invention relates to the field of virus detection, and particularly relates to a primer, a probe and a detection method for identifying grass carp hemorrhagic fever virus.
Background
Grass carp hemorrhagic fever virus belongs to reoviridae, and various organ tissues of infected fishes show congestion type hemorrhage with different degrees. At present, the most effective and reliable detection method of the viruses is fluorescence pcr, relevant detection research of the viruses is not referred in literature, and grass carp hemorrhagic fever virus suppliers on the market comprise Shanghai Kanglang biological science and technology limited company, Shanghai Xuan Yay biological science and technology limited company and the like. The invention aims to develop a method for rapidly and sensitively detecting grass carp hemorrhagic fever viruses, help to find the viruses in the early stage of aquaculture and timely treat and reduce loss.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a detection method capable of effectively evaluating grass carp hemorrhagic fever viruses, a characteristic sequence is found from a gene sequence of the grass carp hemorrhagic fever viruses, a group of upstream and downstream primers which are sensitively combined with target fragment amplification are designed aiming at the sequence, a Fam luminescent group with high fluorescence intensity is marked on a probe, and the detection sensitivity is improved.
The technical scheme of the invention is realized as follows:
a primer for identifying grass carp hemorrhagic fever virus is characterized by comprising the following components:
an upstream primer: 5'-CTTCCGCTATTCCGTCTC-3'
A downstream primer: 5'-CTCCATCATCGTGCTGTT-3'
The further improvement lies in that: the characteristic sequence corresponding to the primer is shown as SEQ ID NO. 1.
The further improvement lies in that: the nucleotide sequence of the probe of the SEQ ID NO.1 characteristic sequence corresponding to the primer is as follows:
5′-Fam CTCTTTCCCCACGTCCACTTTTCC BHQ1-3′
the further improvement lies in that: the probe carries a label.
The further improvement lies in that: the marker is a Fam luminescent group with high fluorescence intensity.
A detection method for identifying grass carp hemorrhagic fever viruses is characterized by comprising the following detection methods:
the method comprises the following steps: taking 200 mul of sample to be detected by using a pipette, and adding the sample into a 1.5ml centrifuge tube;
step two: adding 500 mul of lysis solution into a centrifuge tube, shaking and uniformly mixing, and standing at room temperature;
step three: transferring all the solution in the centrifugal tube into an adsorption column, covering a tube cover, discarding the waste liquid, and returning the adsorption column into a collection tube;
step four: opening the cover of the adsorption column, adding 600 μ l of washing solution, covering the tube cover, centrifuging at 12000 r for 30s, discarding the waste liquid, and returning the adsorption column to the collection tube;
step five: continuously repeating the step 4, opening the cover of the adsorption column, adding 600 μ l of washing solution, covering the tube cover, centrifuging at 12000 r for 30s, discarding the waste liquid, and returning the adsorption column to the collection tube;
step six: placing the adsorption column back into the collection tube, and centrifuging for 2min at 12000 r to remove residual liquid;
step seven: placing the adsorption column into a new 1.5ml centrifuge tube, suspending and dripping 50 μ L of eluent into the middle part of the adsorption membrane, standing at room temperature for 1min, and centrifuging at 12000 r for 30s to obtain RNA/DNA nucleic acid template;
step eight: taking four PCR tubes, and sequentially adding 5 mul of enzyme solution, 18 mul of reaction solution and 2 mul of negative reference/positive reference/extraction sample;
step nine: after the sample is added, the mixture is shaken and mixed evenly, and is centrifuged in a 500g centrifuge for 30s, and finally, the detection result is obtained.
The further improvement is that in the second step, 500 mul of lysis solution is added into a centrifuge tube, after shaking and mixing uniformly, a user selects to stand for 5-10 minutes at room temperature or 5-10 minutes in a 60 ℃ hot bath according to conditions.
In the third step, the solution in the centrifuge tube is completely transferred to the adsorption column and the tube cover is covered, and the centrifuge is kept for 12000 r for 30 s; if flocculent or granular precipitate exists in the solution in the centrifuge tube, centrifuging at 8000 rpm for 2min, and transferring the supernatant to an adsorption column to prevent filter membrane blockage.
In a further improvement, in step seven, 50 μ L of the eluent is added dropwise to the filter of the adsorption column.
Compared with the prior art, the invention has the following advantages.
The invention finds a section of characteristic sequence from the gene sequence of grass carp hemorrhagic fever virus, designs a group of upstream and downstream primers which are sensitively combined with target segment amplification aiming at the sequence, and marks Fam luminescent group with high fluorescence intensity on a probe, thereby improving the detection sensitivity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a table showing the results of PCR amplification according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," "fourth," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Example one
The embodiment of the invention discloses a primer for identifying grass carp hemorrhagic fever viruses, which is characterized by comprising the following components:
an upstream primer: 5'-CTTCCGCTATTCCGTCTC-3'
A downstream primer: 5'-CTCCATCATCGTGCTGTT-3'
The characteristic sequence corresponding to the primer is shown as SEQ ID NO. 1.
The nucleotide sequence of the probe of the SEQ ID NO.1 characteristic sequence corresponding to the primer is as follows:
5′-Fam CTCTTTCCCCACGTCCACTTTTCC BHQ1-3′
the probe carries a label.
The marker is a Fam luminescent group with high fluorescence intensity.
Example two
As shown in fig. 1, a detection method for identifying grass carp hemorrhagic fever virus is characterized by comprising the following detection methods:
the method comprises the following steps: taking 200 mul of sample to be detected by using a pipette, and adding the sample into a 1.5ml centrifuge tube;
step two: adding 500 mul of lysis solution into a centrifuge tube, shaking and uniformly mixing, and standing at room temperature;
step three: transferring all the solution in the centrifugal tube into an adsorption column, covering a tube cover, discarding the waste liquid, and returning the adsorption column into a collection tube;
step four: opening the cover of the adsorption column, adding 600 μ l of washing solution, covering the tube cover, centrifuging at 12000 r for 30s, discarding the waste liquid, and returning the adsorption column to the collection tube;
step five: continuously repeating the step 4, opening the cover of the adsorption column, adding 600 μ l of washing solution, covering the tube cover, centrifuging at 12000 r for 30s, discarding the waste liquid, and returning the adsorption column to the collection tube;
step six: placing the adsorption column back into the collection tube, and centrifuging for 2min at 12000 r to remove residual liquid;
step seven: placing the adsorption column into a new 1.5ml centrifuge tube, suspending and dripping 50 μ L of eluent into the middle part of the adsorption membrane, standing at room temperature for 1min, and centrifuging at 12000 r for 30s to obtain RNA/DNA nucleic acid template;
step eight: taking four PCR tubes, and sequentially adding 5 mul of enzyme solution, 18 mul of reaction solution and 2 mul of negative reference/positive reference/extraction sample;
step nine: after the sample is added, the mixture is shaken and mixed evenly, and is centrifuged in a 500g centrifuge for 30s, and finally, the detection result is obtained.
And in the second step, adding 500 mu l of lysis solution into the centrifuge tube, shaking and uniformly mixing, and allowing the user to stand for 5-10 minutes at room temperature or 5-10 minutes in a 60 ℃ hot bath according to the selection conditions.
In the third step, the solution in the centrifuge tube is completely transferred to the adsorption column and the tube cover is covered, and the centrifuge is kept for 12000 r for 30 s; if flocculent or granular precipitate exists in the solution in the centrifuge tube, centrifuging at 8000 rpm for 2min, and transferring the supernatant to an adsorption column to prevent filter membrane blockage.
In step seven, 50 μ L of the eluent was added dropwise onto the filter of the adsorption column.
The PCR amplification procedure is shown in the following table:
EXAMPLE III
A kit for detecting grass carp hemorrhagic fever virus comprises a reaction solution, an enzyme solution positive sample and a negative sample, wherein the reaction solution contains a probe primer, the enzyme solution is prepared from 50% glycerol, the positive sample contains plasmid, and the negative sample mainly contains water.
Wherein the probe primers and plasmid information are as follows:
the loading of each test sample is shown in the following table:
reaction procedure: cycle 95 ℃ 3min → 35 (95 ℃ 10s → 55 ℃ 10s → 63 ℃ 10s)
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A primer for identifying grass carp hemorrhagic fever virus is characterized by comprising the following components:
an upstream primer: 5'-CTTCCGCTATTCCGTCTC-3'
A downstream primer: 5'-CTCCATCATCGTGCTGTT-3'
2. The primer for identifying the grass carp hemorrhagic fever virus as claimed in claim 1, wherein the characteristic sequence corresponding to the primer is shown as SEQ ID NO. 1.
3. The primer for identifying the grass carp hemorrhagic fever virus as claimed in claim 2, wherein the nucleotide sequence of the probe of the SEQ ID NO.1 characteristic sequence corresponding to the primer is as follows:
5′-Fam CTCTTTCCCCACGTCCACTTTTCC BHQ1-3′
4. the primer for identifying the grass carp hemorrhagic fever virus as claimed in claim 3, wherein the probe is provided with a label.
5. The primer for identifying the hemorrhagic fever viruses in grass carps as claimed in claim 3, wherein the marker is a Fam luminescent group with high fluorescence intensity.
6. A detection method for identifying grass carp hemorrhagic fever viruses is characterized by comprising the following detection methods:
the method comprises the following steps: taking 200 mul of sample to be detected by using a pipette, and adding the sample into a 1.5ml centrifuge tube;
step two: adding 500 mul of lysis solution into a centrifuge tube, shaking and uniformly mixing, and standing at room temperature;
step three: transferring all the solution in the centrifugal tube into an adsorption column, covering a tube cover, discarding the waste liquid, and returning the adsorption column into a collection tube;
step four: opening the cover of the adsorption column, adding 600 μ l of washing solution, covering the tube cover, centrifuging at 12000 r for 30s, discarding the waste liquid, and returning the adsorption column to the collection tube;
step five: continuously repeating the step 4, opening the cover of the adsorption column, adding 600 μ l of washing solution, covering the tube cover, centrifuging at 12000 r for 30s, discarding the waste liquid, and returning the adsorption column to the collection tube;
step six: placing the adsorption column back into the collection tube, and centrifuging for 2min at 12000 r to remove residual liquid;
step seven: placing the adsorption column into a new 1.5ml centrifuge tube, suspending and dripping 50 μ L of eluent into the middle part of the adsorption membrane, standing at room temperature for 1min, and centrifuging at 12000 r for 30s to obtain RNA/DNA nucleic acid template;
step eight: taking four PCR tubes, and sequentially adding 5 mul of enzyme solution, 18 mul of reaction solution and 2 mul of negative reference/positive reference/extraction sample;
step nine: after the sample is added, the mixture is shaken and mixed evenly, and is centrifuged in a 500g centrifuge for 30s, and finally, the detection result is obtained.
7. The detection method for effectively evaluating the hemorrhagic fever viruses of grass carps as claimed in claim 1, wherein in the second step, 500 μ l of lysis solution is added into a centrifuge tube, and after shaking and uniform mixing, a user selects to stand for 5-10 minutes at room temperature or 5-10 minutes in a 60 ℃ hot bath according to conditions.
8. The assay method of claim 1, wherein in step three, the whole solution in the centrifuge tube is transferred to the adsorption column and covered with the tube cover, and the centrifuge is maintained at 12000 rpm for 30 s; if flocculent or granular precipitate exists in the solution in the centrifuge tube, centrifuging at 8000 rpm for 2min, and transferring the supernatant to an adsorption column to prevent filter membrane blockage.
9. The assay method as claimed in claim 1, wherein 50 μ L of the eluate is applied onto the filter of the adsorption column in step seven.
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| CN202011153888.4A CN112048575A (en) | 2020-10-26 | 2020-10-26 | Primer, probe and detection method for identifying grass carp hemorrhagic fever virus |
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| CN202011153888.4A CN112048575A (en) | 2020-10-26 | 2020-10-26 | Primer, probe and detection method for identifying grass carp hemorrhagic fever virus |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831507A (en) * | 2010-05-10 | 2010-09-15 | 中国水产科学研究院长江水产研究所 | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I |
| WO2011041790A1 (en) * | 2009-10-02 | 2011-04-07 | The Trustees Of Columbia University In The City Of New York | Piscine reovirus diagnostic compositions |
| CN102876809A (en) * | 2012-10-18 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | GCRV (grass carp reovirus) RT-PCR (reverse transcription-polymerase chain reaction) detection reagent kit and detection method |
| CN108411041A (en) * | 2018-05-22 | 2018-08-17 | 山东农业大学 | A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application |
| CN108411031A (en) * | 2017-02-09 | 2018-08-17 | 转导精进（武汉）生物技术有限公司 | A kind of detection kit and detection method of grass carp hemorrhage virus |
| CN110894551A (en) * | 2018-09-13 | 2020-03-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type I virus (GCRV-I) |
-
2020
- 2020-10-26 CN CN202011153888.4A patent/CN112048575A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011041790A1 (en) * | 2009-10-02 | 2011-04-07 | The Trustees Of Columbia University In The City Of New York | Piscine reovirus diagnostic compositions |
| CN101831507A (en) * | 2010-05-10 | 2010-09-15 | 中国水产科学研究院长江水产研究所 | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I |
| CN102876809A (en) * | 2012-10-18 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | GCRV (grass carp reovirus) RT-PCR (reverse transcription-polymerase chain reaction) detection reagent kit and detection method |
| CN108411031A (en) * | 2017-02-09 | 2018-08-17 | 转导精进（武汉）生物技术有限公司 | A kind of detection kit and detection method of grass carp hemorrhage virus |
| CN108411041A (en) * | 2018-05-22 | 2018-08-17 | 山东农业大学 | A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application |
| CN110894551A (en) * | 2018-09-13 | 2020-03-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type I virus (GCRV-I) |
Non-Patent Citations (5)
| Title |
|---|
| QING WANG ET AL.: "Comparative Study on Physical–Chemical and Biological Characteristics of Grass Carp Reovirus From Different Genotypes", 《JOURNAL OF THE WORLD AQUACULTURE SOCIETY》 * |
| 刘宝芹 等: "草鱼呼肠孤病毒JX-0901株FQ-PCR检测方法", 《广东农业科学》 * |
| 周勇等: "草鱼呼肠孤病毒TaqManreal-time PCR检测方法的建立", 《水产学报》 * |
| 殷亮 等: "基因Ⅰ型草鱼呼肠孤病毒TaqMan Real-Time PCR检测方法的建立及应用", 《水产学报》 * |
| 范玉顶等: "不同基因型草鱼呼肠孤病毒通用RT-PCR检测方法的建立及应用", 《淡水渔业》 * |
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