CN108130376B - Loop-mediated isothermal amplification detection primer group, detection method and detection kit for vibrio winogrisei - Google Patents
Loop-mediated isothermal amplification detection primer group, detection method and detection kit for vibrio winogrisei Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification detection primer group, a detection method and a detection kit of vibrio wenshui. The detection primer group is as follows: front outer primer F3: 5'-CATACAGCTCTGCGTCTG-3', respectively; front inner primer FIP: 5'-GCGG ATAGCGTCTGCCCACTTCACCGTGCTTATGTA-3', respectively; and (3) a rear inner primer BIP: 5'-GCCAAGGCCGTGTA GTTATCTTGCTGCTGAATCTGTAGTGAA-3', respectively; front loop primer LF: 5'-CGGAATTGCAGACATTAC GC-3', respectively; rear loop primer LB: 5'-CTCTGCAGGTACTGGTAACC-3' are provided. The detection primer group, the detection kit and the detection method have the advantages of strong specificity, convenient and simple operation, short detection time and the like, do not need expensive instruments and equipment in the reaction process, have visual reaction results, can be judged directly by visual observation, and are suitable for popularization and application of on-site rapid detection.
Description
The technical field is as follows:
the invention belongs to the field of microbial detection, and particularly relates to a loop-mediated isothermal amplification detection primer group, a detection method and a detection kit for vibrio wenshuni.
Background art:
vibrio euryphi (Vibrio owensii) is an intestinal pathogenic bacterium with strong toxicity in the sea, can cause acute hepatopancreas necrosis of prawns, can also induce the rapid pathological change death of malpighia virens in the period of phylliform larvae, is also a pathogenic bacterium of rosaceous corals, can infect the corals, and causes the death of the corals in albino. The reports on the pathogenic bacteria and the detection and control of the vibrio winii are not common, and the early detection method of the vibrio winii only stays in some more traditional microorganism classification and identification and traditional PCR detection technologies. Recently, a new development is made on a detection method of vibrio euryphi, and an isothermal recombinant polymerase amplification method can accurately and quickly detect the vibrio euryphi, but the method needs an isothermal amplification fluorescent detection system to detect and record the change of a fluorescent signal, and a quick and simple method is often needed for field base layer field detection, so that expensive instruments and equipment and a complicated operation process are avoided as much as possible.
The loop-mediated isothermal amplification (LAMP) technology is a constant temperature nucleic acid amplification method developed by Notomi et al in 2000. The method is mainly characterized in that the LAMP primer design mainly aims at 6 different regions of a target gene, 4 specific primers are designed, and partial software can be improved to design 6 specific primers, so that the synthesis speed of nucleic acid in the reaction is accelerated. The DNA template is amplified for 30-60min at the constant temperature of 65 ℃ under the action of strand displacement DNA Polymerase (Bst DNA Polymerase), so that a large amount of nucleic acid can be synthesized, and the method has the advantages of simplicity and convenience in operation, rapidness, strong specificity, capability of judging results by naked eyes and the like, and has a very wide application prospect.
The invention content is as follows:
the invention aims to overcome the defects of the prior art and provide a vibrio avii loop-mediated isothermal amplification detection primer group, a detection method and a detection kit, which have strong specificity and are convenient and simple to operate and do not need expensive instruments and equipment.
The invention utilizes the loop-mediated isothermal amplification technology, takes the pryH gene (Genbank accession number is GU111253.1) of the vibrio wenshuni as a specific target gene, designs, screens and optimizes a specific primer, establishes an LAMP detection method, optimizes reaction conditions, adds a positive control group and a negative control group in the reaction process, and analyzes and detects the amplification products of a sample group and other two groups of controls by a calcein color development method and gel electrophoresis to judge whether the sample contains the vibrio wenshuni or not, thereby achieving the purposes of convenience, simplicity, economy, rapidness and sensitivity.
The first purpose of the invention is to provide a loop-mediated isothermal amplification detection primer group of vibrio wenshuni, wherein the detection primer group is shown as follows:
front outer primer F3: 5'-GCGTAGTAGGCGACCA-3' (shown in SEQ ID NO. 1);
rear outer primer B3: 5'-CATACAGCTCTGCGTCTG-3' (shown in SEQ ID NO. 2);
front inner primer FIP: 5'-GCGGATAGCGTCTGCCCACTTCACCGTGCTTATGTA-3' (shown in SEQ ID NO. 3);
and (3) a rear inner primer BIP: 5'-GCCAAGGCCGTGTAGTTATCTTGCTGCTGAATCTGTAGTGAA-3' (shown in SEQ ID NO. 4);
front loop primer LF: 5'-CGGAATTGCAGACATTACGC-3' (shown in SEQ ID NO. 5);
rear loop primer LB: 5'-CTCTGCAGGTACTGGTAACC-3' (shown in SEQ ID NO. 6).
The second purpose of the invention is to provide a loop-mediated isothermal amplification detection kit for vibrio Erwinii, which comprises loop-mediated isothermal amplification reaction liquid, Bst DNA polymerase and MnCl2The kit comprises a solution, a calcein color development solution, a positive control quality control substance, a negative control quality control substance and a detection primer group, wherein the detection primer group is as follows:
front outer primer F3: 5'-GCGTAGTAGGCGACCA-3' (shown in SEQ ID NO. 1);
rear outer primer B3: 5'-CATACAGCTCTGCGTCTG-3' (shown in SEQ ID NO. 2);
front inner primer FIP: 5'-GCGGATAGCGTCTGCCCACTTCACCGTGCTTATGTA-3' (shown in SEQ ID NO. 3);
and (3) a rear inner primer BIP: 5'-GCCAAGGCCGTGTAGTTATCTTGCTGCTGAATCTGTAGTGAA-3' (shown in SEQ ID NO. 4);
front loop primer LF: 5'-CGGAATTGCAGACATTACGC-3' (shown in SEQ ID NO. 5);
rear loop primer LB: 5'-CTCTGCAGGTACTGGTAACC-3' (shown in SEQ ID NO. 6).
The positive control quality control substance is preferably plasmid DNA containing pryH gene (Genbank accession number GU111253.1) of Vibrio eurwenshuri, and can be obtained by artificial synthesis or amplification from genomic DNA of Vibrio eurwenshuii.
The negative control quality control product is preferably DNA or ultrapure water which does not contain the pryH gene of the vibrio wengii. The DNA not containing the pryH gene of Vibrio euryphii may be the pryH gene of other bacteria or ultrapure water. Such as Vibrio corallini (Vibrio coralliilyticus); vibrio lonicera (Vibrio shilonii); vibrio alginolyticus (Vibrio alginolyticus); vibrio furnissi (Vibrio furnissi); vibrio fluvialis (Vibrio fluvialis); anti-quarius; (ii) a bacillus aerophilus; pseudomonas fluorescens; staphylococcus aureus bacteria; ultrapure water.
The third purpose of the invention is to provide a loop-mediated isothermal amplification detection method of vibrio wenshuni for non-disease diagnosis and treatment purposes, which comprises the following steps:
(1) enrichment culture is carried out on the sample to obtain enrichment liquid, and genome DNA in the enrichment liquid is extracted to be used as a template;
(2) the primer group for loop-mediated isothermal amplification detection of the vibrio Erwinii, the loop-mediated isothermal amplification reaction solution, Bst DNA polymerase and MnCl are used2Mixing the solution, the calcein color development solution and the sample genome DNA to form a loop-mediated isothermal amplification reaction system, and carrying out loop-mediated isothermal amplification reaction;
(3) and after the amplification reaction is finished, detecting whether the sample contains the vibrio wenshuriei or not by calcein fluorescence color development method and/or gel electrophoresis analysis.
The detection of whether the sample contains the vibrio oweringi through the calcein fluorescence color development method specifically comprises the following steps: if the color of the amplification product is bright green, the sample contains vibrio erwinii; if the color of the amplification product remains the same as the orange yellow, the sample does not contain Vibrio ohwii.
The specific steps for detecting whether the sample contains the vibrio oweringi through gel electrophoresis analysis are as follows: performing gel electrophoresis on the amplification product, wherein if a ladder-shaped strip exists, the sample contains vibrio oweringi; if no ladder-shaped strip exists, the sample does not contain Vibrio ohwii.
Preferably, the total volume of the loop-mediated Isothermal amplification reaction system is 25 mu L, and the loop-mediated Isothermal amplification reaction system comprises 1 xIsotermal Amp Buffer and 6mmol/L MgSO4、1.4mmol/L dNTP Mix、1.6μmol/L FIP、1.6μmoL/L BIP, 0.2 mu mol/L F3, 0.2 mu mol/L B3, 0.4 mu mol/L LF, 0.4 mu mol/L LB, 320U/mL Bst 2.0DNA Polymerase, 1.2mol/L betaine, 1mmol/L manganese chloride, 1mmol/L calcein 2 mu L, and 1 mu L genomic DNA template, the balance being deionized water.
The reaction conditions of the loop-mediated isothermal amplification reaction are preferably as follows: the reaction was carried out at 65 ℃ for 60min and at 80 ℃ for 10 min.
The invention designs a specific primer group according to the housekeeping gene pyrH of the Vibrio wenshui (Vibrio owensii), has good specificity and can be used for detecting the Vibrio wenshui. The invention designs and screens a set of specific detection primer group, a detection kit containing the primer group and a detection method using the detection kit through loop-mediated isothermal amplification, and further determines whether vibrio ovirens exists in a detected sample. The detection primer group, the detection kit and the detection method have the advantages of strong specificity, convenient and simple operation, short detection time, no need of expensive instruments and equipment and the like, are particularly suitable for field detection of field substrates, and have wide application prospect.
Description of the drawings:
FIG. 1 is a color development analysis result diagram of LAMP amplification products in a pure bacteria specificity experiment; 1. a recombinant plasmid containing the pyrH gene of Vibrio euryphii; 2. vibrio ornithopteri (Vibrio owensii); 3. vibrio corallini (Vibrio coralliilyticus); 4. vibrio lonicera (Vibrio shilonii); 5. vibrio alginolyticus (Vibrio alginolyticus); 6. vibrio furnissi (Vibrio furnissi); 7. vibrio fluvialis (Vibrio fluvialis); vibrio anticiquaria; 9. (ii) a bacillus aerophilus; 10. pseudomonas fluorescens; 11. staphylococcus aureus bacteria; 12. ultrapure water; wherein 1 and 2 are bright green, and 3-12 are orange yellow.
FIG. 2 is a gel electrophoresis result of LAMP amplification products in a pure bacteria specificity experiment; m. standard molecular weight DL 2000; 1. a recombinant plasmid containing the pyrH gene of Vibrio euryphii; 2. vibrio ornithopteri (Vibrio owensii); 3. vibrio corallini (Vibrio coralliilyticus); 4. vibrio lonicera (Vibrio shilonii); 5. vibrio alginolyticus (Vibrio alginolyticus); 6. vibrio furnissi (Vibrio furnissi); 7. vibrio fluvialis (Vibrio fluvialis); v.aniquarius; 9. (ii) a bacillus aerophilus; 10. pseudomonas fluorescens; 11. staphylococcus aureus bacteria; 12. ultrapure water.
FIG. 3 is a result diagram of the chromogenic analysis of the LAMP amplification product in the sensitivity test of the recombinant plasmid pClone 007-pyrH; 1 to 12.6.19 x 1011～6.19×100(ii) a And N, ultrapure water.
FIG. 4 is a diagram showing the results of gel electrophoresis of LAMP amplification products in a sensitivity experiment of the recombinant plasmid pClone 007-pyrH; m. standard molecular weight DL 2000; 1 to 12.6.19 x 1011～2.6.19×100(ii) a And N, ultrapure water.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: design and Synthesis of primers
According to the design principle of the loop-mediated isothermal amplification primers, a target gene sequence of Vibrio euryphi (Vibrio owensii) suitable for loop-mediated isothermal amplification is searched in an NCBI Genebank database, and finally a pyrH gene (Genebnak accession number GU111253.1) with stronger specificity is selected as a target gene. According to the specific region of the pyrH gene of the vibrio wengii and the design principle of the loop-mediated isothermal amplification primers, 6 sets of primers are designed by LAMP Primer design software Primer Designer, and a Primer group with the best specificity is screened out.
The loop-mediated isothermal amplification detection primer of the vibrio wenshuii designed by the method comprises the following steps:
front outer primer F3: 5'-GCGTAGTAGGCGACCA-3' (shown in SEQ ID NO. 1);
rear outer primer B3: 5'-CATACAGCTCTGCGTCTG-3' (shown in SEQ ID NO. 2);
front inner primer FIP: 5'-GCGGATAGCGTCTGCCCACTTCACCGTGCTTATGTA-3' (shown in SEQ ID NO. 3);
and (3) a rear inner primer BIP: 5'-GCCAAGGCCGTGTAGTTATCTTGCTGCTGAATCTGTAGTGAA-3' (shown in SEQ ID NO. 4);
front loop primer LF: 5'-CGGAATTGCAGACATTACGC-3' (shown in SEQ ID NO. 5);
rear loop primer LB: 5'-CTCTGCAGGTACTGGTAACC-3' (shown in SEQ ID NO. 6).
Example 2: nucleic acid extraction
Respectively inoculating Vibrio euryphi (Vibrio owensis) and Vibrio corallini (Vibrio corallinicus), Vibrio lonicera (Vibrio shilonii), Vibrio alginolyticus (Vibrio alginolyticus), Vibrio furnissii (Vibrio furnissii), Vibrio fluvialis (Vibrio fluvialis) and V.antiadiquad into a TSB liquid medium containing 1.5% NaCl, and culturing at 30 ℃ overnight; inoculating Bacillus aerophilus, Pseudomonas fluorescens and Staphylococcus aureus to LB liquid culture medium, and culturing at 37 deg.C overnight. Absorbing 1mL of bacterial liquid, centrifuging at 12000rpm for 2min, removing supernatant, adding 100 mu L of sterile deionized water for resuspension, placing in a metal bath at 100 ℃ for 10min, then performing ice bath for 2min, and centrifuging at 12000rpm for 2min, wherein the supernatant is DNA liquid containing the genome DNA of the bacterial strain and can be used as a template for LAMP amplification reaction.
Example 3: construction of the pyrH recombinant plasmid
The preparation method of the artificially constructed recombinant plasmid DNA containing the pyrH gene of the vibrio Erwinii comprises the following steps: the method comprises the steps of using pClone007 simple vector as a vector, using a pyrH gene of Vibrio erwinii as a DNA target fragment, connecting the pyrH target fragment to the pClone007 simple vector by catalysis of ligase to construct a recombinant plasmid pClone007-pyrH, transforming the recombinant plasmid into E.coli DH5 alpha competent cells, screening out positive clones by 100mg/mL ampicillin, extracting a recombinant plasmid by using a plasmid kit, finally detecting by PCR (polymerase chain reaction) and sending a sequence to detect whether the recombinant plasmid is successfully constructed.
Example 4: specificity of LAMP method
The loop-mediated isothermal amplification reaction system of the sample is 25 mu L, and comprises: 1 × Isothermmal Amp Buffer (final concentration of reaction system), 6mmol/L MgSO4(final concentration of reaction system), 1.4mmol/L dNTP Mix (final concentration of reaction system), 1.6. mu. mol/L FIP (final concentration of reaction system), 1.6. mu. mol/L BIP (final concentration of reaction system), 0.2. mu. mol/L F3 (final concentration of reaction system), 0.2. mu. mol/L B3 (final concentration of reaction system), 0.4. mu. mol/L LF (final concentration of reaction system), 0.4. mu. mol/L LB (final concentration of reaction system), 320U/mL Bst 2.0DNA Polymerase (final concentration of reaction system), 1.2mol/L betaine (final concentration of reaction system),1mmol/L manganese chloride (final concentration of reaction system), 2. mu.L of 1mol/L calcein, and 1. mu.L of bacterial template DNA (each bacterial genomic DNA obtained in example 2), and deionized water was added to make up to 25. mu.L.
The loop-mediated isothermal amplification system of the positive control quality control product is 25 mu L, and comprises: 1 × Isothermmal Amp Buffer (final concentration of reaction system), 6mmol/L MgSO4(final concentration of reaction system), 1.4mmol/L dNTP Mix (final concentration of reaction system), 1.6. mu. mol/L FIP (final concentration of reaction system), 1.6. mu. mol/L BIP (final concentration of reaction system), 0.2. mu. mol/L F3 (final concentration of reaction system), 0.2. mu. mol/L B3 (final concentration of reaction system), 0.4. mu. mol/L LF (final concentration of reaction system), 0.4. mu. mol/L LB (final concentration of reaction system), 320U/mL Bst 2.0DNA Polymerase (final concentration of reaction system), 1.2mol/L betaine (final concentration of reaction system), 1mmol/L manganese chloride (final concentration of reaction system), 1mol/L calcein 2. mu.L and artificially constructed plasmid DNA 1. mu.L containing pyrH gene of Vibrio ohwii, and deionized water was supplemented to 25. mu.L.
The loop-mediated isothermal amplification reaction system of the negative control quality control product is 25 mu L, and comprises: 1 × Isothermmal Amp Buffer (final concentration of reaction system), 6mmol/L MgSO4(final concentration of reaction system), 1.4mmol/L dNTP Mix (final concentration of reaction system), 1.6. mu. mol/L FIP (final concentration of reaction system), 1.6. mu. mol/L BIP (final concentration of reaction system), 0.2. mu. mol/L F3 (final concentration of reaction system), 0.2. mu. mol/L B3 (final concentration of reaction system), 0.4. mu. mol/L LF (final concentration of reaction system), 0.4. mu. mol/L LB (final concentration of reaction system), 320U/mL Bst 2.0DNA Polymerase (final concentration of reaction system), 1.2mol/L betaine (final concentration of reaction system), 1mmol/L manganese chloride (final concentration of reaction system), 1mol/L calcein 2. mu.L and 1. mu.L of genomic DNA of other bacteria than Vibrio ohwii, and deionized water was added to 25. mu.L.
Respectively uniformly mixing the 3 loop-mediated isothermal amplification systems, and putting the mixture into a constant-temperature water bath box under the reaction conditions of 65 ℃ and 60 min; the reaction was stopped at 80 ℃ for 10 min.
MnCl is added into a reaction system2Observing whether the color of the reaction product changes after the reaction of the solution and the calcein color development solution is finished, if the color is bright green,is positive; the color was orange and negative. As shown in fig. 1, the results of the calcein fluorescence color development method show that: the amplified products of the Vibrio wenshuni (Vibrio owensii) and the positive control quality control substance pryH recombinant plasmid DNA are bright green and positive; and amplification reaction products of Vibrio corallinus (Vibrio corallilyticus), Vibrio shilonii (Vibrio shilonii), Vibrio alginolyticus (Vibrio algorityticus), Vibrio furnisi (Vibrio furnisii), Vibrio fluvialis and Vibrio antarius, Bacillus aerophilus, Pseudomonas fluorescens, Staphylococcus aureus, ultrapure water and the like are orange yellow and negative.
After the loop-mediated isothermal amplification reaction is finished, 4.5 mu L of amplification product is mixed with 2 mu L of 6 XLodding buffer, the mixture is spotted on 1.5% agarose gel for electrophoresis detection, 130V is electrified for 35min, as shown in figure 2, the electrophoresis analysis and detection result shows that the electrophoresis band is a specific ladder-shaped band (positive) of the amplification product of Vibrio ovi owensis (Vibrio owensii) and the positive control substance pryH recombinant plasmid DNA; and amplification products of Vibrio corallinus (Vibrio corallilyticus), Vibrio shilonii (Vibrio shilonii), Vibrio alginolyticus (Vibrio algyrinolyticus), Vibrio furnissii (Vibrio furnissii), Vibrio fluvialis and Vibrio antarius, as well as Bacillus aerophilus, Pseudomonas fluorescens and Staphylococcus aureus, and ultrapure water, which have no specific band (negative) as electrophoresis results. The invention has the advantages of high specificity and strong specificity, and can be used for rapidly detecting whether the sample contains the vibrio eurypheni.
Example 5: sensitivity of LAMP method
The pryH recombinant plasmid DNA constructed according to example 3 was tested to have a plasmid concentration of 152 ng/. mu.L. The copy number of the plasmid is 6.19X 10 calculated according to the length and concentration of the plasmid and a specific formula13copies/mL. 10 μ L of plasmid DNA was diluted with ultrapure water in a 10-fold gradient to obtain plasmid DNA at each dilution concentration, and the obtained plasmid DNA was used as a DNA template for LAMP sensitivity detection.
Loop-mediated isothermal amplification reaction system for LAMP sensitivity detection25 μ L, comprising: 1 × Isothermmal Amp Buffer (final concentration of reaction system), 6mmol/L MgSO4(reaction system final concentration), 1.4mmol/L dNTP Mix (reaction system final concentration), 1.6. mu. mol/L FIP (reaction system final concentration), 1.6. mu. mol/L BIP (reaction system final concentration), 0.2. mu. mol/L F3 (reaction system final concentration), 0.2. mu. mol/L B3 (reaction system final concentration), 0.4. mu. mol/L LF (reaction system final concentration), 0.4. mu. mol/L LB (reaction system final concentration), 320U/mL Bst 2.0DNA Polymerase (reaction system final concentration), 1.2mol/L betaine (reaction system final concentration), 1mmol/L manganese chloride (reaction system final concentration), 1mol/L calcein 2. mu.L and plasmid DNA 2. mu.L of each dilution concentration, and deionized water is supplemented to 25. mu.L. After the LAMP amplification system is uniformly mixed, putting the mixture into a constant-temperature water bath box, and reacting for 60min under the conditions of 65 ℃; the reaction was stopped at 80 ℃ for 10 min.
As shown in FIGS. 3 and 4, the sensitivity of the loop-mediated isothermal amplification detection method of the present invention is: 6.19X 103copies/mL. The detection result of calcein by fluorescence color development method is 6.19 multiplied by 1011～6.19×103copies/mL are bright green (positive), 6.19X 103～6.19×100copies/mL is orange yellow (negative), N is ultrapure water, orange yellow (negative); the result of 1.5% gel electrophoresis was 6.19X 1011～6.19×103copies/mL have a specific band (positive), 6.19X 102～6.19×100A copies/mL non-specific strip (negative), N is ultrapure water, and the non-specific strip (negative); the results of the fluorescence color development (FIG. 3) and the results of the gel electrophoresis (FIG. 4) agree, and thus it was demonstrated that the sensitivity of the loop-mediated isothermal amplification detection method of the present invention was 6.19X 103copies/mL。
The results of the calcein fluorescence color development method and the gel electrophoresis analysis are consistent, and the method is proved to have reliable data. Can directly judge whether the sample contains the vibrio erwinii or not through the color of the amplification product, thereby greatly shortening the detection time.
In conclusion, the loop-mediated isothermal amplification detection primer group and the detection method of the vibrio erwinii can quickly, simply and sensitively detect the vibrio erwinii, and have strong specificity and high accuracy.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> primer group, detection method and detection kit for loop-mediated isothermal amplification of Vibrio euryphi
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> DNA
<213> Vibrio ohvenicus (Vibrio owensii)
<400> 1
gcgtagtagg cgacca 16
<210> 2
<211> 18
<212> DNA
<213> Vibrio ohvenicus (Vibrio owensii)
<400> 2
catacagctc tgcgtctg 18
<210> 3
<211> 36
<212> DNA
<213> Vibrio ohvenicus (Vibrio owensii)
<400> 3
gcggatagcg tctgcccact tcaccgtgct tatgta 36
<210> 4
<211> 42
<212> DNA
<213> Vibrio ohvenicus (Vibrio owensii)
<400> 4
gccaaggccg tgtagttatc ttgctgctga atctgtagtg aa 42
<210> 5
<211> 20
<212> DNA
<213> Vibrio ohvenicus (Vibrio owensii)
<400> 5
cggaattgca gacattacgc 20
<210> 6
<211> 20
<212> DNA
<213> Vibrio ohvenicus (Vibrio owensii)
<400> 6
ctctgcaggt actggtaacc 20
Claims (9)
1. A loop-mediated isothermal amplification detection primer group of Vibrio euryphi is characterized in that: the detection primer group is as follows:
front outer primer F3: 5'-GCGTAGTAGGCGACCA-3', respectively;
rear outer primer B3: 5'-CATACAGCTCTGCGTCTG-3', respectively;
front inner primer FIP: 5'-GCGGATAGCGTCTGCCCACTTCACCGTGCTTATGTA-3', respectively;
and (3) a rear inner primer BIP: 5'-GCCAAGGCCGTGTAGTTATCTTGCTGCTGAATCTGTAGTGAA-3', respectively;
front loop primer LF: 5'-CGGAATTGCAGACATTACGC-3', respectively;
rear loop primer LB: 5'-CTCTGCAGGTACTGGTAACC-3' are provided.
2. A loop-mediated isothermal amplification detection kit for Vibrio euryphi comprises loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, and MnCl2The kit comprises a solution, a calcein color development solution, a positive control quality control substance, a negative control quality control substance and a detection primer group, and is characterized in that the detection primer group is as follows:
front outer primer F3: 5'-GCGTAGTAGGCGACCA-3', respectively;
rear outer primer B3: 5'-CATACAGCTCTGCGTCTG-3', respectively;
front inner primer FIP: 5'-GCGGATAGCGTCTGCCCACTTCACCGTGCTTATGTA-3', respectively;
and (3) a rear inner primer BIP: 5'-GCCAAGGCCGTGTAGTTATCTTGCTGCTGAATCTGTAGTGAA-3', respectively;
front loop primer LF: 5'-CGGAATTGCAGACATTACGC-3', respectively;
rear loop primer LB: 5'-CTCTGCAGGTACTGGTAACC-3' are provided.
3. The loop-mediated isothermal amplification assay kit of vibrio erwinii according to claim 2, wherein the positive control quality control is plasmid DNA containing the pryH gene of vibrio erwinii.
4. The loop-mediated isothermal amplification assay kit of vibrio avenae according to claim 2, wherein the negative control quality control is DNA or ultrapure water not containing the pryH gene of vibrio avenae.
5. A loop-mediated isothermal amplification detection method of Vibrio euryphi for non-disease diagnosis and treatment purposes, comprising the steps of:
(1) enrichment culture is carried out on the sample to obtain enrichment liquid, and genome DNA in the enrichment liquid is extracted to be used as a template;
(2) the primer set for detecting the loop-mediated isothermal amplification of Vibrio euryphi according to claim 1, a loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, MnCl2Mixing the solution, the calcein color development solution and the sample genome DNA to form a loop-mediated isothermal amplification reaction system, and carrying out loop-mediated isothermal amplification reaction;
(3) and after the amplification reaction is finished, detecting whether the sample contains the vibrio wenshuriei or not by calcein fluorescence color development method and/or gel electrophoresis analysis.
6. The LAMP detection method of Vibrio erwinii for non-disease diagnosis and treatment according to claim 5, wherein the detection of whether the sample contains Vibrio erwinii by calcein fluorescence color development specifically comprises: if the color of the amplification product is bright green, the sample contains vibrio erwinii; if the color of the amplification product remains the same as the orange yellow, the sample does not contain Vibrio ohwii.
7. The loop-mediated isothermal amplification detection method of Vibrio harveyi for non-disease diagnosis and treatment according to claim 5, wherein the detection of whether the sample contains Vibrio harveyi by gel electrophoresis analysis specifically comprises: performing gel electrophoresis on the amplification product, wherein if a ladder-shaped strip exists, the sample contains vibrio oweringi; if no ladder-shaped strip exists, the sample does not contain Vibrio ohwii.
8. The method for detecting the LAMP for the Vibrio euryphenii for the non-disease diagnosis and treatment according to claim 5, wherein the LAMP comprises a reaction system with a total volume of 25 μ L, including 1 × Isotermal Amp Buffer, 6mmol/L MgSO41.4mmol/L dNTP Mix, 1.6. mu. mol/L FIP, 1.6. mu. mol/L BIP, 0.2. mu. mol/L F3, 0.2. mu. mol/L B3, 0.4. mu. mol/L LF, 0.4. mu. mol/L LB, 320U/mL Bst 2.0DNA Polyme ase, 1.2mol/L betaine, 1mmol/L manganese chloride, 1mmol/L calcein 2. mu.L and 1. mu.L of genome DNA template, and the balance of deionized water.
9. The method for detecting the loop-mediated isothermal amplification of Vibrio euryphenii for non-disease diagnosis and treatment according to claim 5, wherein the reaction conditions of the loop-mediated isothermal amplification reaction are as follows: the reaction was carried out at 65 ℃ for 60min and at 80 ℃ for 10 min.
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