CN103898108A - Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof - Google Patents
Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof Download PDFInfo
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Abstract
The invention relates to a nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 serotypes and an application thereof. The nucleotide comprises 1) at least one of nucleotides shown in SEQIDNO: 1-14; and 2) at least one of nucleotides shown in SEQIDNO: 1-14. The nucleotides can be used for preparing PCR (polymerase chain reaction) kits and gene chips for detecting Vibrio fluvialis. The nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 serotypes disclosed by the invention, as well as a PCR kit and a gene chip which contain the nucleotide, are strong in practicability, and the PCR kit is simple in preparation method, short in detection cycle, rapid in speed, strong in maneuverability, convenient for industrialized production, and low in detection cost; the accuracy is high; and the sensitivity is high.
Description
Technical field
The present invention relates to vibrio fluvialis O2, O4, O13, O15 and the special Nucleotide of O18 serotype, relate in particular to vibrio fluvialis O2, O4, O13, special Nucleotide and the application thereof of individual gene in O15 and O18 serotype O antigen gene cluster.
Background technology
Vibrio fluvialis is Gram-negative bacteria, be ocean environment mainly perch one of bacterium, extensively be present in river and environment waters, marine outfall, also be one of main bacterial pathogen of the mankind and hydrobiont, can play the disease of the multiple cultivated animals such as fish, shrimp, shellfish, bring serious financial loss to aquaculture.Vibrio fluvialis can also cause the epidemic diarrhea that the mankind are serious by various food, is the kinds of pathogenic vibrio that is only second to vibrio cholerae and Vibrio parahaemolyticus in Vibrio, is considered to a kind of novel pathogenic bacteria of global Zoonosis.Its somatotype is one of important prerequisite of ocean strain library foundation with evaluation.
Typing of bacteria and authentication method mainly contain traditional phenotype method, serological method and molecular assay method.But along with molecular biological development, traditional serotype and authentication method exist certain problem, diagnostic method as this in serotype needs a large amount of antiserum(antisera)s, and antiserum(antisera) general classes is incomplete, quantity not sufficient, also there are some difficulties in preparation with in storing in a large amount of antiserum(antisera)s.On the other hand serotype method length consuming time, sensitivity is low, loss is high, poor accuracy, between the antiserum(antisera) that different O antigen produces, often has cross reaction.Therefore the serological diagnosis method of, setting up based on Protocols in Molecular Biology becomes developing direction.
The Molecular Identification of vibrio fluvialis is more and more subject to people's attention, and becomes the important evidence that vibrio fluvialis bacterial classification and plant type are identified, therefore many new bacterium also produce.For vibrios, biochemical reaction is mainly the performance of various zymograms, belongs to the content of formalness, and the similarity of nucleic acid is only the defining the most at all of vibrios kind, the most direct feature.Therefore, the somatotype of vibrio fluvialis with identify the most effectively, the most direct mode should trigger from the research of nucleic acid, determine the ownership of vibrio fluvialis by the similarity of nucleic acid (comprising genome and nucleic acid fragment) relatively.
In recent years, increasing molecular engineering, for somatotype, evaluation, detection and the disease screening of pathogenic bacteria, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) analysis, rDNA restriction fragment length polymorphism (RFLP) analysis etc.Molecular biology method not only can be used for the quick serotype examination of vibrio fluvialis, and stable qualification result can make up the deficiency of phenotypic characteristic authentication method.Compare with traditional detection technology, these molecular detection technologies based on polymerase chain reaction (PCR), do not need the process such as separation, pure culture through pathogenic bacteria, and have the advantages such as quick, sensitive, high specificity.
Polymerase chain reaction technology (Polymerase chain reaction, be called for short round pcr) admitted at present and promoted as microorganism detection technology, this technology has the advantages such as high-throughput, detection speed are fast, high specificity, sensitivity height with respect to traditional method, only need carry out simple pre-bacterium or the increasing bacterium process of increasing to sample, again by the centrifugal and detailed bacterium DNA profiling of cracking, the target sequence that just can increase in the PCR process under high specific primer mediation, reaches and detects the object that whether contains invasive organism to be measured in sample.The amplification procedure of PCR only needs 1 and a half hours.This has greatly improved undoubtedly working speed and has reduced job costs inspection and quarantine department and Clinical Laboratory.
No matter from internal and international angle, identify quickly and accurately serum type, be very important for the prevention and control of vibrio fluvialis provide effect technique support.
Summary of the invention
The object of the present invention is to provide and the present invention relates to vibrio fluvialis O2, O4, O13, O15 and the special Nucleotide of O18 serotype, is characterized in that described Nucleotide has:
1) at least one in the Nucleotide shown in SEQ ID NO:1-14;
2) at least one and in the Nucleotide of the Nucleotide complementation shown in SEQ ID NO:1-14; Described SEQ ID NO:1-14 is as follows:
The present invention also provides a kind of PCR test kit, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer is at least one in the Nucleotide as shown in SEQ ID NO:1-4.Described test kit, also comprises following reagent: 10 mM dNTP 30 μ l; 10 × enzyme spcificity reaction buffer, 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; Primer mixture 10 μ l; Positive reference substance 10 μ l; Negative control product 10 μ l; ddH
2o 5ml.
Wherein said PCR primer is preferably at least one in described Nucleotide as shown in SEQ ID NO:1-14.
The present invention further discloses vibrio fluvialis O2, O4, O13, the special SEQ ID NO:1-14 Nucleotide of O15 and O18 serotype is for the preparation of detecting vibrio fluvialis vibrio fluvialis O2, O4, O13, the application of PCR test kit, gene chip or the microarray aspect of O15 and O18 serotype.
Vibrio fluvialis of the present invention can sample in the crude extract of the culture of tap water, river water, seawater, or the crude extract of the pure growth of vibrio fluvialis etc.
Collecting vibrio fluvialis extraction genome is to adopt ordinary method to prepare.
For the PCR test kit of vibrio fluvialis, whole detecting step comprises sample pretreatment-amplification-electrophoresis detection result.Primer and the needed reagent of PCR reaction system add in amplification pipe in advance, and user only need add pretreated sample amplification pipe to start amplified reaction, simple and quick complete testing.
The present invention also provides a kind of gene chip, comprises solid phase carrier and is fixed on the oligonucleotide probe on solid phase carrier, and wherein said oligonucleotide probe comprises above-mentioned Nucleotide; Wherein said Nucleotide is preferably the Nucleotide as shown in SEQ ID NO:1-14.
The present invention also provides a kind of micro-array, and it comprises above-mentioned Nucleotide; Wherein said Nucleotide is preferably the Nucleotide as shown in SEQ ID NO:1-14.
Disclosed by the invention to vibrio fluvialis O2, O4, O13, compared with prior art, tool of the present invention has the following advantages the special Nucleotide of O15 and O18 serotype:
(1) practical
A kind of PCR reaction system that the present invention sets up, can detect vibrio fluvialis, provides serotype to detect special primer used, utilizes this PCR method to detect clinical samples.
(2) accuracy is high
The present invention is by the PCR reaction to the special gene of each serotype of vibrio fluvialis, and each sample obtains the band of an entry, will obtain object fragment and compare with known length, just can obtain the affiliated serotype of vibrio fluvialis.
(3) testing cost is relatively low
Can be applied to the fields such as Food Hygiene Surveillance, environmental monitoring, still product supervision and inspection quarantine, and provide technology mode for other different pathogenic microbes detects combine.
The above, only operation of the present invention and implementation method, not the present invention is done to any pro forma restriction, any simple modification, equivalent variations and modification that every foundation technical spirit of the present invention is done above embodiment, all still belong in the scope of technical solution of the present invention.
Accompanying drawing explanation:
Fig. 1 represents that O2 serotype special primer of the present invention detects other serotype reference culture electrophoresis result of vibrio fluvialis figure,
wzythe screening of gene P1 and P2 primer, object band is 470bp, remaining serotype is without any band;
orfthe screening of 9 gene P3 and P4 primer, object band is 336bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Fig. 2 represents the species specific evaluation electrophoresis result of O2 serotype special primer of the present invention figure, has wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Fig. 3 represents O4 serotype of the present invention
wzxgene P5 and P6 primer detect other serotype reference culture electrophoresis result of vibrio fluvialis figure,
wzxthe screening of gene P5 and P6 primer, object band is 863bp, remaining serotype is without any band.Concrete bacterial strain information is in table 2
Fig. 4 represents O4 serotype wzx gene P5 of the present invention and the species specific evaluation electrophoresis result of P6 primer figure, has wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, all without any band.Concrete bacterial strain information is in table 2.
Fig. 5 represents O13 serotype of the present invention
wzxgene P7 and P8 primer detect other serotype reference culture electrophoresis result of vibrio fluvialis figure,
wzxthe screening of gene P7 and P8 primer, object band is 430bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Fig. 6 represents O13 serotype of the present invention
wzxgene P7 and the species specific evaluation electrophoresis result of P8 primer figure, wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Fig. 7 represents O15 serotype of the present invention
orf13gene P9 and P10 primer detect other serotype reference culture electrophoresis result of vibrio fluvialis figure,
orf13the screening of gene P9 and P10 primer, object band is 313bp, remaining serotype is without any band.Concrete bacterial strain information is in table 2
Fig. 8 represents O15 serotype of the present invention
orf13gene P9 and the species specific evaluation electrophoresis result of P10 primer figure, wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, all without any band.Concrete bacterial strain information is in table 2.
Fig. 9 represents O18 serotype of the present invention
wzxgene P11 and P12 primer detect other serotype reference culture electrophoresis result of vibrio fluvialis figure,
wzxthe screening of gene P11 and P12 primer, object band is 338bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Figure 10 represents O18 serotype of the present invention
wzxgene P11 and the species specific evaluation electrophoresis result of P12 primer figure, wherein use
wzxgene P11 and P12 primer have detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Figure 11 represents O18 serotype of the present invention
orf18gene P13 and P14 primer detect other serotype reference culture electrophoresis result of vibrio fluvialis figure,
orf18the screening of gene P13 and P14 primer, object band is 590bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Figure 12 represents O18 serotype of the present invention
orf18gene P13 and the species specific evaluation electrophoresis result of P14 primer figure, wherein use
orf18gene P13 and P14 primer have detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Figure 13 represents to use respectively O2, O4, and O13, the increase electrophoresis result figure of corresponding serotype of O15 and O18 special primer, concrete bacterial strain information is in table 2;
Wherein: O2, O4, O13, O15, O18 and negative contrast (-) represent corresponding serotype primer amplification result, and first swimming lane H and last swimming lane are 2000bp ladder marker.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: the condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).Wherein vibrio fluvialis derives from Japan Collection of Microorganisms(JCM).
embodiment 1: genomic extraction
37 ℃ of nutrient broth mediums are cultivated vibrio fluvialis, collect bacterium, extract genome concrete steps as follows:
With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, then add the N,O-Diacetylmuramidase of 10ul 10mg/ml to continue insulation 20 minutes.Add afterwards Proteinase K, the 15ul 10%SDS of 3ul 20mg/ml, 50 ℃ of incubations 2 hours, then add the RNase of 3 ul 10mg/ml, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, then use isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) solution extracting twice, get supernatant liquor, then with isopyknic ether extraction to remove remaining phenol.For supernatant liquor, 2 times of volume ethanol precipitation DNA, roll out DNA with glass yarn and also wash DNA with 70% ethanol, finally DNA are resuspended in 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
embodiment 2:sequence is decoded
Extract the genome of vibrio fluvialis O2,04,013,015 and 018 serotype reference culture, by Solexa pair-end sequencing technologies, each serotype genome of vibrio fluvialis is carried out genome sequencing and is obtained the sequence of this serotype, use Blast and PSI-Blast to carry out sequence alignment, adopt TMHMM 2.0 program to carry out cross-film structure prediction, use ClustalW program carry out sequence alignment and screen conservative and specific gene fragment, finally obtain the O antigen gene cluster sequence of each serotype of vibrio fluvialis and decode result.
embodiment 3: design of primers
The O antigen gene cluster sequence of vibrio fluvialis O2, O4, O13, O15 and each serotype of O18 is to test oneself in this laboratory, by compare of analysis, we choose Blast comparison result identity and the relatively low gene specific section design primer of similarity value.Wherein O2 serotype
wzygene comparison result identity value and similarity value are 44% and 68%,
orf9gene comparison result identity value and similarity value are 44% and 65%; O4 serotype
wzxgene comparison result identity value and similarity value are 22% and 42%; O13 serotype
wzxgene comparison result identity value and similarity value are 26% and 48%; O15 serotype
orf13gene comparison result identity value and similarity value are 27% and 52%; O18 serotype
wzxgene comparison result identity value and similarity value are 29% and 53%,
orf18gene comparison result identity value and similarity value are 47% and 64%.So each serotype is chosen respectively the gene of above-mentioned correspondence as the specific target gene of this serotype, designs respectively special primer for the gene specific section of each serotype.
Design of primers is the core of this invention.Said gene is imported to Primer Premier 5 and carry out design of primers, each gene design pair of primers, has single purpose band.
After design of primers, in Genbank, carry out BLAST, the primer of design can not be too high with the sequence similarity of other nearly edge bacterium, so just can guarantee that this primer only increases in the predetermined position of oneself, and not with other nearly edge bacterium or the environment of collect specimen in nearly edge bacterium do not produce positive reaction.This point is very important for avoiding the generation of non-specific band and the success or failure of experiment.
The primer of designing is as shown in table 1.
Table 1 is for the primer sequence of PCR
embodiment 4: the screening of special primer
The reference culture of vibrio fluvialis 02, O4, O13, O15 and O18 serotype and the each strain of the reference culture of other 14 kinds of serotypes are collected, 11 strains are the vibrio fluvialis of somatotype not, 6 other bacterial strains of strain Vibrio, the specificity of 1 strain Salmonella bacterial strain and 1 strain coli strain checking primer, strain number and source are in table 2.
Table 2 is for the bacterial strain of specific detection
The PCR system that gene identification primer screening is used be the testing sample template of 5 μ M primer 0.4 μ l, 10 × enzyme spcificity reaction buffer, 2.5 μ l, 10mM dNTP 0.25 μ l, 5U/ μ l hot resistant DNA polymerase 0.2 μ l and 3 μ l in the thin-walled PCR pipe of 0.2ml, last ddH
2o supplies 25 μ l.All primers all, obtaining positive findings in corresponding serotype separately, do not obtain any PCR product band in other groups.
Reaction cycle parameter on PCR instrument in this step comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially:
Early stage for make sex change can reach required temperature essential early stage treating processes a circulation be 95 ℃, 5 minutes;
Denaturation temperature and time are 95 ℃, 45 seconds;
Renaturation temperature and time is 55 ℃/68 ℃, 1 minute;
Elongating temperature and time are 72 ℃, 1 minute;
The cycle index of sex change, renaturation, extension is 35 circulations;
The temperature and time that carries out a circulation for stablizing amplified production is 72 ℃, 5 minutes
Wherein, O2 uses 68 ℃ of amplifications, and O4 uses 61 ℃ of amplifications, and O13 uses 55-65 ℃ of amplification, O15 use 55-58 ℃ of amplification all can, O18 uses 65 ℃ of amplifications.
Above-mentioned steps is electrophoresis amplified production in electrophoresis equipment, and the concrete steps that record result are:
Work by primer screening after basic PC R reaction finishes substantially, and necessary length adjustment is little on the impact of W-response condition, and the primer sequence of using in the present invention is all summarised in table 1.
Table 1 is for the primer sequence of PCR
preparation and the application of embodiment 6:PCR detection kit
1, the composition of PCR test kit:
dNTP(10mM) 30μl；
10 × Buffer (10 × enzyme spcificity reaction buffer), 50 μ l;
Taq polysaccharase (5U/ μ l hot resistant DNA polymerase) 5 μ l;
PCR primer mixture (5 μ M) 10 μ l;
Positive reference substance (KP) 10 μ l;
Negative control product (KN) 10 μ l;
ddH
2O 5ml；
Each test kit can be used for detecting 10 samples.
Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by precious biotechnology company limited; It is synthetic that primer mixture is that the sequence of designed, designed offers Shanghai Ying Jun biotech company; Positive reference substance, negative control product and ddH
2o is prepared voluntarily by us.
2, plant and instrument
Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by the raw work in Shanghai; It is synthetic that primer mixture is that the sequence of designed, designed offers Shanghai Ying Jun biotech company; Positive reference substance, negative control product and ddH2O are prepared voluntarily by us.Equipment PCR instrument (having another name called DNA thermal cycling amplification instrument), electrophoresis equipment (comprising electrophoresis apparatus and electrophoresis chamber), gel imaging instrument ,-20 ℃ of refrigerators, supercentrifuge, micropipet and 0.2ml PCR thin-walled tubes of experiment.
3, the use specific examples of PCR test kit
The PCR detection method that uses above-mentioned PCR test kit to detect vibrio fluvialis comprises the steps:
(1) extract environmental sample template to be measured;
(2) in PCR thin-walled tube, add, dNTP, 10 × Buffer, Taq polysaccharase, primer, testing sample template and ddH
2o mixes;
(3) mixture mixing in thin-walled PCR pipe is increased on PCR instrument;
(4) electrophoresis amplified production in electrophoresis equipment, records result;
(5) analyze and carry out result judgement.
Environmental sample template in above-mentioned steps (1) is the crude extract of the culture of tap water, river water, seawater etc., or the crude extract of the pure growth of vibrio fluvialis or pure dna, or positive reference substance and negative control product.
The extracting method of the environmental sample template in above-mentioned steps (1) is:
get 1.5ml culture, under 12000rpm condition centrifugal 1 minute, remove supernatant liquor;
5. get 3 μ l middle layer supernatant as pcr template
Reaction cycle parameter on PCR instrument in above-mentioned steps (3) comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially:
Early stage for make sex change can reach required temperature essential early stage treating processes a circulation be 95 ℃, 5 minutes;
Denaturation temperature and time are 95 ℃, 45 seconds;
Renaturation temperature and time is 55 ℃/68 ℃, 1 minute;
Elongating temperature and time are 72 ℃, 1 minute;
The cycle index of sex change, renaturation, extension is 35 circulations;
The temperature and time that carries out a circulation for stablizing amplified production is 72 ℃, 5 minutes.
Above-mentioned steps (4) electrophoresis amplified production in electrophoresis equipment, the concrete steps that record result are:
mixed solution is splined on 1.0% sepharose;
The present invention is by configuring a kind of PCR test kit of can industrialization producing that detects vibrio fluvialis, the combination of components that PCR detection method need to be used together, when use, extract testing sample, just can carry out quick, sensitive, easy detection through comparatively simple operation sequence simultaneously, in test kit, the consumption of each component and concentration are test gained, and the testing installation using with this test kit detection vibrio fluvialis is simple, and testing cost is low.
Using the object of positive and negative control product is for the whole operating process of Quality Control, to draw judgement accurately.If contain vibrio fluvialis object O antigenic type, from electrophoresis result, can observe the band with positive reference substance same position; If do not contain vibrio fluvialis object O antigenic type, the same with negative control product do not have this band.
The amount of reagent that one-time detection of the present invention is tested in the test kit using sees the following form shown in 3, and DNA profiling amount is 3 μ l
Table 3 one-time detection is tested the amount of reagent in the test kit using
| Composition | Concentration | (μ l) for application of sample amount |
| ddH 2O | 18.65 | |
| 10 × PCR damping fluid | 10× | 2.5 |
| 10mM dNTP | 10mM | 0.25 |
| Primer mixture | 5mM | 0.4 |
| Taq enzyme | 5U/μl | 0.2 |
| Cumulative volume | 25 |
Hot resistant DNA polymerase in the present invention is Taq enzyme.
Above-mentioned positive reference substance is to have determined it is the sample of each O antigenic type of vibrio fluvialis, and negative control product are not the samples of vibrio fluvialis for determining through laboratory.
If this PCR test kit carries out pcr amplification with the bacteria suspension of vibrio fluvialis, and through extracting the DNA that obtains as template acquired results always.Susceptibility and specificity indifference, like this, can save the extraction step of template DNA, and working method is simplified.Meanwhile, compare routine biochemistry detection method, the testing sample that present method adopts can be directly clinical sample nutrient solution, or detection sample is carried out to simple separation cultivation and just can detect, thereby has saved manpower and materials.
4, providing of testing sample
Collect vibrio fluvialis O2, O4, O13, O15 and O18 serotype reference culture, 6 other bacterial strains of strain Vibrio, the specificity of 1 strain Salmonella bacterial strain and 1 strain coli strain checking primer, strain number and source are in table 2.
SEQUENCE LISTING
<110> Nankai University
<120> is to vibrio fluvialis O2, O4, O13, the Nucleotide that O15 and O18 are special and application thereof
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> artificial sequence
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gggtatcaat gacggcg 17
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<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 18
<212> DNA
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<211> 18
<212> DNA
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<210> 5
<211> 18
<212> DNA
<213> artificial sequence
<400> 5
<210> 6
<211> 19
<212> DNA
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<400> 6
tcgctaaaac ttcattgcc 19
<210> 7
<211> 18
<212> DNA
<213> artificial sequence
<400> 7
<210> 8
<211> 22
<212> DNA
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<400> 8
aataaataag agcacagacg ag 22
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<400> 9
tggaatggaa ataacgaat 19
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tcacagatgt agcaggcag 19
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tggcattgtc atccatac 18
Claims (7)
1. couple vibrio fluvialis O2, O4, O13, O15 and the special Nucleotide of O18 serotype, is characterized in that described Nucleotide has:
1) at least one in the Nucleotide shown in SEQ ID NO:1-14;
2) at least one and in the Nucleotide of the Nucleotide complementation shown in SEQ ID NO:1-14.
2. above-mentioned Nucleotide can be for the preparation of detecting vibrio fluvialis O2, O4, O13, PCR test kit, gene chip or microarray for O15 and O18 serotype; Described vibrio fluvialis can sample in the crude extract of the culture of tap water, river water, seawater, or the crude extract of the pure growth of vibrio fluvialis.
3. a PCR test kit, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer is at least one in the Nucleotide as shown in SEQ ID NO:1-14.
4. test kit claimed in claim 2, also comprises following reagent: 10 mM dNTP 30 μ l; 10 × enzyme spcificity reaction buffer, 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; Primer mixture 10 μ l; Positive reference substance 10 μ l; Negative control product 10 μ l; ddH
2o 1ml.
5. SEQ ID NO:1-14 Nucleotide claimed in claim 1 is in the application aspect detection vibrio fluvialis PCR test kit.
6. the application of SEQ ID NO:1-14 Nucleotide claimed in claim 1 aspect the gene chip for the preparation of detection vibrio fluvialis.
7. SEQ ID NO:1-14 Nucleotide claimed in claim 1 is in the application aspect detection vibrio fluvialis microarray.
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| CN105177133A (en) * | 2015-09-07 | 2015-12-23 | 南开大学 | Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide |
| CN105200044A (en) * | 2015-08-03 | 2015-12-30 | 南开大学 | Nucleotides specific to vibrio fluvialis O1, O6, O7, O8 and O9 as well as application of nucleotides |
| CN105200045A (en) * | 2015-08-03 | 2015-12-30 | 南开大学 | Nucleotides specific to vibrio fluvialis O11, O14, O16 and O17 as well as application of nucleotides |
| CN105219769A (en) * | 2015-10-20 | 2016-01-06 | 南开大学 | The Nucleotide special to citric acid bacillus O6 and O9 and application thereof |
| CN105255871A (en) * | 2015-11-02 | 2016-01-20 | 南开大学 | Specific nucleotide for aeromonas hydrophila O7, O8, O9 and O10 and application thereof |
| CN112375836A (en) * | 2020-11-30 | 2021-02-19 | 南开大学 | Real-time fluorescence PCR detection method for vibrio fluvialis and application |
| CN112375835A (en) * | 2020-11-30 | 2021-02-19 | 南开大学 | Loop-mediated isothermal amplification detection method for 4O antigen serotypes of vibrio fluvialis and application |
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| CN105200045B (en) * | 2015-08-03 | 2018-02-23 | 南开大学 | The nucleotides special to vibrio fluvialis O11, O14, O16 and O17 and its application |
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| CN105200045A (en) * | 2015-08-03 | 2015-12-30 | 南开大学 | Nucleotides specific to vibrio fluvialis O11, O14, O16 and O17 as well as application of nucleotides |
| CN105200044B (en) * | 2015-08-03 | 2018-02-27 | 南开大学 | The nucleotides special to vibrio fluvialis O1, O6, O7, O8 and O9 and its application |
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| CN105177133B (en) * | 2015-09-07 | 2018-08-17 | 南开大学 | The nucleotide special to comma bacillus O6, O4, O7 and O15 and its application |
| CN105219769A (en) * | 2015-10-20 | 2016-01-06 | 南开大学 | The Nucleotide special to citric acid bacillus O6 and O9 and application thereof |
| CN105219769B (en) * | 2015-10-20 | 2018-05-15 | 南开大学 | The nucleotide special to citric acid bacillus O6 and O9 and its application |
| CN105255871A (en) * | 2015-11-02 | 2016-01-20 | 南开大学 | Specific nucleotide for aeromonas hydrophila O7, O8, O9 and O10 and application thereof |
| CN105255871B (en) * | 2015-11-02 | 2018-10-26 | 南开大学 | The nucleotide special to aeromonas hydrophila O7, O8, O9 and O10 and application |
| CN112375836A (en) * | 2020-11-30 | 2021-02-19 | 南开大学 | Real-time fluorescence PCR detection method for vibrio fluvialis and application |
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| CN112375836B (en) * | 2020-11-30 | 2023-03-31 | 南开大学 | Real-time fluorescence PCR detection method for vibrio fluvialis and application |
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