CN103898108B - The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof - Google Patents
The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof Download PDFInfo
- Publication number
- CN103898108B CN103898108B CN201410158813.3A CN201410158813A CN103898108B CN 103898108 B CN103898108 B CN 103898108B CN 201410158813 A CN201410158813 A CN 201410158813A CN 103898108 B CN103898108 B CN 103898108B
- Authority
- CN
- China
- Prior art keywords
- nucleotide
- vibrio fluvialis
- serotype
- primer
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 serotype and application thereof, described nucleotide includes 1) at least one in the nucleotide shown in SEQ ID NO:1 14；2) with at least one in the nucleotide of the nucleotide complementary shown in SEQ ID NO:1 14.These nucleotide can be used for preparation detection vibrio fluvialis PCR kit and gene chip.The present invention to vibrio fluvialis O2, O4, O13, nucleotide that O15 and O18 serotype is special and comprise the PCR kit of this nucleotide, gene chip practical, PCR kit compound method is easy, and the detection cycle is short, speed fast, workable, it is prone to industrialization produce, and testing cost is relatively low；Accuracy is high；Highly sensitive.
Description
Technical field
The present invention relates to the nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 serotype, particularly relate to river
Flow nucleotide and application thereof that in vibrio O2, O4, O13, O15 and O18 serotype O antigen gene cluster, individual gene is special.
Background technology
Vibrio fluvialis is gram negative bacteria, be marine environment mainly perch one of bacterium, be widely present in river and going out
Environment waters, Haikou, is also one of mankind's bacterial pathogen main with aquatile, can play the multiple cultivation such as fish, shrimp, shellfish
The disease of animal, carrys out serious economic loss to cultivation industrial belt.Vibrio fluvialis can also cause the mankind serious by various foods
Epidemic diarrhea, be vibrio to be only second to the kinds of pathogenic vibrio of vibrio cholera and vibrio parahaemolytious it is considered to be a kind of complete
The novel pathogen of the Zoonosis of ball.Its typing and qualification are one of important prerequisites of ocean strain library foundation.
Typing of bacteria and authentication method mainly have traditional phenotypic approach, serological method and method for identifying molecules.So
And, along with the development of molecular biology, traditional serotype and authentication method also exist certain problem, as serotype this
Kind of diagnostic method needs substantial amounts of antiserum, and antiserum general classes is complete, lazy weight, substantial amounts of antiserum in preparation and
Storage there is also some difficulties.On the other hand serotype method is time-consumingly grown, sensitivity is low, loss is high, poor accuracy, no
It is frequently present of cross reaction between the antiserum that same O antigen produces.Therefore, serum based on Protocols in Molecular Biology mirror is set up
The method of determining becomes developing direction.
The Molecular Identification of vibrio fluvialis is increasingly subject to people's attention, and becomes the weight that vibrio fluvialis strain is identified with plant type
Will foundation, the most therefore many new bacterium produce.For vibrio, biochemical reaction is mainly the performance of various zymogram, belongs to outside shape
The content of state, the similarity of nucleic acid is only the defining the most at all of vibrio kind, the most direct feature.Therefore, the typing of vibrio fluvialis
Should trigger from the research of nucleic acid with qualification mode most effective, the most direct, (include genome and nucleic acid sheet by comparing nucleic acid
Section) similarity determine the ownership of vibrio fluvialis.
In recent years, increasing molecular engineering for pathogen typing, identify, detect and disease screening, including turning
Record spacer (ITS) sequence analysis, random amplification length polymorphism (RAPD) are analyzed, rDNA Restriction Fragment Length is many
State property (RFLP) analysis etc..Molecular biology method cannot be only used for the rapid serum typing examination of vibrio fluvialis, stable mirror
Determine result and can make up the deficiency of phenotypic characteristic authentication method.Comparing with traditional sensing techniques, these are anti-based on polymerase chain type
Should the molecular detection technology of (PCR), it is not necessary to through processes such as the separation of pathogen, pure cultures, and have quick, sensitive,
The advantages such as high specificity.
Polymerase chain reaction technology (Polymerase chain reaction is called for short round pcr) is examined as microorganism
Survey technology is currently admitted and is promoted, and this technology has relative to traditional method that high flux, detection speed is fast, specificity
By force, sensitivity advantages of higher, only need to carry out sample simple pre-increasing bacterium or increasing bacterium process, then by centrifugal and cracking preparation is thin
Bacterium DNA profiling, it is possible to expand target sequence during the PCR under high specific primer mediates, reaches to detect in sample and is
The no purpose containing invasive organism to be measured.The amplification procedure of PCR only needs 1 and a half hours.This to inspection and quarantine department and
Clinical Laboratory greatly improves operating rate undoubtedly and reduces job costs.
No matter from the perspective of internal and international, identify serum type quickly and accurately, for the prevention and control of vibrio fluvialis
It is highly important for being provided with effect technique support.
Summary of the invention
Object of the present invention is to provide and the present invention relates to vibrio fluvialis O2, O4, O13, O15 and O18 serotype spy
Different nucleotide, it is characterised in that described nucleotide has:
1) at least one in the nucleotide shown in SEQ ID NO:1-14；
2) with at least one in the nucleotide of the nucleotide complementary shown in SEQ ID NO:1-14；Described SEQ ID
NO:1-14 is as follows:
Present invention also offers a kind of PCR kit, including PCR primer, dNTP, buffer and archaeal dna polymerase, described
PCR primer is at least one in the nucleotide as shown in SEQ ID NO:1-4.Described test kit, also includes following reagent:
10 mM dNTP 30μl；10 × enzyme spcificity reaction buffer 50 μ l；5U/ μ l hot resistant DNA polymerase 5 μ l；Primer mixture
10μl；Positive reference substance 10 μ l；Negative controls 10 μ l；ddH2O 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 the SEQ ID NO special to vibrio fluvialis O2, O4, O13, O15 and O18 serotype:
1-14 nucleotide preparation be used for detecting the PCR kit of vibrio fluvialis vibrio fluvialis O2, O4, O13, O15 and O18 serotype,
Application in terms of gene chip or microarray.
Vibrio fluvialis of the present invention can sample in tap water, river water, the crude extract of culture of sea water, or river
The crude extract etc. of the pure culture of stream vibrio.
Collecting vibrio fluvialis and extracting genome is to use conventional method to prepare.
For the PCR kit of vibrio fluvialis, whole detecting step includes sample pretreatment amplification electrophoresis detection knot
Really.Reagent required for primer and PCR reaction system has been previously added in amplification pipe, and user only need to be by pretreated sample
Adding amplification pipe and start amplified reaction, simple and quick completes detection work.
The present invention also provides for a kind of gene chip, visits including solid phase carrier and the oligonucleotide being fixed on solid phase carrier
Pin, wherein said oligonucleotide probe includes above-mentioned nucleotide；Wherein said nucleotide is preferably such as SEQ ID NO:1-14
Shown nucleotide.
The present invention also provides for a kind of micro-array, and it includes above-mentioned nucleotide；Wherein said nucleotide is preferably such as SEQ
Nucleotide shown in ID NO:1-14.
The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 serotype disclosed by the invention and prior art
Compare, present invention have the advantage that
(1) practical
A kind of PCR reaction system that the present invention sets up, can detect vibrio fluvialis, it is provided that the spy used in serotype detection
Different primer, utilizes this PCR method can detect clinical samples.
(2) accuracy is high
The present invention is reacted by the PCR of the special gene of each serotype to vibrio fluvialis, and each sample obtains an entry
Band, purpose fragment will be obtained compared with known length, it is possible to obtain the serotype belonging to vibrio fluvialis.
(3) testing cost is relatively low
Can be applied to the field such as Food Hygiene Surveillance, environmental monitoring, the still quarantine of product supervision and inspection, and be other not
Technology mode is provided with pathogenic bacterium detection combination.
The above, be only operation and the implementation of the present invention, and the present invention not makees any pro forma limit
System, every any simple modification, equivalent variations and modification above example made according to the technical spirit of the present invention, the most still
Belong in the range of technical solution of the present invention.
Accompanying drawing illustrates:
Fig. 1 represents that O2 serotype special primer of the present invention detects other serotype reference culture electrophoresis result of vibrio fluvialis
Figure,wzyGene P1 and the screening of P2 primer, purpose band is 470bp, and remaining serotype does not has any band；orf9 genes
The screening of P3 and P4 primer, purpose band is 336bp, and remaining serotype does not has any band, concrete bacterial strain information to be shown in Table 2；
Fig. 2 represents O2 serotype special primer of the present invention species specific qualification electrophoresis result figure, wherein have detected 6 strain arcs
Bacterium and 1 strain salmonella, 1 strain escherichia coli, all without any band, concrete bacterial strain information is shown in Table 2；
Fig. 3 represents O4 serotype of the present inventionwzxGene P5 and P6 primer detection other serotype reference cultures of vibrio fluvialis
Electrophoresis result figure,wzxGene P5 and the screening of P6 primer, purpose band is 863bp, and remaining serotype does not has any band.
Concrete bacterial strain information is shown in Table 2
Fig. 4 represents O4 serotype wzx gene P5 of the present invention and P6 primer species specific qualification electrophoresis result figure, Qi Zhongjian
6 strain vibrios and 1 strain salmonella, 1 strain escherichia coli are surveyed, all without any band.Concrete bacterial strain information is shown in Table 2.
Fig. 5 represents O13 serotype of the present inventionwzxGene P7 and P8 primer detection other serotype reference cultures of vibrio fluvialis
Electrophoresis result figure,wzxGene P7 and the screening of P8 primer, purpose band is 430bp, and remaining serotype does not has any band,
Concrete bacterial strain information is shown in Table 2；
Fig. 6 represents O13 serotype of the present inventionwzxGene P7 and P8 primer species specific qualification electrophoresis result figure, wherein
Have detected 6 strain vibrios and 1 strain salmonella, 1 strain escherichia coli, all without any band, concrete bacterial strain information is shown in Table 2；
Fig. 7 represents O15 serotype of the present inventionorf13Gene P9 and P10 primer detection other serotype standards of vibrio fluvialis
Bacterial strain electrophoresis result figure,orf13Gene P9 and the screening of P10 primer, purpose band is 313bp, and remaining serotype is not appointed
What band.Concrete bacterial strain information is shown in Table 2
Fig. 8 represents O15 serotype of the present inventionorf13Gene P9 and P10 primer species specific qualification electrophoresis result figure, its
In have detected 6 strain vibrios and 1 strain salmonella, 1 strain escherichia coli, all without any band.Concrete bacterial strain information is shown in Table 2.
Fig. 9 represents O18 serotype of the present inventionwzxGene P11 and P12 primer detection other serotype standard bacteria of vibrio fluvialis
Strain electrophoresis result figure,wzxGene P11 and the screening of P12 primer, purpose band is 338bp, and remaining serotype does not has any
Band, concrete bacterial strain information is shown in Table 2；
Figure 10 represents O18 serotype of the present inventionwzxGene P11 and P12 primer species specific qualification electrophoresis result figure, its
Middle usewzxGene P11 and P12 primer have detected 6 strain vibrios and 1 strain salmonella, 1 strain escherichia coli, all without any band, and tool
Body bacterial strain information is shown in Table 2；
Figure 11 represents O18 serotype of the present inventionorf18Gene P13 and P14 primer detection other serotype marks of vibrio fluvialis
Quasi-bacterial strain electrophoresis result figure,orf18Gene P13 and the screening of P14 primer, purpose band is 590bp, and remaining serotype does not has
Any band, concrete bacterial strain information is shown in Table 2；
Figure 12 represents O18 serotype of the present inventionorf18Gene P13 and P14 primer species specific qualification electrophoresis result figure,
Wherein useorf18Gene P13 and P14 primer have detected 6 strain vibrios and 1 strain salmonella, 1 strain escherichia coli, all without any
Band, concrete bacterial strain information is shown in Table 2；
Figure 13 represents the electrophoresis result figure expanding corresponding serotype respectively with O2, O4, O13, O15 and O18 special primer, tool
Body bacterial strain information is shown in Table 2；
Wherein: O2, O4, O13, O15, O18 and negative control (-) represent corresponding serotype primer amplification result, first swimming
Road H and last swimming lane are 2000bp ladder marker.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in.Wherein vibrio fluvialis derives from Japan Collection of Microorganisms
(JCM).
Embodiment 1: the extraction of genome
37 DEG C of nutrient broth mediums cultivate vibrio fluvialis, collect antibacterial, extract genome and specifically comprise the following steps that
With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 DEG C of incubations 20 minutes,
The lysozyme being subsequently adding 10ul 10mg/ml continues insulation 20 minutes.Add the E.C. 3.4.21.64 of 3ul 20mg/ml, 15ul afterwards
10%SDS, 50 DEG C of incubations 2 hours, add the RNase of 3 ul 10mg/ml, 65 DEG C of incubations 30 minutes.Add the extracting of equal-volume phenol
Mixture, takes supernatant, then uses isopyknic phenol: chloroform: isoamyl alcohol (25:24:1) solution extracts twice, takes supernatant, then uses
Isopyknic ether extraction is to remove remaining phenol.Supernatant, with 2 times of volume ethanol precipitation DNA, rolls out DNA with glass fiber and uses
DNA washed by 70% ethanol, is finally resuspended in 30ul TE by DNA.Genomic DNA is detected by the agarose gel electrophoresis of 0.4%.
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 carries out genome sequencing to each serotype genome of vibrio fluvialis and obtains the sequence of this serotype, uses
Blast and PSI-Blast carries out sequence alignment, uses TMHMM 2.0 program to carry out transmembrane structure prediction, uses
ClustalW program carries out sequence alignment and screening is conservative and specific gene fragment, final acquisition each serum of vibrio fluvialis
The O antigen gene cluster sequence of type and decoding result.
Embodiment 3: design of primers
The O antigen gene cluster sequence of vibrio fluvialis each serotype of O2, O4, O13, O15 and O18 is that this laboratory is tested oneself
, being analyzed by comparison, we choose Blast comparison result identity and the relatively low gene specific of similarity value
Section design primer.Wherein O2 serotypewzyGene comparison result identity value and similarity value are 44% and 68%,orf9Gene comparison result identity value and similarity value are 44% and 65%；O4 serotypewzxGene comparison result
Identity value and similarity value are 22% and 42%；O13 serotypewzxGene comparison result identity value and
Similarity value is 26% and 48%；O15 serotypeorf13Gene comparison result identity value and similarity value are
27% and 52%；O18 serotypewzxGene comparison result identity value and similarity value are 29% and 53%,orf18Base
Because comparison result identity value and similarity value are 47% and 64%.So each serotype chooses above-mentioned correspondence respectively
Gene separately designs special primer as the specific target gene of this serotype, the gene specific section for each serotype.
Design of primers is the core of this invention.Said gene imports Primer Premier 5 carry out primer and set
Meter, each gene design pair of primers, there is single goal band.
Carrying out BLAST after design of primers in Genbank, the primer of design can not be with the sequence phase of other nearly edge antibacterial
Too high like property, thus can ensure that this primer only expands in the precalculated position of oneself, and not with other nearly edge bacterium or
Nearly edge bacterium in the environment of collect specimen does not produce positive reaction.This point is for avoiding the generation of non-specific band and experiment
Success or failure are particularly significant.
The primer designed is as shown in table 1.
Table 1 is for the primer sequence of PCR
Embodiment 4: the screening of special primer
Have collected vibrio fluvialis 02, the reference culture of O4, O13, O15 and O18 serotype and the mark of other 14 kinds of serotypes
The each strain of quasi-bacterial strain, the vibrio fluvialis of the 11 non-typings of strain, 6 other bacterial strains of strain vibrio, 1 strain Salmonella strain and 1 strain are big
The specificity of enterobacteria host strains primer, strain number and source are shown in Table 2.
Table 2 is for the bacterial strain of specific detection
The PCR system that gene identification primer screening is used is 5 μMs of primer 0.4 μ l, 10 × enzyme spcificity reaction buffers 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 testing sample template to the thin-walled of 0.2ml
In PCR pipe, last ddH2O supplies 25 μ l.All primers all obtain positive findings, at it in the most corresponding serotype
His group does not obtain any PCR primer band.
Reaction cycle parameter in PCR instrument in the step for of include the degeneration of DNA, renaturation, the temperature and time of extension,
Cycle-index, particularly as follows:
Early stage is to enable degeneration to reach required temperature and a circulation of required early stage processing procedure is 95 DEG C, and 5
Minute；
Denaturation temperature and time are 95 DEG C, 45 seconds；
Renaturation temperature and time is 55 DEG C/68 DEG C, 1 minute；
Elongating temperature and time are 72 DEG C, 1 minute；
Degeneration, renaturation, the cycle-index of extension are 35 circulations；
The temperature and time carrying out a circulation for stable amplified production is 72 DEG C, 5 minutes
Wherein, O2 uses 68 DEG C of amplifications, and O4 uses 61 DEG C of amplifications, and O13 uses 55-65 DEG C of amplification, and O15 uses 55-58 DEG C
Amplification, O18 uses 65 DEG C of amplifications.
Above-mentioned steps is electrophoresis amplified production in electrophoresis equipment, concretely comprising the following steps of record result:
Take 2～5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer to mix with the volume ratio of 1:1；
Mixed liquor is splined on the agarose gel of 1.0%；
By agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 20 minutes, compare with DL2000 Marker；
Observe and record result.
After being reacted by basic PC R, the work of primer screening terminates substantially, and necessary length adjustment is to W-response bar
Part impact is little, and the primer sequence used in the present invention is all summed up in Table 1.
Table 1 is for the primer sequence of PCR
The preparation of embodiment 6:PCR detection kit and application
1, the composition of PCR kit:
dNTP(10mM) 30μl；
10 × Buffer (10 × enzyme spcificity reaction buffer) 50 μ l；
Taq polymerase (5U/ μ l hot resistant DNA polymerase) 5 μ l；
PCR primer mixture (5 μMs) 10 μ l；
Positive reference substance (KP) 10 μ l；
Negative controls (KN) 10 μ l；
ddH2O 5ml；
Each test kit can be used for detecting 10 samples.
Wherein 10 × Buffer, dNTP, Taq polymerase are provided by precious biological engineering company limited；Primer mixture is certainly
The sequence of row design is supplied to the synthesis of Shanghai Ying Jun biotech company；Positive reference substance, negative controls and ddH2O is by us
Prepare voluntarily.
2, instrument and equipment
Wherein 10 × Buffer, dNTP, Taq polymerase are provided by the raw work in Shanghai；Primer mixture is the sequence of designed, designed
Row are supplied to the synthesis of Shanghai Ying Jun biotech company；Positive reference substance, negative controls and ddH2O are prepared voluntarily by us.
The equipment PCR instrument (having another name called DNA thermal cycling amplification instrument) of experiment, electrophoresis equipment (including electrophresis apparatus and electrophoresis tank), gel imaging
Instrument ,-20 DEG C of refrigerators, high speed centrifuge, micropipettor and 0.2ml PCR light-wall pipe.
3, the use instantiation of PCR kit
The PCR detection method using above-mentioned PCR kit detection vibrio fluvialis comprises the steps:
(1) environmental sample template to be measured is extracted；
(2) addition in PCR light-wall pipe, dNTP, 10 × Buffer, Taq polymerase, primer, testing sample template and
ddH2O mixes；
(3) mixture of mixing in thin-walled PCR pipe is expanded in 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, sea water etc., or
It is crude extract or the pure dna of the pure culture of vibrio fluvialis, or positive reference substance and negative controls.
The extracting method of the environmental sample template in above-mentioned steps (1) is:
Take 1.5ml culture, be centrifuged 1 minute under the conditions of 12000rpm, remove supernatant；
Take the ddH of 500 μ l2The resuspended precipitation of O, is centrifuged 5 minutes under the conditions of 8000rpm, removes supernatant, dries；
Take 100 μ lddH2The resuspended precipitation of O, water-bath 10 minutes in 100 DEG C of boiling water；
It is placed on ice after 10 minutes again, is centrifuged 2 minutes under the conditions of 12000rpm；
5. 3 μ l middle layer supernatant are taken as pcr template
The reaction cycle parameter in PCR instrument in above-mentioned steps (3) include the degeneration of DNA, renaturation, the temperature of extension and time
Between, cycle-index, particularly as follows:
Early stage is to enable degeneration to reach required temperature and a circulation of required early stage processing procedure is 95 DEG C, and 5
Minute；
Denaturation temperature and time are 95 DEG C, 45 seconds；
Renaturation temperature and time is 55 DEG C/68 DEG C, 1 minute；
Elongating temperature and time are 72 DEG C, 1 minute；
Degeneration, renaturation, the cycle-index of extension are 35 circulations；
The temperature and time carrying out a circulation for stable amplified production is 72 DEG C, 5 minutes.
Above-mentioned steps (4) electrophoresis amplified production in electrophoresis equipment, concretely comprising the following steps of record result:
Take 2～5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer to mix with the volume ratio of 5:1；
Mixed liquor is splined on the agarose gel of 1.0%；
By agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 20 minutes, compare with DL2000 Marker；
Observe and record result.
The present invention by configure a kind of detect vibrio fluvialis can industrialization produce PCR kit, by PCR detection side
Method needs the component used to combine, and during use, extracts testing sample, just may be used through relatively simple operation sequence simultaneously
To carry out quick, sensitive, easy detection, in test kit, consumption and the concentration of each component are test gained, use this test kit
The testing equipment that detection vibrio fluvialis is used is simple, and testing cost is low.
Using positive and negative controls purpose is for the whole operating process of Quality Control, in order to draws and judges accurately.
If containing vibrio fluvialis purpose O antigenic type, then from electrophoresis result it is observed that with the bar of positive reference substance same position
Band；If not containing vibrio fluvialis purpose O antigenic type, then not there is as negative controls this band.
Amount of reagent in the test kit that one-time detection of the present invention test is used see table shown in 3, and DNA profiling amount is 3 μ l
Amount of reagent in the test kit that the test of table 3 one-time detection is used
Hot resistant DNA polymerase in the present invention is Taq enzyme.
Above-mentioned positive reference substance is to have determined that it is the sample of each O antigenic type of vibrio fluvialis, and negative controls is then
Determine it is not the sample of vibrio fluvialis through laboratory.
If this PCR kit bacteria suspension of vibrio fluvialis carries out PCR amplification, with the extracted DNA obtained as template
Acquired results is always.Sensitivity and specificity indifference, so, can save the extraction step of template DNA, make operational approach be able to
Simplify.Meanwhile, for comparing routine biochemistry detection method, the testing sample that this method is used can be directly clinical sample training
Nutrient solution, or detection sample is carried out simple separation cultivate and can be carried out detection, thus save manpower and materials.
4, the offer of testing sample
Have collected vibrio fluvialis O2, O4, O13, O15 and O18 serotype reference culture, 6 other bacterial strains of strain vibrio, 1 strain
Salmonella strain and the specificity of 1 strain coli strain checking primer, strain number and source are shown in Table 2.
SEQUENCE LISTING
<110>Nankai University
<120>to the special nucleotide of vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>artificial sequence
<400> 1
gggtatcaat gacggcg 17
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
gcaaggaaat gcgaacag 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
ggaaaacttc aggcacag 18
<210> 4
<211> 18
<212> DNA
<213>artificial sequence
<400> 4
agaaacccca acaccaac 18
<210> 5
<211> 18
<212> DNA
<213>artificial sequence
<400> 5
atccgttctg atgctgct 18
<210> 6
<211> 19
<212> DNA
<213>artificial sequence
<400> 6
tcgctaaaac ttcattgcc 19
<210> 7
<211> 18
<212> DNA
<213>artificial sequence
<400> 7
tgttaccgat gaccagga 18
<210> 8
<211> 22
<212> DNA
<213>artificial sequence
<400> 8
aataaataag agcacagacg ag 22
<210> 9
<211> 19
<212> DNA
<213>artificial sequence
<400> 9
tggaatggaa ataacgaat 19
<210> 10
<211> 19
<212> DNA
<213>artificial sequence
<400> 10
tcacagatgt agcaggcag 19
<210> 11
<211> 20
<212> DNA
<213>artificial sequence
<400> 11
aaacagagga atcaccgaac 20
<210> 12
<211> 20
<212> DNA
<213>artificial sequence
<400> 12
gcgtattaaa tgcaacacgt 20
<210> 13
<211> 19
<212> DNA
<213>artificial sequence
<400> 13
ggaactttta acttagcgg 19
<210> 14
<211> 18
<212> DNA
<213>artificial sequence
<400> 14
tggcattgtc atccatac 18
Claims (7)
1. pair combination nucleotide that vibrio fluvialis O2, O4, O13, O15 and O18 serotype is special, it is characterised in that described combination
Nucleotide is shown in SEQ ID NO:1-14.
2. the combination nucleotide described in claim 1 is used in preparation detection vibrio fluvialis O2, O4, O13, O15 and O18 serotype
Application in terms of PCR kit, gene chip or microarray；Described vibrio fluvialis samples in tap water, river water, the training of sea water
Support the crude extract of thing, or the crude extract of the pure culture of vibrio fluvialis.
3. a PCR kit, including PCR primer, dNTP, buffer and archaeal dna polymerase, described PCR primer is such as SEQ ID
Nucleotide shown in NO:1-14.
4. the test kit described in claim 3, also includes following reagent: 10 mM dNTP 30 μ l；10 × enzyme spcificity is reacted
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 controls 10 μ
l；ddH2O 1ml。
5. described in claim 1, the combination nucleotide of SEQ ID NO:1-14 is used for detecting vibrio fluvialis PCR kit in preparation
The application of aspect.
6. the combination nucleotide of SEQ ID NO:1-14 described in claim 1 is used for detecting the gene chip of vibrio fluvialis in preparation
The application of aspect.
7. the combination nucleotide of SEQ ID NO:1-14 described in claim 1 is in terms of preparation is used for detecting vibrio fluvialis microarray
Application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410158813.3A CN103898108B (en) | 2014-04-21 | 2014-04-21 | The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410158813.3A CN103898108B (en) | 2014-04-21 | 2014-04-21 | The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103898108A CN103898108A (en) | 2014-07-02 |
| CN103898108B true CN103898108B (en) | 2016-09-28 |
Family
ID=50989679
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410158813.3A Active CN103898108B (en) | 2014-04-21 | 2014-04-21 | The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103898108B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN105154438B (en) * | 2015-08-04 | 2018-10-26 | 南开大学 | To Hafnia alvei G5907, G5908, G5913, nucleotide special G5916 and its application |
| 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 |
| 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 |
| 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 |
| 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 |
| CN112375836B (en) * | 2020-11-30 | 2023-03-31 | 南开大学 | Real-time fluorescence PCR detection method for vibrio fluvialis and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560273A (en) * | 2004-02-26 | 2005-01-05 | 中山大学 | Kit for diagnosing gene of pathogenic bacterial and river vibrion of aquatic animal and human and testing method thereof |
| CN101020922A (en) * | 2007-01-18 | 2007-08-22 | 中国科学院南海海洋研究所 | Reagent kit and process for detecting river vibrio in circular mediated constant temperature amplification method |
-
2014
- 2014-04-21 CN CN201410158813.3A patent/CN103898108B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560273A (en) * | 2004-02-26 | 2005-01-05 | 中山大学 | Kit for diagnosing gene of pathogenic bacterial and river vibrion of aquatic animal and human and testing method thereof |
| CN101020922A (en) * | 2007-01-18 | 2007-08-22 | 中国科学院南海海洋研究所 | Reagent kit and process for detecting river vibrio in circular mediated constant temperature amplification method |
Non-Patent Citations (2)
| Title |
|---|
| Species-specific identification of Vibrio fluvialis by PCR targeted to the conserved transcriptional activation and variable membrane tether regions of the toxR gene;C Rupa,et al;《Journal of medical microbiology》;20061231;第55卷(第6期);805-8 * |
| 基于tox R 基因的河流弧菌PCR 快速检测方法的建立;文万侥 等;《水产科学》;20091031;第28卷(第10期);575-578 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103898108A (en) | 2014-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103898108B (en) | The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof | |
| CN104059975B (en) | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof | |
| CN104862393A (en) | Nucleotide specific to Cronobacter O antigen and application thereof | |
| CN103993090B (en) | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof | |
| CN103451310B (en) | Gene chip capable of simultaneously detecting various vibrios and method for detecting vibrios | |
| CN105200044B (en) | The nucleotides special to vibrio fluvialis O1, O6, O7, O8 and O9 and its application | |
| CN105200045B (en) | The nucleotides special to vibrio fluvialis O11, O14, O16 and O17 and its application | |
| CN102154270B (en) | Cronobacter sakazakii O antigen specific nucleotides and use thereof | |
| CN105256041B (en) | The nucleotide special to aeromonas hydrophila O44, O24, O25 and O28 and application | |
| CN105154559A (en) | Specific nucleotide for vibrio parahaemolyticus K36, K37 and K68 and application thereof | |
| CN105256042B (en) | The nucleotide special to aeromonas hydrophila O13, O36, O16 and O19 and application | |
| CN105112406B (en) | To Hafnia alvei G5897, G5898, G5900 special nucleotide and its application | |
| CN105154438B (en) | To Hafnia alvei G5907, G5908, G5913, nucleotide special G5916 and its application | |
| CN105154439B (en) | To Hafnia alvei G5902, G5903, G5904, G5906 special nucleotide sequence | |
| CN105087569B (en) | To vibrio cholerae O 18, O19, O23 and the special nucleotides of O12 and its application | |
| CN105177133B (en) | The nucleotide special to comma bacillus O6, O4, O7 and O15 and its application | |
| CN105256028B (en) | The nucleotide special to citric acid bacillus 017 and O39 and its application | |
| CN105256043B (en) | The nucleotide special to aeromonas hydrophila O29, O30, O33 and O35 and application | |
| CN105255871B (en) | The nucleotide special to aeromonas hydrophila O7, O8, O9 and O10 and application | |
| CN108715899A (en) | Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence | |
| CN105177144A (en) | Nucleotide for specificity of K4, K32 and K34 of vibrio parahaemolyticus and application thereof | |
| CN108715897A (en) | Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence | |
| AU2020104336A4 (en) | Application of Riemerella anatipestifer Imp gene and the PCR Assay Kit and Method thereof | |
| CN110791579B (en) | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method for identifying chlamydophila abortus | |
| CN102337261A (en) | Neisseria meningitidis antigen-specific nucleotide and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |