CN105256028B - The nucleotide special to citric acid bacillus 017 and O39 and its application - Google Patents
The nucleotide special to citric acid bacillus 017 and O39 and its application Download PDFInfo
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- CN105256028B CN105256028B CN201510681496.8A CN201510681496A CN105256028B CN 105256028 B CN105256028 B CN 105256028B CN 201510681496 A CN201510681496 A CN 201510681496A CN 105256028 B CN105256028 B CN 105256028B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The present invention relates to citric acid bacillus 017 and O39 serotypes special nucleotide and its application, the nucleotide is SEQ ID NO:At least one of nucleotide shown in 1-4.These nucleotide can be used for preparing detection citric acid bacillus PCR kit.The citric acid bacillus can be sampled in the crude extract of alimentary canal, urine, blood and wound culture, can also be isolated from human and animal's excrement environment.The present invention nucleotide special to citric acid bacillus 017 and O39 serotypes and comprising the PCR kit of the nucleotide, PCR kit preparation method is easy, and detection cycle is short, speed is fast, and operability is strong, is easy to industrialization production, and testing cost is relatively low；Accuracy is high；High sensitivity.
Description
Technical field
The present invention relates to the nucleotide special to citric acid bacillus 017 and O39 serotypes, more particularly to citric acid bacillus
Individual gene special nucleotide and its application in 017 and O39 serotype O antigen gene clusters.
Background technology
Citric acid bacillus（Citrobacter）It is a kind of common Gram-negative bacteria in enterobacteriaceae, it usually can profit
It uses citric acid as unique carbon source, belongs to facultative anaerobic bacteria.Citric acid bacillus and salmonella and Escherichia coli affiliation
Recently, it is a kind of conditioned pathogen.
Citric acid bacillus can be divided into 11 gene kinds, respectively citrobacter freundii by the method for DNA hybridization
(C.freundii), Coriolis citric acid bacillus (C.koseri), no malonate citric acid bacillus
(C.amalonatIzcus), Fa Shi citric acid bacillus (C.farmeri), Young citric acid bacillus (C.youngae), Bu Shi
Citric acid bacillus (C.braakii) irrigates gram Mans citric acid bacillus (C.werkmanii), Sai Lakeshi citric acid bacillus
(C.sedlakif), rodent citric acid bacillus (C.rodentium), lucky Lun Shi citric acid bacillus (C.gillenii), mouse
Class citric acid bacillus (C.murliniae), wherein to the mankind have it is apparent it is pathogenic be citrobacter freundii
(C.freundii) and Coriolis citric acid bacillus (C.koseri).
Citrobacter freundii can cause the peritonitis of people；Coriolis citric acid bacillus can be in baby and immunologic inadequacy person
In cause meningitis, central nervous system abscess and septicemia, respiratory tract and the urinary tract infection of the elderly can be caused, moreover it is possible to cause
The infection etc. of skin；Some other citric acid bacillus can be infected in the intraperitoneal of immunosuppressed patient.In addition, rodent
Citric acid bacillus without apparent pathogenic, but is the important mucous membrane pathogenic bacteria of mouse, multiple pathogenic mechanisms and enteron aisle to human body
Enteropathogenic E. Coli（EPEC）And enterohemorrhagic escherichia coli（EHEC）It is identical to the pathogenic mechanism of people, so, rodent lemon
Lemon acidfast bacilli is often used as models of the research EPEC and EHEC to human body cause illness to the pathogenic of mouse.
Citric acid bacillus is mainly colonized in human and animal's enteron aisle, exists in the environment such as soil, water, sewage, food
In, it also can be isolated from alimentary canal, urine, blood and wound, it can also divide from the environment with human and animal's excrement
From obtaining.
Typing of bacteria mainly has traditional phenotypic approach, serological method and method for identifying molecules with identification method.However,
There is certain limitations for traditional serotype and identification method, and classifying method needs to prepare a system such as serotype
Row specific antisera, and antiserum general classes are not complete, lazy weight, the antiserum that obtain specificity is cumbersome,
It prepares and storage all has some difficulties；Time-consuming, sensitivity is low, omission factor is high for another aspect serotype method, accuracy
Difference, different O antigens generate antiserum between be frequently present of cross reaction.Therefore, it establishes based on Protocols in Molecular Biology
Serological diagnosis method becomes developing direction.
The Molecular Identification of citric acid bacillus is increasingly valued by people, and is become citric acid bacillus strain and is identified with plant type
Important evidence, also therefore produce many new serotypes.Molecular Biological Detection technology has speed fast and is easy to advise greatly
The advantages of mould uses is the developing direction for the pathogenic bacteria detection generally acknowledged in the world at present, but current difficult point is DNA target mark point
Sub- specificity is relatively low.The diversity of bacterial surface polysaccharides antigen is caused by the genetic diversity for being responsible for its gene cluster synthesized
's.For a long time, people always strive to explore the diversity of this important virulence factor of the surface polysaccharide antigen of citric acid bacillus
Situation and corresponding genetic evolution basis.But macro-progress is very slow in the research direction, for its polysaccharide antigen diversity
With the understanding in terms of variation law is basic or blank, such as：People do not know about in citric acid bacillus that there are how many kinds of surfaces still
Polysaccharide antigen and which surface polysaccharide antigen are of great significance for the pathogenic and popular of the bacterium, and it is more to be responsible for its surface
The gene cluster of sugar antigens synthesis is not yet positioned.The main reason for causing above-mentioned phenomenon be：Polysaccharide antigen synthetic gene cluster group
Difficulty etc. is identified at more difficult pre-, the sugared synthetic gene of complicated, gene function.In addition, the unit for having carried out part correlative study is most
Lack early-stage study basis..
In this project, we will be on the basis for obtaining citric acid bacillus all surfaces polysaccharide antigen gene cluster sequence information
On, citric acid bacillus molecule serological typing system and rapid detection method are established, there is important scientific meaning and stronger
Application value.
In recent years, more and more molecular engineerings are used for parting, identification, detection and the disease screening of pathogen, including turn
Record spacer region (ITS) sequence analysis, random amplification length polymorphism (RAPD) is analyzed, rDNA Restriction Fragment Length is more
State property (RFLP) analysis etc..Molecular biology method cannot be only used for the rapid serum parting screening of citric acid bacillus, stablize
Qualification result can make up the deficiency of phenotypic characteristic identification method.It is compared with traditional sensing techniques, these are based on polymerase chain type
The molecular detection technology for reacting (PCR) needs not move through the processes such as separation, the pure culture of pathogen, and with quick, spirit
The advantages that quick, high specificity.
Polymerase chain reaction technology (Polymerase chain reaction, abbreviation round pcr) is examined as microorganism
Survey technology is currently accepted and is promoted, which has high throughput, detection speed fast, specific relative to traditional method
By force, the advantages that high sensitivity, simply pre- increasing bacterium need to be only carried out to sample or increases bacterium process, then is prepared carefully by centrifuging and cracking
Bacterium DNA profiling, so that it may with high specific primer mediation under PCR during expand target sequence, reach detection sample in be
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 examination undoubtedly greatly improves operating rate and reduces job costs.No matter from the perspective of internal and international, soon
Speed accurately identifies serum type, and for the prevention and control of citric acid bacillus, to provide effective technical support be highly important.
Invention content
The object of the present invention is to provide the present invention relates to the nucleosides special to citric acid bacillus 017 and O39 serotypes
Acid, it is characterised in that the nucleotide has：
1）SEQ ID NO:At least one of nucleotide shown in 1-4；
2）With SEQ ID NO:The nucleotide of nucleotide complementation, i.e. SEQ ID NO shown in 1-4:At least one in 5-8
Kind；
The SEQ ID NO:1-4 is as follows：
The SEQ ID NO:5-8 is as follows：
It is described the present invention also provides a kind of PCR kit, including PCR primer, dNTP, buffer solution and archaeal dna polymerase
PCR primer is such as SEQ ID NO:At least pair of primers in nucleotide shown in 1-4.The kit further includes as follows
Reagent：10 mM dNTP 30 μl；10 × enzyme spcificity reaction buffer, 50 μ l；5 μ l of 5U/ μ l hot resistant DNA polymerases；Draw
10 μ l of object mixture；10 μ l of positive reference substance；10 μ l of negative controls；ddH2O 5 ml.Wherein the PCR primer is excellent
It is selected as described such as SEQ ID NO:At least one of nucleotide shown in 1-4.
The present invention further discloses the SEQ ID NOs special to citric acid bacillus 017 and O39 serotypes:1-4 nucleotide
Application in terms of preparing the PCR kit for detecting citric acid bacillus 017 and O39 serotypes.Citric acid of the present invention
Bacillus can sample crude extract or human and animal's excrement environment training in alimentary canal, urine, blood and wound culture
The crude extract etc. that foster object obtains.Collecting citric acid bacillus extraction genome is prepared using conventional method.
For the PCR kit of citric acid bacillus, entire detecting step includes sample pretreatment-amplification-electrophoresis detection
As a result.Primer and the required reagent of PCR reaction systems have been previously added in amplification pipe, and user only need to be by pretreated sample
This addition expands pipe and starts amplified reaction, and simple and quick completion detects work.
The nucleotide special to citric acid bacillus 017 and O39 serotypes disclosed by the invention compared with prior art, this hair
It is bright to have the following advantages that：
（1）It is highly practical
A kind of PCR reaction systems that the present invention establishes can detect citric acid bacillus, provide used in serotype detection
Special primer can be detected clinical samples using the PCR method.
（2）Accuracy is high
The present invention is reacted by the PCR of the special gene of each serotype to citric acid bacillus, and each sample obtains one
Purpose band will obtain target fragment and compare with known length, so that it may to obtain the serotype belonging to citric acid bacillus.
（3）Testing cost is relatively low
Food Hygiene Surveillance, environmental monitoring, the still fields such as product supervision and inspection quarantine can be applied to, and not for other
Technology mode is provided with pathogenic bacteria detection combination.
Description of the drawings：
Fig. 1 shows 017 serotypes of the inventionwzmGene P1 and P2 primer detection citric acid bacillus and other serotype standards
Bacterial strain electrophoresis result figure,wzmThe screening of gene P1 and P2 primer, purpose band are 327 bp, remaining serotype is not any
Band, specific bacterial strain information are shown in Table 3；
Fig. 2 indicates 017 serotype of the inventionwzmThe species specific identification electrophoresis result figure of gene P1 and P2 primer, wherein
4 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli are had detected, without any band, specific bacterial strain information is shown in Table 3；
Fig. 3 indicates O39 serotypes of the present inventionwzyOther serotype standard bacterias of gene P3 and P4 primer detection citric acid bacillus
Strain electrophoresis result figure,wzyThe screening of gene P3 and P4 primer, purpose band are 284 bp, remaining serotype does not have any
Band, specific bacterial strain information are shown in Table 3；
Fig. 4 indicates O39 serotypes of the present inventionwzyThe species specific identification electrophoresis result figure of gene P3 and P4 primer, wherein
4 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli are had detected, without any band, specific bacterial strain information is shown in Table 3；
Fig. 5 indicates to expand the electrophoresis result figure of corresponding serotype, specific bacterial strain information with 017 and O39 special primers respectively
It is shown in Table 3.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as Sambrook et al., molecular cloning：Laboratory manual（New York: Cold Spring Harbor Laboratory
Press,1989）Described in condition.Wherein citric acid bacillus derives from Polish Academy of Sciences.
Embodiment 1：The extraction of genome
37 DEG C of brain heart infusion broth medium culture citric acid bacillus collect bacterium, with bacterial genomes extracts kit
（Health is century CW0552）Genome is extracted, is as follows：
1. taking bacterial cultures 1-5 ml（106-108 cell, no more than 2 × 109 cells）, 10000
Rpm centrifuges 1 minute, abandons net supernatant.
2. 180 μ l Buffer GTL are added into precipitation, concussion makes thalline be resuspended.
3. 20 μ l Proteinase K are added, vortex mixing, 56 DEG C of incubations crack completely up to thalline, are incubated process
In at regular intervals overturn or shake centrifuge tube so that sample is disperseed.
4. the RNase solution As of a concentration of 100 mg/ml of 4 μ l are added（Article No. CW0601）, vortex mixing, room temperature incubates
It educates 5 minutes, then carries out step 5.
5. adding 200 μ l Buffer GL, the concussion that is vortexed mixes well；Add 200 μ l absolute ethyl alcohols, the concussion that is vortexed is abundant
Mixing.
6. by previous step acquired solution（Precipitation including formation）All it is added in Spin Column DM（Spin
Column DM have been put into Collection Tube）, 10000 rpm, centrifuge 1 minute, waste liquid is abandoned, by Spin Column DM
It puts back in Collection Tube.
7. 500 μ l Buffer GW1 are added into Spin Column DM（It is please first checked whether before use and nothing has been added
Water-ethanol）, 10000 rpm, centrifuge 1 minute, abandon waste liquid, Spin Column DM put back in Collection Tube.
8. 500 μ l Buffer GW2 are added into Spin Column DM（It is please first checked whether before use and nothing has been added
Water-ethanol）, 10,000 rpm（~20,000 x g）, centrifuge 1 minute, abandon waste liquid, Spin Column DM are put back to
In Collection Tube.
9. 12,000 rpm centrifuge 2 minutes, abandon waste liquid.Spin Column DM are placed in and are placed at room temperature for several minutes, with
Thoroughly dry Buffer GW2 remaining in sorbing material.
Pay attention to：The purpose of this step is to remove rinsing liquid remaining in Spin Column DM, ethyl alcohol in rinsing liquid
Residual can influence subsequent enzymatic reaction（Digestion, PCR etc.）.
10. Spin Column DM are transferred in a new centrifuge tube, 80 μ l are vacantly added dropwise to the intermediate position of film
Buffer GE or aqua sterilisa are placed at room temperature for 1-5 minutes, 10000 rpm, centrifuge 1 minute, solution is collected into centrifuge tube.-
20 DEG C of preservation DNA.,
Pay attention to：1）If downstream experiment is sensitive to pH value or EDTA, sterilizing water elution can be used.The pH value pair of eluent
Elution efficiency has a significant impact, and should ensure that its pH value in 7.0-8.5 if doing eluent with water（It can be with NaOH by the pH value tune of water
To this range）, elution efficiency is not high when pH value is less than 7.0.
2）Incubation at room temperature can increase yield in 5 minutes before centrifugation.
3）Yield can be increased by being eluted again with 200 other μ l Buffer GE or aqua sterilisa.
4）If improving the final concentration of DNA, the DNA eluents obtained by step 9 can be added on adsorbed film again,
Repeat step 9；If elution volume is less than 200 μ l, the final concentration of DNA can be increased, but total output may be reduced.If DNA
Amount be less than 1 μ g, recommend with 50 μ l Buffer GE or sterilize water elution.
5）Because the DNA preserved in water can be influenced by acidic hydrolysis effect, if you need to long-term preservation, recommend to use
Buffer GE elutions are simultaneously preserved in -20 DEG C.
Embodiment 2:Sequence is decoded
The genome for extracting citric acid bacillus 017 and O39 serotype reference cultures, is sequenced by Solexa pair-end
Technology carries out citric acid bacillus each serotype genome the sequence that genome sequencing obtains the serotype, with Blast
And PSI-Blast carries out sequence alignment, transmembrane structure prediction is carried out using 2.0 program of TMHMM, with ClustalW
Program carries out sequence alignment and screening is conservative and specific gene segment, the final O for obtaining each serotype of citric acid bacillus are anti-
Protogene cluster sequence and decoding result.
Embodiment 3：Design of primers
According to gene cluster decode situation, it has been found that wzm andwzyGene is strictly the special gene of serotype, so choosing
The gene specific section is taken to design special primer.
Design of primers is the core of the invention.Design primer is designed according to the specific gene described in document.wzm
WithwzyGene is gene more special in citric acid bacillus O antigen gene clusters, can be as the target gene of Serotype Identification.
Said gene is imported into Primer Premier 5 and carries out design of primers, the length of primer is preferably between 18~24 bp, Tm values
At 55 DEG C or so.Each gene designs pair of primers, there is single goal band.
BLAST is carried out in Genbank after design of primers, the primer of design cannot be with the sequence phase of other nearly edge bacteriums
It is excessively high like property, ensure that the primer is only expanded in the precalculated position of oneself in this way, without with other nearly edge bacterium or
Nearly edge bacterium in the environment of collect specimen do not generate positive reaction, and this point is for avoiding the generation and experiment of non-specific band
Success or failure are particularly significant.The respective complementary sequence of designed primer can also serve as carrying out specific amplification for PCR primer.
The primer designed is as shown in table 1, and complementary series is as shown in table 2.
Table 1 is used for the primer sequence of PCR
Complementary series of the table 2 for the primer of PCR
Embodiment 4：The screening of special primer
The reference culture of the reference culture and other 10 kinds of serotypes that have collected citric acid bacillus 017 and O39 serotypes is each
One plant, 4 plants of vibrio bacterial strains, the specificity of 1 plant of Salmonella strain and 1 plant of coli strain verification primer, bacterial strain compiles
Number and source be shown in Table 3.
Table 3 is used for the bacterial strain of specific detection
The PCR system that identified for genes primer screening is used is 5 μM of 0.4 μ l of primer, 10 × enzyme spcificity reaction buffers
2.5 μ l, 10 mM dNTP, 0.25 μ l, 5U/ μ l hot resistant DNA polymerases 0.2 μ l and 3 μ l sample to be tested template to 0.2
In the thin-walled PCR pipe of ml, last ddH2O is supplied to 25 μ l.All primers all obtain the positive in respectively corresponding serotype
As a result, not obtaining any PCR product band in other groups.
The reaction cycle parameter in PCR instrument in the step include the denaturation of DNA, renaturation, extension temperature and time,
Cycle-index, specially：
Early period is so that denaturation is reached required temperature and a cycle of required pre-processing process is 95 DEG C, 5
Minute；
Denaturation temperature and time are 95 DEG C, 30 seconds；
Renaturation temperature and time is 58 DEG C, 30 seconds；
Elongating temperature and time are 72 DEG C, 45 seconds；
Denaturation, renaturation, the cycle-index of extension are 35 cycles；
The temperature and time that a cycle is carried out to stablize amplified production is 72 DEG C, 10 minutes.
Above-mentioned steps electrophoresis amplified production in electrophoresis equipment, record result the specific steps are：
Take 2~5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer with 1：1 volume ratio mixing；
Mixed liquor is splined on 2.0% Ago-Gel；
By 120 v voltage stabilizings electrophoresis of agarose gel electrophoresis about 20 minutes, compareed with DL2000 Marker；
Observe and record result.
Terminated substantially by the work of primer screening after basic PC R reactions, necessary length adjustment is to W-response item
Part influences less, and the primer sequence used in the present invention is all summarised in table 1, and complementary series is all summarised in table 2.
Table 1 is used for the primer sequence of PCR
The complementary series of 2 PCR primer of table
Embodiment 5：The preparation and application of PCR detection kit
1, the composition of PCR kit：
dNTP(10mM) 30 μl；
10 × Buffer (10 × enzyme spcificity reaction buffer), 50 μ l；
Taq polymerase（5U/ μ l hot resistant DNA polymerases） 5 μl；
PCR primer mixture（5μM） 10 μl；
10 μ l of positive reference substance (KP)；
Negative controls（KN） 10 μl；
ddH2O 5 ml；
Each kit can be used for detecting 10 samples.
Wherein 10 × Buffer, dNTP, Taq polymerase are provided by precious bioengineering Co., Ltd；Primer mixture is certainly
The sequence of row design is supplied to Shanghai Ying Jun biotech companies to synthesize；Positive reference substance, negative controls and ddH2O is by us
Voluntarily prepare.
2, instrument and equipment
Wherein 10 × Buffer, dNTP, Taq polymerase are provided by Shanghai life work；Primer mixture is the sequence of designed, designed
Row are supplied to Shanghai Ying Jun biotech companies to synthesize；Positive reference substance, negative controls and ddH2O is voluntarily prepared by us.
The equipment PCR instrument of experiment（Also known as DNA thermal cycling amplification instrument）, electrophoresis equipment（Including electrophoresis apparatus and electrophoresis tank）, gel imaging
Instrument, -20 DEG C of refrigerators, supercentrifuge, micropipettor and 0.2 ml PCR thin-wall tubes.
3, the use specific example of PCR kit
The PCR detection method that citric acid bacillus is detected using above-mentioned PCR kit is included the following steps：
（1）Extract environmental sample template to be measured；
（2）In PCR thin-wall tubes addition, dNTP, 10 × Buffer, Taq polymerase, primer, sample to be tested template and
ddH2O mixings；
（3）The mixture of mixing in thin-walled PCR pipe is expanded in PCR instrument；
（4）The electrophoresis amplified production in electrophoresis equipment records result；
（5）It analyzes and carries 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
It is the crude extract or pure dna or positive reference substance and negative controls of the pure culture of citric acid bacillus.Optimization citric acid
The crude extract or pure dna of the pure culture of bacillus.
Above-mentioned steps（1）In the extracting method of environmental sample template be：
1.5 ml cultures are taken, is centrifuged 1 minute under the conditions of 12000 rpm, removes supernatant；
Take the ddH of 500 μ l2Precipitation is resuspended in O, is centrifuged 5 minutes under the conditions of 8000 rpm, removes supernatant, dries；
Take 100 μ lddH2Precipitation, water-bath 10 minutes in 100 DEG C of boiling water are resuspended in O；
It is placed on ice after ten minutes, is centrifuged 2 minutes under the conditions of 12000 rpm again；
5. taking 3 μ l middle layer supernatants as pcr template
Above-mentioned steps（3）In PCR instrument on reaction cycle parameter include the denaturation of DNA, renaturation, the temperature of extension and when
Between, cycle-index, specially：
Early period is so that denaturation is reached required temperature and a cycle of required pre-processing process is 95 DEG C, 5
Minute；
Denaturation temperature and time are 95 DEG C, 30 seconds；
Renaturation temperature and time is 58 DEG C, 30 seconds；
Elongating temperature and time are 72 DEG C, 45 seconds；
Denaturation, renaturation, the cycle-index of extension are 35 cycles；
The temperature and time that a cycle is carried out to stablize amplified production is 72 DEG C, 10 minutes.
Above-mentioned steps（4）The electrophoresis amplified production in electrophoresis equipment, record result the specific steps are：
Take 2~5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer with 5：1 volume ratio mixing；
Mixed liquor is splined on 1.0% Ago-Gel；
By 120 v voltage stabilizings electrophoresis of agarose gel electrophoresis about 20 minutes, compareed with DL2000 Marker；
Observe and record result：As a result see attached drawing, be described as follows：
Fig. 1 shows 017 serotypes of the inventionwzmGene P1 and P2 primer detection citric acid bacillus and other serotype standards
Bacterial strain electrophoresis result figure,wzmThe screening of gene P1 and P2 primer, purpose band are 327 bp, remaining serotype is not any
Band, specific bacterial strain information are shown in Table 3；
Fig. 2 indicates 017 serotype of the inventionwzmThe species specific identification electrophoresis result figure of gene P1 and P2 primer, wherein
4 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli are had detected, without any band, specific bacterial strain information is shown in Table 3；
Fig. 3 indicates O39 serotypes of the present inventionwzyOther serotype standard bacterias of gene P3 and P4 primer detection citric acid bacillus
Strain electrophoresis result figure,wzyThe screening of gene P3 and P4 primer, purpose band are 284 bp, remaining serotype does not have any
Band, specific bacterial strain information are shown in Table 3；
Fig. 4 indicates O39 serotypes of the present inventionwzyThe species specific identification electrophoresis result figure of gene P3 and P4 primer, wherein
4 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli are had detected, without any band, specific bacterial strain information is shown in Table 3；
Fig. 5 indicates to expand the electrophoresis result figure of corresponding serotype, specific bacterial strain information with 017 and O39 special primers respectively
It is shown in Table 3.
The present invention by configure a kind of detectable citric acid bacillus can industrialization production PCR kit, PCR is detected
Method needs component to be used to combine, in use, extraction sample to be tested, while just by relatively simple operation sequence
It can carry out quick, sensitive, easy detection, the dosage and concentration of each component are experiment gained in kit, with the reagent
Testing equipment is simple used in box detection citric acid bacillus, and testing cost is low.
The use of positive and negative controls purpose is to be used for Quality Control whole operation process, to obtain accurate judgement.
If containing citric acid bacillus purpose O antigenic types, from electrophoresis result it is observed that with positive reference substance same position
Band；If not containing citric acid bacillus purpose O antigenic types, do not have this band as negative controls.
Amount of reagent in kit used in one-time detection experiment of the present invention see the table below shown in 4, and DNA profiling amount is 3 μ l
Amount of reagent in kit used in the experiment of 4 one-time detection of table
Hot resistant DNA polymerase in the present invention is Taq enzyme.
Above-mentioned positive reference substance be have determined that be each O antigenic types of citric acid bacillus sample, negative controls are then
To determine the sample for not being citric acid bacillus through laboratory.
If the bacteria suspension of this PCR kit citric acid bacillus carries out PCR amplification, with extracted obtained DNA as mould
Plate acquired results are consistent.Susceptibility and specific indifference make operating method obtain in this way, the extraction step of template DNA can be saved
With simplification.Meanwhile compared to for routine biochemistry detection method, sample to be tested used by this method can directly be clinical sample
Culture solution, or simple separation culture is carried out to detection sample and can be carried out detecting, thus save manpower and materials.
4, the offer of sample to be tested
Have collected citric acid bacillus 017 and O39 serotype reference cultures, 4 plants of vibrio other bacterial strains, 1 plant of Salmonella
The specificity of bacterial strain and 1 plant of coli strain verification primer, strain number and source are shown in Table 3.
The above is only operation and the implementation of the present invention, not makees limit in any form to the present invention
System, it is every according to the technical essence of the invention to any simple modification, equivalent change and modification made by above example, still
Belong in the range of technical solution of the present invention.
SEQUENCE LISTING
<110>Nankai University
<120>The nucleotide special to citric acid bacillus 017 and O39 and its application
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
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ctggcatctt tggactacat ta 22
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ttatggcaaa gcatcatgaa 20
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence
<400> 3
agagtggaag ctgcggg 17
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
gcagaaccca gcaaagac 18
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<211> 22
<212> DNA
<213>Artificial sequence
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gaccgtagaa acctgatgta at 22
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
aataccgttt cgtagtactt 20
<210> 7
<211> 17
<212> DNA
<213>Artificial sequence
<400> 7
tctcaccttc gacgccc 17
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<400> 8
cgtcttgggt cgtttctg 18
Claims (3)
1. the special nucleotide combination of 017 serotype of pair citric acid bacillus, it is characterised in that the nucleotide combination is：SEQ
ID NO: 1-2 ,SEQ ID NO:Nucleotide shown in 5-6.
2. a kind of PCR kit, including PCR primer, dNTP, buffer solution and archaeal dna polymerase, the PCR primer is SEQ ID
NO: 1-2 ,SEQ ID NO:Nucleotide shown in 5-6.
3. the nucleotide combination special to 017 serotype of citric acid bacillus described in claim 1 is being prepared for detecting lemon
Application in terms of acidfast bacilli PCR kit；The citric acid bacillus is sampled in alimentary canal, urine, blood and wound culture
Crude extract or the crude extract that is obtained from human and animal's excrement environment culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| EP1927664A1 (en) * | 2006-11-28 | 2008-06-04 | Canon Kabushiki Kaisha | Primer for bacterium genome amplification reaction |
| CN102181547A (en) * | 2011-04-13 | 2011-09-14 | 北京三元食品股份有限公司 | PCR (polymerase chain reaction) synchronous detection kit for staphylococcus aureus enterotoxin A and B genes |
| CN103898227A (en) * | 2014-04-16 | 2014-07-02 | 南开大学 | Nucleotide specific to cronobacter O antigen and application of nucleotide |
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| EP1927664A1 (en) * | 2006-11-28 | 2008-06-04 | Canon Kabushiki Kaisha | Primer for bacterium genome amplification reaction |
| CN102181547A (en) * | 2011-04-13 | 2011-09-14 | 北京三元食品股份有限公司 | PCR (polymerase chain reaction) synchronous detection kit for staphylococcus aureus enterotoxin A and B genes |
| CN103898227A (en) * | 2014-04-16 | 2014-07-02 | 南开大学 | Nucleotide specific to cronobacter O antigen and application of nucleotide |
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