CN104059975B - To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof - Google Patents
To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof Download PDFInfo
- Publication number
- CN104059975B CN104059975B CN201410281515.3A CN201410281515A CN104059975B CN 104059975 B CN104059975 B CN 104059975B CN 201410281515 A CN201410281515 A CN 201410281515A CN 104059975 B CN104059975 B CN 104059975B
- Authority
- CN
- China
- Prior art keywords
- providence
- nucleotide
- serotype
- primer
- pcr
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 serotype is special and application thereof, does is described Nucleotide SEQ? ID? at least one in Nucleotide shown in NO:1-12.These Nucleotide can be used for preparation and detect Providence PCR kit and gene chip.Described Providence can sample the crude extract of the culture in diarrhea stool, urinary tract infection, wound, burn and microbemia sample, or the crude extract etc. of the pure growth of Providence.The present invention is to Providence O3, O4, O8, the Nucleotide that O12, O13 and O20 serotype is special and comprise PCR kit, the gene chip of this Nucleotide, PCR kit compound method is easy, sense cycle is short, speed is fast, workable, be easy to industrialization and produce, and testing cost is relatively low; Accuracy is high; Highly sensitive.
Description
Technical field
The present invention relates to Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 serotype is special, particularly relate to Providence O3, the Nucleotide that in O4, O8, O12, O13 and O20 serotype O antigen gene cluster, individual gene is special and application thereof.
Background technology
Providence (Providencia) is a kind of common Gram-negative bacteria in enterobacteriaceae, is a kind of opportunistic pathogen.Infect this bacterium mainly through ward infection or edible seafood, can respiratory tract infection be caused, also can cause septicemia, urinary system infection, Secondary cases meningitis etc.Be located away from diarrhea stool, urinary tract infection, wound, burn and microbemia sample.
Typing of bacteria and authentication method mainly contain traditional phenotypic approach, serological method and molecular assay method.But along with molecular biological development, traditional serotype and authentication method also 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, a large amount of antiserum(antisera)s is in preparation and also there are some difficulties in storing.On the other hand serotype method length consuming time, sensitivity is low, loss is high, poor accuracy, often there is cross reaction between the antiserum(antisera) that different O antigen produces.Therefore, the serological diagnosis method set up based on Protocols in Molecular Biology becomes developing direction.
The Molecular Identification of Providence is more and more subject to people's attention, and becomes the important evidence that Providence bacterial classification and plant type are identified, therefore many new bacterium also produce.It is fast and be easy to the extensive advantage used that molecular Biological Detection technology has speed, be the developing direction of the pathogenic microbes detect of generally acknowledging in the world at present, but current difficult point is that DNA target molecules specificity is lower.The diversity of bacterial surface polysaccharides antigen is caused by the genetic diversity of the gene cluster being responsible for its synthesis.For a long time, people attempt to explore the diversity situation of this important virulence factor of surface polysaccharide antigen be present in Providence and corresponding genetic evolution basis always.But macro-progress slowly in this research direction; understanding for its polysaccharide antigen diversity and variation law aspect is basic or blank; such as: people still do not understand in Providence exist how many kinds of surface polysaccharide antigen and which surface polysaccharide antigen for the pathogenic of this bacterium and popularity significant, the gene cluster being responsible for the synthesis of its surface polysaccharide antigen not yet locate (the part surface polysaccharide antigen gene of other unit-prediction all can not be corresponding very well with the polysaccharide antigen chemical structure of parsing).The major cause of above-mentioned phenomenon is caused to be: polysaccharide antigen synthetic gene bunch composition is complicated, more difficult pre-, the sugared synthetic gene qualification difficulty of gene function etc.In addition, the unit carrying out part correlative study lacks early-stage Study basis mostly.
In early-stage Study, it is theoretical that our specific gene proposed on the basis analysing in depth bacterial surface polysaccharides Evolution of Antigen mechanism in surface antigen gene bunch has high specificity for the surface antigen that it encode, and establish the technology of to screen special molecular and identifying from bacterial surface antigen gene cluster based on this.We establish the maximum pathogenic microorganism special molecular home banking of current capacity in the world (comprising the special molecular mark that more than 300 plant different bacterium) accordingly and establish comparatively perfect bacteria molecule type detection system.These technology have been comprised USDA both at home and abroad, disease prevention and control center of the U.S., french food are affixed one's name to safely, tens unit widespread uses such as Dutch food and Consumer Product Safety mechanism.
In this project, we, by the basis obtaining Providence all surfaces polysaccharide antigen gene cluster sequence information, set up Providence molecule serological typing system and method for quick, have important scientific meaning and stronger using value.
In recent years, increasing molecular engineering is used for the somatotype of pathogenic bacteria, qualification, detection and disease screening, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) is analyzed, rDNA restriction fragment length polymorphism (RFLP) is analyzed.Molecular biology method not only can be used for the rapid serum somatotype examination of Providence, and stable qualification result can make up the deficiency of phenotypic characteristic authentication method.Compare with traditional sensing techniques, these are based on the molecular detection technology of 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 (Polymerasechainreaction, be called for short round pcr) admitted at present as microorganism detection technology and promoted, this technology has relative to traditional method that high-throughput, detection speed are fast, high specificity, sensitivity advantages of higher, only need carry out simple pre-increasing bacterium to sample or increase bacterium process, again by the centrifugal and detailed bacterium DNA profiling of cracking, just can to increase target sequence in the PCR process under the mediation of high specific primer, reach the object detected in sample whether containing invasive organism to be measured.The amplification procedure of PCR only needs 1 and a half hours.This greatly improves working speed undoubtedly to inspection and quarantine department and Clinical Laboratory and reduces job costs.No matter from internal and international angle, identify serum type quickly and accurately, it is very important that the prevention and control for Providence provide effect technique support.
Summary of the invention
The object of the present invention is to provide and the present invention relates to Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 serotype is special, it is characterized in that described Nucleotide has:
1) at least one in the Nucleotide shown in SEQIDNO:1-12;
2) with in the Nucleotide of the nucleotide complementary shown in SEQIDNO:1-12 at least one (such as, have can with O31 serotype special primer amplify the nucleotide sequence that complementary matches);
Described SEQIDNO:1-12 is as follows:
Present invention also offers a kind of PCR kit, comprise PCR primer, dNTP, damping fluid and archaeal dna polymerase, described PCR primer is at least one in the Nucleotide such as shown in SEQIDNO:1-12.Described test kit, also comprises following reagent: 10mMdNTP30 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 controls 10 μ l; ddH
2o5ml.Wherein said PCR primer is preferably at least one in described Nucleotide as shown in SEQIDNO:1-12.
The present invention further discloses Providence O3, O4, O8, O12, the special SEQIDNO:1-12 Nucleotide of O13 and O20 serotype for the preparation of detection Providence Providence O3, O4, O8, the application of the PCR kit of O12, O13 and O20 serotype, gene chip or microarray aspect.Providence of the present invention can sample the crude extract of the culture in diarrhea stool, urinary tract infection, wound, burn and microbemia sample, or the crude extract etc. of the pure growth of Providence.Collecting Providence extraction genome is adopt ordinary method to prepare.
For the PCR kit of Providence, whole detecting step comprises sample pretreatment-amplification-electrophoresis detection result.Primer and the reagent required for PCR reaction system add in amplification pipe in advance, and pretreated sample only need be added amplification pipe and start amplified reaction by user, and simple and quick completes testing.
The present invention also provides a kind of Liquid Detection chip, comprises liquid phase magnetic bead and is connected to the oligonucleotide probe on liquid phase magnetic bead, and the corresponding primer corresponding to the fragment of correspondent probe place; Wherein said Nucleotide is preferably the Nucleotide as shown in SEQIDNO:1-12.
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 SEQIDNO:1-12.
Disclosed by the invention to Providence O3, compared with prior art, tool of the present invention has the following advantages the special Nucleotide of O4, O8, O12, O13 and O20 serotype:
(1) practical
A kind of PCR reaction system that the present invention sets up, can detect Providence, provides the special primer used by serotype detection, utilizes this PCR method can detect clinical samples.
(2) accuracy is high
The present invention passes through the PCR reaction to the special gene of each serotype of Providence, and each sample obtains the band of an entry, will obtain object fragment compared with known length, just can obtain the serotype belonging to Providence.
(3) testing cost is relatively low
The field such as Food Hygiene Surveillance, environmental monitoring, the still quarantine of product supervision and inspection can be applied to, and provide technology mode for other different pathogenic microbes detects combine.
Accompanying drawing illustrates:
Fig. 1 represents O3 serotype of the present invention
wzygene P1 and P2 primer detect other serotype reference cultures of Providence electrophoresis result figure,
wzythe screening of gene P1 and P2 primer, object band is 216bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Fig. 2 represents O3 serotype wzy gene the P1 of the present invention and species specific qualification electrophoresis result figure of P2 primer, and wherein have 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. 3 represents O4 serotype of the present invention
wzygene P3 and P4 primer detect other serotype reference cultures of Providence electrophoresis result figure,
wzythe screening of gene P3 and P4 primer, object band is 181bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Fig. 4 represents O4 serotype of the present invention
wzygene P3 and the species specific qualification electrophoresis result figure of P4 primer, wherein have 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 O8 serotype of the present invention
wzygene P5 and P6 primer detect other serotype reference cultures of Providence electrophoresis result figure,
wzythe screening of gene P5 and P6 primer, object band is 150bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Fig. 6 represents O8 serotype of the present invention
wzygene P5 and the species specific qualification electrophoresis result figure of P6 primer, wherein have 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. 7 represents O12 serotype of the present invention
wzygene P7 and P8 primer detect other serotype reference cultures of Providence electrophoresis result figure,
wzythe screening of gene P7 and P8 primer, object band is 170bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Fig. 8 represents O12 serotype of the present invention
wzygene P7 and P8 primer species specific qualification electrophoresis result figure, Qi Zhongyong
wzygene P7 and P8 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;
Fig. 9 represents O13 serotype of the present invention
wzygene P9 and P10 primer detect other serotype reference cultures of Providence electrophoresis result figure,
wzythe screening of gene P9 and P10 primer, object band is 240bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Figure 10 represents O13 serotype of the present invention
wzygene P9 and P10 primer species specific qualification electrophoresis result figure, Qi Zhongyong
wzygene P9 and P10 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 O20 serotype of the present invention
wzygene P11 and P12 primer detect other serotype reference cultures of Providence electrophoresis result figure,
wzythe screening of gene P11 and P12 primer, object band is 140bp, and remaining serotype is without any band, and concrete bacterial strain information is in table 2;
Figure 12 represents O20 serotype of the present invention
wzygene P11 and P12 primer species specific qualification electrophoresis result figure, Qi Zhongyong
wzygene 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 13 represents and uses O3 respectively, and the electrophoresis result figure of the corresponding serotype of O4, O8, O12, O13 and O20 specific primers amplify, concrete bacterial strain information is in table 2.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition as people such as Sambrook, molecular cloning: the condition described in laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989).Wherein Providence derives from Polish Lodz university.
embodiment 1: genomic extraction
37 DEG C of nutrient broth mediums cultivate Providence, collect bacterium, extract genome concrete steps as follows:
With 500ul50mMTris-HCl (pH8.0) and 10ul0.4MEDTA re-suspended cell, 37 DEG C of incubations 20 minutes, the N,O-Diacetylmuramidase then adding 10ul10mg/ml continues insulation 20 minutes.Add the Proteinase K of 3ul20mg/ml, 15ul10%SDS afterwards, 50 DEG C of incubations 2 hours, then add the RNase of 3ul10mg/ml, 65 DEG C 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.Supernatant liquor 2 times of volume ethanol precipitation DNA, roll out DNA with glass yarn and wash DNA with 70% ethanol, being finally resuspended in 30ulTE by DNA.Genomic dna is detected by the agarose gel electrophoresis of 0.4%.
embodiment 2:sequence is decoded
Extract Providence O3, O4, O8, O12, the genome of O13 and O20 serotype reference culture, by Solexapair-end sequencing technologies, the sequence that genome sequencing obtains this serotype is carried out to each serotype genome of Providence, Blast and PSI-Blast is used to carry out sequence alignment, TMHMM2.0program is adopted to carry out transmembrane structure prediction, ClustalWprogram is used to carry out sequence alignment and screen conservative and specific gene fragment, final O antigen gene cluster sequence and the decoding result obtaining each serotype of Providence.
embodiment 3: design of primers
Decode situation according to gene cluster, we find
wzywith
wzxgene is the special gene of serotype really, so choose this gene specific section design special primer.Due to
wzymore special, thus mainly with
wzygene is target gene.
Design of primers is the core of this invention.Design primer designs according to the specific gene described in document.
wzxwith
wzythese two genes are genes more special in Providence O antigen gene cluster, can as the target gene of Serotype Identification.Said gene is imported PrimerPremier5 and carry out design of primers, the length of primer is preferably between 18 ~ 24bp, and Tm value is at 50 ~ 55 DEG C.Each gene design pair of primers, has single purpose band.
In Genbank, BLAST is carried out after design of primers, 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 produce positive reaction with the nearly edge bacterium in the environment of other nearly edge bacterium or collect specimen.This point is very important for the success or failure of the generation and experiment of avoiding non-specific band.
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 Providence O3, O4, O8, O12, the reference culture of O13 and O20 serotype and each strain of the reference culture of other 12 kinds of serotypes, the Providence of the non-somatotype of 10 strain, 6 strain Vibrio bacterial strains, the specificity of 1 strain Salmonella 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 5 μMs of primer 0.4 μ l, 10 × enzyme spcificity reaction buffer 2.5 μ l, 10mMdNTP0.25 μ l, 5U/ μ l hot resistant DNA polymerase 0.2 μ l and 3 μ l testing sample template in the thin-walled PCR pipe of 0.2ml, last ddH
2o supplies 25 μ l.All primers all obtain positive findings in serotype corresponding separately, do not obtain any PCR primer band in other groups.
Reaction cycle parameter in PCR instrument in this step comprises the sex change of DNA, renaturation, the temperature and time of extension, cycle index, is specially:
Early stage for enable sex change reach required temperature and required early stage treating processes a circulation be 95 DEG C, 5 minutes;
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;
The cycle index of sex change, renaturation, extension is 35 circulations;
For stable amplified production and carry out one circulation temperature and time be 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 of amplification, and O18 uses 65 DEG C of amplifications.
Above-mentioned steps is electrophoresis amplified production in electrophoresis equipment, and the concrete steps of record result are:
get 2 ~ 5 μ l amplified productions to mix with the volume ratio of 1:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;
mixed solution is splined on the sepharose of 1.0%;
by agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 20 minutes, contrast with DL2000Marker;
observe and record result.
Substantially terminated by the work of primer screening after basic PC R reaction, necessary length adjustment is little on the impact of W-response condition, 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 preparations and applicatio of embodiment 6:PCR detection kit
1, the composition of PCR 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 μMs) 10 μ l;
Positive reference substance (KP) 10 μ l;
Negative controls (KN) 10 μ l;
ddH
2O5ml；
Each test kit can be used for detection 10 samples.
Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by precious biotechnology company limited; Primer mixture is that the sequence of designed, designed is supplied to the synthesis of Shanghai Ying Jun biotech company; Positive reference substance, negative controls 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; Primer mixture is that the sequence of designed, designed is 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 (comprising electrophoresis apparatus and electrophoresis chamber), gel imaging instrument ,-20 DEG C of refrigerators, supercentrifuge, micropipet and 0.2mlPCR thin-walled tubes.
3, the use specific examples of PCR kit
The PCR detection method using above-mentioned PCR kit to detect Providence comprises the steps:
(1) environmental sample template to be measured is extracted;
(2) add in PCR thin-walled tube, dNTP, 10 × Buffer, Taq polysaccharase, primer, testing sample template and ddH
2o mixes;
(3) mixture mixed in thin-walled PCR pipe is increased in PCR instrument;
(4) electrophoresis amplified production in electrophoresis equipment, record 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 Providence or pure dna, or positive reference substance and negative controls.The crude extract of the pure growth of preferred Providence or pure dna.
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;
get the ddH of 500 μ l
2the resuspended precipitation of O, under 8000rpm condition centrifugal 5 minutes, removes supernatant liquor, dries;
get 100 μ lddH
2the resuspended precipitation of O, water-bath 10 minutes in 100 DEG C of boiling water;
be placed on ice after 10 minutes again, under 12000rpm condition centrifugal 2 minutes;
5. 3 μ l middle layer supernatant are got as pcr template
Reaction cycle parameter in PCR instrument in above-mentioned steps (3) comprises the sex change of DNA, renaturation, the temperature and time of extension, cycle index, is specially:
Early stage for enable sex change reach required temperature and required early stage treating processes a circulation be 95 DEG C, 5 minutes;
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;
The cycle index of sex change, renaturation, extension is 35 circulations;
For stable amplified production and carry out one circulation temperature and time be 72 DEG C, 5 minutes.
Above-mentioned steps (4) electrophoresis amplified production in electrophoresis equipment, the concrete steps of record result are:
get 2 ~ 5 μ l amplified productions to mix with the volume ratio of 5:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;
mixed solution is splined on the sepharose of 1.0%;
by agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 20 minutes, contrast with DL2000Marker;
observe and record result.
The present invention by configuration a kind of detect Providence can industrialization produce PCR kit, PCR detection method is needed the combination of components of use together, during use, extract testing sample, just quick, sensitive, easy detection can be carried out through comparatively simple operation sequence simultaneously, in test kit, the consumption of each component and concentration are test gained, and detect with this test kit the testing installation that Providence uses simple, testing cost is low.
Use object that is positive and negative controls to be for the whole operating process of Quality Control, judge accurately to draw.If containing Providence object O antigenic type, then can observe the band with positive reference substance same position from electrophoresis result; If not containing Providence object O antigenic type, then the same with negative controls do not have this band.
The amount of reagent that one-time detection of the present invention is tested in the test kit used sees the following form shown in 3, and DNA profiling amount is 3 μ l
Table 3 one-time detection tests the amount of reagent in the test kit used
| Composition | Concentration | Application of sample amount (μ l) |
| 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 determined it is the sample of each O antigenic type of Providence, and negative controls is then for determining it is not the sample of Providence through laboratory.
If this PCR kit bacteria suspension of Providence carries out pcr amplification, 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, 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 to detection sample carry out simple separation cultivate just can detect, thus save manpower and materials.
4, the providing of testing sample
Have collected Providence O3, O4, O8, O12, O13 and O20 serotype reference culture, 6 other bacterial strains of strain Vibrio, the specificity of 1 strain Salmonella strain and 1 strain coli strain checking primer, strain number and source are in table 2.
The above, only operation of the present invention and implementation method, not any pro forma restriction is done to the present invention, every above embodiment is done according to technical spirit of the present invention any simple modification, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
SEQUENCELISTING
<110> Nankai University
<120> to Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof
<160>12
<170>PatentInversion3.5
<210>1
<211>20
<212>DNA
<213> artificial sequence
<400>1
ttcaatttcaggagctcgcc20
<210>2
<211>26
<212>DNA
<213> artificial sequence
<400>2
aacgtactttcaaatatacgacctgt26
<210>3
<211>23
<212>DNA
<213> artificial sequence
<400>3
aaatatagtttttggcgtgggtt23
<210>4
<211>23
<212>DNA
<213> artificial sequence
<400>4
aattaaaccacctaaaccaacgt23
<210>5
<211>24
<212>DNA
<213> artificial sequence
<400>5
atgcgagactatggttctggatta24
<210>6
<211>24
<212>DNA
<213> artificial sequence
<400>6
gctaacacagcactcgaagcatat24
<210>7
<211>28
<212>DNA
<213> artificial sequence
<400>7
aggtacagatttaggagctcatctatta28
<210>8
<211>21
<212>DNA
<213> artificial sequence
<400>8
ataggctggaaagcctacagg21
<210>9
<211>20
<212>DNA
<213> artificial sequence
<400>9
gctgttcgatattcagccgg20
<210>10
<211>28
<212>DNA
<213> artificial sequence
<400>10
tgtgtattttattaatggtgcagtatca28
<210>11
<211>24
<212>DNA
<213> artificial sequence
<400>11
acaccaagccttacgctagtattt24
<210>12
<211>22
<212>DNA
<213> artificial sequence
<400>12
ctgtttgtcgtgcataaaatgc22
Claims (6)
1. couple Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 serotype is special, is characterized in that described Nucleotide is for the Nucleotide shown in SEQIDNO:1-12.
2. a PCR kit, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer is the Nucleotide such as shown in SEQIDNO:1-12.
3. test kit according to claim 2, is characterized in that comprising following reagent: 10mMdNTP30 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 controls 10 μ l; ddH
2o1ml.
4. described in claim 1 to Providence O3, the special Nucleotide of O4, O8, O12, O13 and O20 serotype is for the preparation of the application detected in Providence PCR kit.
5. application according to claim 4, wherein said detection Providence refers to detection and causes respiratory tract infection, septicemia, urinary system infection, the meningitic bacterium of Secondary cases.
6. application according to claim 4, wherein said Providence refers to and samples in diarrhea stool, urinary tract infection, wound, burn and the crude extract of microbemia sample culture or the crude extract of the pure growth of Providence.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410281515.3A CN104059975B (en) | 2014-06-23 | 2014-06-23 | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof |
| PCT/CN2015/000305 WO2015196778A1 (en) | 2014-06-23 | 2015-05-04 | Providencia o3, o4, o8, o12, o13 and o20 specific nucleotide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410281515.3A CN104059975B (en) | 2014-06-23 | 2014-06-23 | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104059975A CN104059975A (en) | 2014-09-24 |
| CN104059975B true CN104059975B (en) | 2015-12-02 |
Family
ID=51547905
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410281515.3A Active CN104059975B (en) | 2014-06-23 | 2014-06-23 | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN104059975B (en) |
| WO (1) | WO2015196778A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993090B (en) * | 2014-05-29 | 2016-05-11 | 南开大学 | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof |
| CN104059975B (en) * | 2014-06-23 | 2015-12-02 | 南开大学 | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof |
| CN104988144A (en) * | 2015-06-26 | 2015-10-21 | 南开大学 | Gene liquid chip for detecting 10 kinds of common pathogenic microorganisms in soil and detection method of gene liquid chip |
| CN105154561A (en) * | 2015-09-29 | 2015-12-16 | 南开大学 | Nucleotide specific to providencia O5 and O21 and application thereof |
| CN105200138A (en) * | 2015-09-29 | 2015-12-30 | 南开大学 | Nucleotide specific to Providencia vermicola O34 and O46 and application of nucleotide |
| CN111748641A (en) * | 2020-07-08 | 2020-10-09 | 南开大学 | Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191146A (en) * | 2006-11-28 | 2008-06-04 | 佳能株式会社 | Primer for bacterium genome amplification reaction |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5267293B2 (en) * | 2009-04-13 | 2013-08-21 | 島根県 | Comprehensive rapid detection method for foodborne pathogens by real-time PCR |
| CN104059975B (en) * | 2014-06-23 | 2015-12-02 | 南开大学 | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof |
-
2014
- 2014-06-23 CN CN201410281515.3A patent/CN104059975B/en active Active
-
2015
- 2015-05-04 WO PCT/CN2015/000305 patent/WO2015196778A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191146A (en) * | 2006-11-28 | 2008-06-04 | 佳能株式会社 | Primer for bacterium genome amplification reaction |
Non-Patent Citations (2)
| Title |
|---|
| Localization and molecular characterization of putative O antigen gene clusters of Providencia species;Olga G. Ovchinnikova et al.;《Microbiology》;20120126;第158卷;1024-1036 * |
| Structual, serological, and genetic characterization of the O-antigen of Providencia alcalifaciens O40;Olga G. Ovchinnikova et al.;《FEMS Immunology & Medical Microbiology》;20121017;第66卷;382-392 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2015196778A1 (en) | 2015-12-30 |
| CN104059975A (en) | 2014-09-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104059975B (en) | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof | |
| CN103898108B (en) | The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof | |
| CN104862393A (en) | Nucleotide specific to Cronobacter O antigen and application thereof | |
| CN109468395A (en) | A kind of primer, kit, detection method and application detecting mycoplasma | |
| CN103993090B (en) | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof | |
| CN104232783A (en) | Quick detection method for cow brucella attenuated vaccine strain A19 | |
| CN103571943A (en) | Nucleic acid detection method for salmonella typhimurium | |
| CN105154559A (en) | Specific nucleotide for vibrio parahaemolyticus K36, K37 and K68 and application thereof | |
| CN102154270B (en) | Cronobacter sakazakii O antigen specific nucleotides and use thereof | |
| 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 | |
| CN105256041A (en) | Specific nucleotide for aeromonas hydrophila O44, O24, O25 and O28 and application thereof | |
| CN115747361A (en) | Real-time fluorescent MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method | |
| CN105112406B (en) | To Hafnia alvei G5897, G5898, G5900 special nucleotide and its application | |
| CN105154439B (en) | To Hafnia alvei G5902, G5903, G5904, G5906 special nucleotide sequence | |
| CN105177144A (en) | Nucleotide for specificity of K4, K32 and K34 of vibrio parahaemolyticus and application thereof | |
| CN105154438B (en) | To Hafnia alvei G5907, G5908, G5913, nucleotide special G5916 and its application | |
| CN111996268A (en) | Vibrio alginolyticus double TaqMan probe real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit and preparation method thereof | |
| CN105256042A (en) | Nucleotide specific to Aeromonas hydrophila O13, O36, O16 and O19 and application | |
| CN111621578A (en) | RU 61-00441 gene-based Salmonella spelt constant-temperature detection kit and method | |
| CN105200138A (en) | Nucleotide specific to Providencia vermicola O34 and O46 and application of nucleotide | |
| CN105154561A (en) | Nucleotide specific to providencia O5 and O21 and application thereof | |
| CN105112505A (en) | Nucleotides with specificity on Klebsiella K44, K59, K61, and K63 serotypes, and applications thereof | |
| CN105112405A (en) | Nucleotides with specificity on Klebsiella K8, K20, K38, and K39 serotypes, and applications thereof | |
| CN102311950A (en) | Nucleotide specific to wzt of Legionella pneumophila serogroup 1 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 |