CN104059975A - Providencia O3, O4, O8, O12, O13 and O20 specific nucleotides and application thereof - Google Patents
Providencia O3, O4, O8, O12, O13 and O20 specific nucleotides and application thereof Download PDFInfo
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Abstract
The present invention relates to Providencia O3, O4, O8, O12, O13 and O20 serotype specific nucleotides and application thereof, wherein the nucleotide is at least one shown in the SEQIDNO:1-12. These nucleotides can be used for preparing a PCR kit and a gene chip for detecting Providencia. The Providencia sample can be taken from crude extract of diarrhea stool, urinary tract infections, wounds, burns and bacteremia specimens, or the crude extract of pure specimens of Providencia. The invention provides the Providencia O3, O4, O8, O12, O13 and O20 serotype specific nucleotides and the PCR kit and gene chip containing the nucleotides; and the PCR kit has the advantages of simple preparation method, short detection period, high speed, strong operability, easiness for industrialization production, low detection cost, high accuracy and high sensitivity.
Description
Technical field
The present invention relates to Providence O3, O4, O8, O12, O13 and the special Nucleotide of O20 serotype, relate in particular to Providence O3, O4, O8, O12, special Nucleotide and the application thereof of individual gene in O13 and O20 serotype O antigen gene cluster.
Background technology
Providence (Providencia) is a kind of common Gram-negative bacteria in enterobacteriaceae, is a kind of opportunistic pathogen.Mainly infect this bacterium by ward infection or edible seafood, can cause respiratory tract infection, also can cause septicemia, urinary system infection, Secondary cases meningitis etc.Be located away from diarrhoea stool, urinary tract infection, wound, burn and microbemia sample.
Typing of bacteria and authentication method mainly contain traditional phenotype method, serological method and molecular assay method.But along with molecular biological development, traditional serotype and authentication method exist certain problem, diagnostic method as this in serotype needs a large amount of antiserum(antisera)s, and antiserum(antisera) general classes is incomplete, quantity not sufficient, also there are some difficulties in preparation with in storing in a large amount of antiserum(antisera)s.On the other hand serotype method length consuming time, sensitivity is low, loss is high, poor accuracy, between the antiserum(antisera) that different O antigen produces, often has cross reaction.Therefore the serological diagnosis method of, setting up based on Protocols in Molecular Biology becomes developing direction.
The Molecular Identification of Providence is more and more subject to people's attention, and becomes the important evidence of Providence bacterial classification and plant type qualification, and therefore many new bacterium also produce.Molecular Biological Detection technology has advantages of that speed is fast and is easy to extensive use, be the current developing direction of generally acknowledged pathogenic microbes detect in the world, but current difficult point is that DNA target molecules specificity is lower.The diversity of bacterium surface polysaccharide antigen is to be caused by the genetic diversity of being responsible for its synthetic gene cluster.For a long time, people attempt to explore the diversity situation and the corresponding genetic evolution basis that are present in surperficial this important virulence factor of polysaccharide antigen in Providence always.But in this research direction, macro-progress is very slow; understanding for its polysaccharide antigen diversity and variation law aspect is basic or blank; for example: people still do not understand and in Providence, have pathogenic and popular significant for this bacterium of how many kinds of surface polysaccharide antigen and which surperficial polysaccharide antigen, be responsible for the synthetic gene cluster of its surperficial polysaccharide antigen be not yet positioned (the part surface polysaccharide antigen gene that other unit predicts all can not be fine corresponding with the polysaccharide antigen chemical structure of parsing) etc.Cause the major cause of above-mentioned phenomenon to be: polysaccharide antigen synthetic gene bunch composition is complicated, more difficult pre-, the sugared synthetic gene qualification of gene function is difficult etc.In addition the unit that, carries out part correlative study lacks early-stage Study basis mostly.
In early-stage Study, the specific gene that we have proposed in surface antigen gene bunch on the basis of analysing in depth bacterium surface polysaccharide antigen evolution mechanism has high specificity theory for the surface antigen of its coding, and has set up based on this technology of screening special molecular mark from bacterium surface antigen gene cluster.We have set up accordingly the pathogenic microorganism special molecular home banking (comprising that more than 300 plant the special molecular mark of different bacterium) of the current maximum of capacity in the world and have set up comparatively perfect bacteria molecule type detection system.Tens unit widespread uses such as these technology have been comprised both at home and abroad, and USDA, disease prevention and control center of the U.S., french food are affixed one's name to safely, Dutch food and consumer's goods safety mechanism.
In this project, we,, by obtaining on the basis of 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, for somatotype, qualification, detection and the disease screening of pathogenic bacteria, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) analysis, rDNA restriction fragment length polymorphism (RFLP) analysis etc.Molecular biology method not only can be used for the quick serotype examination of Providence, and stable qualification result can make up the deficiency of phenotypic characteristic authentication method.Compare with traditional detection technology, these molecular detection technologies based on polymerase chain reaction (PCR), do not need the process such as separation, pure culture through pathogenic bacteria, and have the advantages such as quick, sensitive, high specificity.
Polymerase chain reaction technology (Polymerase chain reaction, be called for short round pcr) admitted at present and promoted as microorganism detection technology, this technology has the advantages such as high-throughput, detection speed are fast, high specificity, sensitivity height with respect to traditional method, only need carry out simple pre-bacterium or the increasing bacterium process of increasing to sample, again by the centrifugal and detailed bacterium DNA profiling of cracking, the target sequence that just can increase in the PCR process under high specific primer mediation, reaches and detects the object that whether contains invasive organism to be measured in sample.The amplification procedure of PCR only needs 1 and a half hours.This has greatly improved undoubtedly working speed and has reduced job costs inspection and quarantine department and Clinical Laboratory.No matter from internal and international angle, identify quickly and accurately serum type, be very important for the prevention and control of 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, O4, O8, O12, O13 and the special Nucleotide of O20 serotype, is characterized in that described Nucleotide has:
1) at least one in the Nucleotide shown in SEQ ID NO:1-12;
2) at least one (for example, the thering is the nucleotide sequence that can amplify with O31 serotype special primer sequence complementary pairing) and in the Nucleotide of the Nucleotide complementation shown in SEQ ID NO:1-12;
Described SEQ ID NO:1-12 is as follows:
The present invention also provides a kind of PCR test kit, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer is at least one in the Nucleotide as shown in SEQ ID NO:1-12.Described test kit, also comprises following reagent: 10 mM dNTP 30 μ l; 10 × enzyme spcificity reaction buffer, 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; Primer mixture 10 μ l; Positive reference substance 10 μ l; Negative control product 10 μ l; ddH
2o 5ml.Wherein said PCR primer is preferably at least one in described Nucleotide as shown in SEQ ID NO:1-12.
The present invention further discloses Providence O3, O4, O8, O12, the special SEQ ID NO:1-12 Nucleotide of O13 and O20 serotype is for the preparation of detecting Providence Providence O3, O4, O8, O12, the application of PCR test kit, gene chip or the microarray aspect of O13 and O20 serotype.Providence of the present invention can sample in the crude extract of the culture of diarrhoea stool, urinary tract infection, wound, burn and microbemia sample, or the crude extract of the pure growth of Providence etc.Collecting Providence extraction genome is to adopt ordinary method to prepare.
For the PCR test kit of Providence, whole detecting step comprises sample pretreatment-amplification-electrophoresis detection result.Primer and the needed reagent of PCR reaction system add in amplification pipe in advance, and user only need add pretreated sample amplification pipe to start amplified reaction, simple and quick complete testing.
The present invention also provides a kind of Liquid Detection chip, and comprise liquid phase magnetic bead and be connected to the oligonucleotide probe on liquid phase magnetic bead, and the corresponding corresponding primer of correspondent probe place fragment; Wherein said Nucleotide is preferably the Nucleotide as shown in SEQ ID NO: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 SEQ ID NO:1-12.
Disclosed by the invention to Providence O3, O4, O8, O12, compared with prior art, tool of the present invention has the following advantages the special Nucleotide of O13 and O20 serotype:
(1) practical
A kind of PCR reaction system that the present invention sets up, can detect Providence, provides serotype to detect special primer used, utilizes this PCR method to detect clinical samples.
(2) accuracy is high
The present invention is by the PCR reaction to the special gene of each serotype of Providence, and each sample obtains the band of an entry, will obtain object fragment and compare with known length, just can obtain the affiliated serotype of Providence.
(3) testing cost is relatively low
Can be applied to the fields such as Food Hygiene Surveillance, environmental monitoring, still product supervision and inspection quarantine, and provide technology mode for other different pathogenic microbes detects combine.
Brief description of the drawings:
Fig. 1 represents O3 serotype of the present invention
wzygene P1 and P2 primer detect other serotype reference culture electrophoresis result of Providence 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 P1 of the present invention and the species specific qualification electrophoresis result of P2 primer figure, has wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Fig. 3 represents O4 serotype of the present invention
wzygene P3 and P4 primer detect other serotype reference culture electrophoresis result of Providence 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 of P4 primer figure, wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Fig. 5 represents O8 serotype of the present invention
wzygene P5 and P6 primer detect other serotype reference culture electrophoresis result of Providence 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 of P6 primer figure, wherein detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Fig. 7 represents O12 serotype of the present invention
wzygene P7 and P8 primer detect other serotype reference culture electrophoresis result of Providence 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 the species specific qualification electrophoresis result of P8 primer 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 culture electrophoresis result of Providence 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 the species specific qualification electrophoresis result of P10 primer figure, Qi Zhongyong
wzygene P9 and P10 primer 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 culture electrophoresis result of Providence 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 the species specific qualification electrophoresis result of P12 primer figure, Qi Zhongyong
wzygene P11 and P12 primer primer have detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, and all without any band, concrete bacterial strain information is in table 2;
Figure 13 represents to use respectively O3, O4, and O8, O12, the increase electrophoresis result figure of corresponding serotype of O13 and O20 special primer, concrete bacterial strain information is in table 2.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: the condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).Wherein Providence derives from Polish Lodz university.
embodiment 1: genomic extraction
37 DEG C of nutrient broth mediums are cultivated Providence, collect bacterium, extract genome concrete steps as follows:
With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 DEG C of incubations 20 minutes, then add the N,O-Diacetylmuramidase of 10ul 10mg/ml to continue insulation 20 minutes.Add afterwards Proteinase K, the 15ul 10%SDS of 3ul 20mg/ml, 50 DEG C of incubations 2 hours, then add the RNase of 3 ul 10mg/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.For supernatant liquor, 2 times of volume ethanol precipitation DNA, roll out DNA with glass yarn and also wash DNA with 70% ethanol, finally DNA are resuspended in 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
embodiment 2:sequence is decoded
Extract Providence O3, O4, O8, O12, the genome of O13 and O20 serotype reference culture, by Solexa pair-end sequencing technologies, each serotype genome of Providence is carried out genome sequencing and is obtained the sequence of this serotype, use Blast and PSI-Blast to carry out sequence alignment, adopt TMHMM 2.0 program to carry out cross-film structure prediction, use ClustalW program carry out sequence alignment and screen conservative and specific gene fragment, finally obtain the O antigen gene cluster sequence of each serotype of Providence and decode result.
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 of narrating in document.
wzxwith
wzythese two genes are more special genes in Providence O antigen gene cluster, can be used as the target gene of Serotype Identification.Said gene is imported to Primer Premier 5 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.
After design of primers, in Genbank, carry out BLAST, the primer of design can not be too high with the sequence similarity of other nearly edge bacterium, so just can guarantee that this primer only increases in the predetermined position of oneself, and not with other nearly edge bacterium or the environment of collect specimen in nearly edge bacterium do not produce positive reaction.This point is very important for avoiding the generation of non-specific band and the success or failure of experiment.
The primer of designing is as shown in table 1.
Table 1 is for the primer sequence of PCR
embodiment 4: the screening of special primer
Collect Providence O3, O4, O8, O12, the each strain of reference culture of the reference culture of O13 and O20 serotype and other 12 kinds of serotypes, 10 strains are the Providence of somatotype not, 6 strain Vibrio bacterial strains, the specificity of 1 strain Salmonella bacterial strain and 1 strain coli strain checking primer, strain number and source are in table 2.
Table 2 is for the bacterial strain of specific detection
The PCR system that gene identification primer screening is used be the testing sample template of 5 μ M primer 0.4 μ l, 10 × enzyme spcificity reaction buffer, 2.5 μ l, 10mM dNTP 0.25 μ l, 5U/ μ l hot resistant DNA polymerase 0.2 μ l and 3 μ l in the thin-walled PCR pipe of 0.2ml, last ddH
2o supplies 25 μ l.All primers all, obtaining positive findings in corresponding serotype separately, do not obtain any PCR product band in other groups.
Reaction cycle parameter on PCR instrument in this step comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially:
Early stage for make sex change can reach required temperature essential early stage treating processes a circulation be 95 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;
The temperature and time that carries out a circulation for stablizing 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, O15 use 55-58 DEG C of amplification all can, O18 uses 65 DEG C of amplifications.
Above-mentioned steps is electrophoresis amplified production in electrophoresis equipment, and the concrete steps that record result are:
getting 2～5 μ l amplified productions mixes with the volume ratio of 1:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;
mixed solution is splined on 1.0% sepharose;
by agarose gel electrophoresis 120v voltage stabilizing electrophoresis approximately 20 minutes, contrast with DL2000 Marker;
observe and record result.
Work by primer screening after basic PC R reaction finishes substantially, and necessary length adjustment is little on the impact of W-response condition, and the primer sequence of using in the present invention is all summarised in table 1.
Table 1 is for the primer sequence of PCR
preparation and the application of embodiment 6:PCR detection kit
1, the composition of PCR test kit:
dNTP(10mM) 30μl；
10 × Buffer (10 × enzyme spcificity reaction buffer), 50 μ l;
Taq polysaccharase (5U/ μ l hot resistant DNA polymerase) 5 μ l;
PCR primer mixture (5 μ M) 10 μ l;
Positive reference substance (KP) 10 μ l;
Negative control product (KN) 10 μ l;
ddH
2O 5ml；
Each test kit can be used for detecting 10 samples.
Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by precious biotechnology company limited; It is synthetic that primer mixture is that the sequence of designed, designed offers Shanghai Ying Jun biotech company; Positive reference substance, negative control product and ddH
2o is prepared voluntarily by us.
2, plant and instrument
Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by the raw work in Shanghai; It is synthetic that primer mixture is that the sequence of designed, designed offers Shanghai Ying Jun biotech company; Positive reference substance, negative control product and ddH2O are prepared voluntarily by us.Equipment PCR instrument (having another name called DNA thermal cycling amplification instrument), electrophoresis equipment (comprising electrophoresis apparatus and electrophoresis chamber), gel imaging instrument ,-20 DEG C of refrigerators, supercentrifuge, micropipet and 0.2ml PCR thin-walled tubes of experiment.
3, the use specific examples of PCR test kit
The PCR detection method that uses above-mentioned PCR test kit to detect Providence comprises the steps:
(1) extract environmental sample template to be measured;
(2) in PCR thin-walled tube, add, dNTP, 10 × Buffer, Taq polysaccharase, primer, testing sample template and ddH
2o mixes;
(3) mixture mixing in thin-walled PCR pipe is increased on PCR instrument;
(4) electrophoresis amplified production in electrophoresis equipment, records result;
(5) analyze and carry out result judgement.
Environmental sample template in above-mentioned steps (1) is the crude extract of the culture of tap water, river water, seawater etc., or the crude extract of the pure growth of Providence or pure dna, or positive reference substance and negative control product.Preferably crude extract or the pure dna of the pure growth of Providence.
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, remove supernatant liquor, dry;
get 100 μ lddH
2the resuspended precipitation of O, water-bath 10 minutes in 100 DEG C of boiling water;
be placed in again on ice after 10 minutes under 12000rpm condition centrifugal 2 minutes;
5. get 3 μ l middle layer supernatant as pcr template
Reaction cycle parameter on PCR instrument in above-mentioned steps (3) comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially:
Early stage for make sex change can reach required temperature essential early stage treating processes a circulation be 95 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;
The temperature and time that carries out a circulation for stablizing amplified production is 72 DEG C, 5 minutes.
Above-mentioned steps (4) electrophoresis amplified production in electrophoresis equipment, the concrete steps that record result are:
getting 2～5 μ l amplified productions mixes with the volume ratio of 5:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;
mixed solution is splined on 1.0% sepharose;
by agarose gel electrophoresis 120v voltage stabilizing electrophoresis approximately 20 minutes, contrast with DL2000 Marker;
observe and record result.
The present invention is by configuring a kind of PCR test kit of can industrialization producing that detects Providence, the combination of components that PCR detection method need to be used together, when use, extract testing sample, just can carry out quick, sensitive, easy detection through comparatively simple operation sequence simultaneously, in test kit, the consumption of each component and concentration are test gained, and the testing installation using with this test kit detection Providence is simple, and testing cost is low.
Using the object of positive and negative control product is for the whole operating process of Quality Control, to draw judgement accurately.If contain Providence object O antigenic type, from electrophoresis result, can observe the band with positive reference substance same position; If do not contain Providence object O antigenic type, the same with negative control product do not have this band.
The amount of reagent that one-time detection of the present invention is tested in the test kit using sees the following form shown in 3, and DNA profiling amount is 3 μ l
Table 3 one-time detection is tested the amount of reagent in the test kit using
| Composition | Concentration | (μ l) for application of sample amount |
| ddH 2O | 18.65 | |
| 10 × PCR damping fluid | 10× | 2.5 |
| 10mM dNTP | 10mM | 0.25 |
| Primer mixture | 5mM | 0.4 |
| Taq enzyme | 5U/μl | 0.2 |
| Cumulative volume | 25 |
Hot resistant DNA polymerase in the present invention is Taq enzyme.
Above-mentioned positive reference substance is to have determined it is the sample of each O antigenic type of Providence, and negative control product are not the samples of Providence for determining through laboratory.
If this PCR test kit carries out pcr amplification with the bacteria suspension of Providence, and through extracting the DNA that obtains as template acquired results always.Susceptibility and specificity indifference, like this, can save the extraction step of template DNA, and working method is simplified.Meanwhile, compare routine biochemistry detection method, the testing sample that present method adopts can be directly clinical sample nutrient solution, or detection sample is carried out to simple separation cultivation and just can detect, thereby has saved manpower and materials.
4, providing of testing sample
Collect Providence O3, O4, O8, O12, O13 and O20 serotype reference culture, 6 other bacterial strains of strain Vibrio, the specificity of 1 strain Salmonella bacterial strain and 1 strain coli strain checking primer, strain number and source are in table 2.
The above, only operation of the present invention and implementation method, not the present invention is done to any pro forma restriction, any simple modification, equivalent variations and modification that every foundation technical spirit of the present invention is done above embodiment, all still belong in the scope of technical solution of the present invention.
SEQUENCE LISTING
<110> Nankai University
<120> is to Providence O3, O4, O8, O12, the Nucleotide that O13 and O20 are special and application thereof
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
ttcaatttca ggagctcgcc 20
<210> 2
<211> 26
<212> DNA
<213> artificial sequence
<400> 2
aacgtacttt caaatatacg acctgt 26
<210> 3
<211> 23
<212> DNA
<213> artificial sequence
<400> 3
aaatatagtt tttggcgtgg gtt 23
<210> 4
<211> 23
<212> DNA
<213> artificial sequence
<400> 4
aattaaacca cctaaaccaa cgt 23
<210> 5
<211> 24
<212> DNA
<213> artificial sequence
<400> 5
atgcgagact atggttctgg atta 24
<210> 6
<211> 24
<212> DNA
<213> artificial sequence
<400> 6
gctaacacag cactcgaagc atat 24
<210> 7
<211> 28
<212> DNA
<213> artificial sequence
<400> 7
aggtacagat ttaggagctc atctatta 28
<210> 8
<211> 21
<212> DNA
<213> artificial sequence
<400> 8
ataggctgga aagcctacag g 21
<210> 9
<211> 20
<212> DNA
<213> artificial sequence
<400> 9
gctgttcgat attcagccgg 20
<210> 10
<211> 28
<212> DNA
<213> artificial sequence
<400> 10
tgtgtatttt attaatggtg cagtatca 28
<210> 11
<211> 24
<212> DNA
<213> artificial sequence
<400> 11
acaccaagcc ttacgctagt attt 24
<210> 12
<211> 22
<212> DNA
<213> artificial sequence
<400> 12
ctgtttgtcg tgcataaaat gc 22
Claims (8)
1. couple Providence O3, O4, O8, O12, O13 and the special Nucleotide of O20 serotype, is characterized in that described Nucleotide has:
1) at least one in the Nucleotide shown in SEQ ID NO:1-12;
2) at least one and in the Nucleotide of the Nucleotide complementation shown in SEQ ID NO:1-12.
2. a PCR test kit, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer is at least one in the Nucleotide as shown in SEQ ID NO:1-12.
3. test kit claimed in claim 2, also comprises following reagent: 10 mM dNTP 30 μ l; 10 × enzyme spcificity reaction buffer, 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; Primer mixture 10 μ l; Positive reference substance 10 μ l; Negative control product 10 μ l; ddH
2o 1ml.
4. SEQ ID NO:1-12 specific nucleotide claimed in claim 1 is in the application aspect detection Providence PCR test kit.
5. the application of SEQ ID NO:1-12 specific nucleotide claimed in claim 1 aspect the gene chip for the preparation of detection Providence.
6. SEQ ID NO:1-12 specific nucleotide claimed in claim 1 is in the application aspect detection Providence microarray.
7. the application described in claim 4-6, wherein said detection Providence refers to detect and causes respiratory tract infection, septicemia, urinary system infection, the meningitic bacterium of Secondary cases.
8. the application described in claim 4-6, wherein said Providence refers to and samples in diarrhoea 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 true CN104059975A (en) | 2014-09-24 |
| CN104059975B CN104059975B (en) | 2015-12-02 |
Family
ID=51547905
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| 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) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2015180484A1 (en) * | 2014-05-29 | 2015-12-03 | 南开大学 | Nucleotides specific to providencia o31, o41, o42, o43 and o50 and uses thereof |
| CN105154561A (en) * | 2015-09-29 | 2015-12-16 | 南开大学 | Nucleotide specific to providencia O5 and O21 and application thereof |
| WO2015196778A1 (en) * | 2014-06-23 | 2015-12-30 | 南开大学 | Providencia o3, o4, o8, o12, o13 and o20 specific nucleotide 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 |
|---|
| OLGA G. OVCHINNIKOVA ET AL.: "Localization and molecular characterization of putative O antigen gene clusters of Providencia species", 《MICROBIOLOGY》, vol. 158, 26 January 2012 (2012-01-26), pages 1024 - 1036 * |
| OLGA G. OVCHINNIKOVA ET AL.: "Structual, serological, and genetic characterization of the O-antigen of Providencia alcalifaciens O40", 《FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY》, vol. 66, 17 October 2012 (2012-10-17), pages 382 - 392 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015180484A1 (en) * | 2014-05-29 | 2015-12-03 | 南开大学 | Nucleotides specific to providencia o31, o41, o42, o43 and o50 and uses thereof |
| WO2015196778A1 (en) * | 2014-06-23 | 2015-12-30 | 南开大学 | Providencia o3, o4, o8, o12, o13 and o20 specific nucleotide 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104059975B (en) | 2015-12-02 |
| WO2015196778A1 (en) | 2015-12-30 |
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