CN102154270A - Cronobacter sakazakii O antigen specific nucleotides and use thereof - Google Patents
Cronobacter sakazakii O antigen specific nucleotides and use thereof Download PDFInfo
- Publication number
- CN102154270A CN102154270A CN 201110004830 CN201110004830A CN102154270A CN 102154270 A CN102154270 A CN 102154270A CN 201110004830 CN201110004830 CN 201110004830 CN 201110004830 A CN201110004830 A CN 201110004830A CN 102154270 A CN102154270 A CN 102154270A
- Authority
- CN
- China
- Prior art keywords
- pcr
- gene
- nucleotides
- seq
- primer
- 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.)
- Granted
Links
- 241001135265 Cronobacter sakazakii Species 0.000 title claims abstract description 30
- 239000002773 nucleotide Substances 0.000 title claims abstract description 27
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 27
- 108091007433 antigens Proteins 0.000 title claims abstract description 24
- 239000000427 antigen Substances 0.000 title claims abstract description 14
- 102000036639 antigens Human genes 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 238000012360 testing method Methods 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 7
- 238000013016 damping Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108020004635 Complementary DNA Proteins 0.000 claims description 2
- 239000011535 reaction buffer Substances 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 37
- 238000001514 detection method Methods 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000009004 PCR Kit Methods 0.000 abstract 2
- 230000000295 complement effect Effects 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 17
- 238000013461 design Methods 0.000 description 14
- 238000012216 screening Methods 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 150000002482 oligosaccharides Polymers 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000002158 endotoxin Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 229920006008 lipopolysaccharide Polymers 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 108700023372 Glycosyltransferases Proteins 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 241000607142 Salmonella Species 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000013350 formula milk Nutrition 0.000 description 3
- 108091008053 gene clusters Proteins 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 201000009906 Meningitis Diseases 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 101150060566 galF gene Proteins 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 101150014950 gnd gene Proteins 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002028 premature Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 208000013223 septicemia Diseases 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000032754 Infant Death Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 206010051606 Necrotising colitis Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- AVVWPBAENSWJCB-DGPNFKTASA-N beta-D-galactofuranose Chemical compound OC[C@@H](O)[C@@H]1O[C@@H](O)[C@H](O)[C@H]1O AVVWPBAENSWJCB-DGPNFKTASA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- -1 comprises lipoid A Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 208000004995 necrotizing enterocolitis Diseases 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 201000006195 perinatal necrotizing enterocolitis Diseases 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 101150088264 pol gene Proteins 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a Cronobacter sakazakii O antigen specific nucleotides and use thereof. The nucleotides comprises: 1) at least one of nucleotides represented by sequences from SEQ ID No.1 to SEQ ID No.14; and 2) at least of nucleotides complementary to the nucleotides represented by the sequences from SEQ ID No.1 to SEQ ID No.14. The nucleotides can be used for preparing polymerase chain reaction (PCR) kits and gene chips for detecting Cronobacter sakazakii. The Cronobacter sakazakii O antigen specific nucleotides, and the PCR kits and the gene chips containing the nucleotides have high practicality; the preparation method of the PCR kits are simple and convenient, the detection period is short, the detection speed is high, the operability of the kits is high, the industrial production of the kits is easy, and the detection cost is relatively low; the accuracy is high; and the sensitivity is high.
Description
Technical field
The present invention relates to a kind ofly, relate in particular to a kind of special Nucleotide and the application thereof of individual gene in the Enterobacter sakazakii O antigen gene bunch to Enterobacter sakazakii O antigen gene bunch special Nucleotide and application thereof.
Background technology
Enterobacter sakazakii is the enteron aisle bacterial parasite and the conditioned pathogen of humans and animals, essential condition pathogenic bacterium that are defined as causing infant's death by the World Health Organization and many countries, the disease that can cause any age level crowd, especially to light baby of premature infant, baby weight or immunocompromised host baby's threat maximum, severe patient can cause septicemia, meningitis or necrotizing enterocolitis, and mortality ratio can reach 40%～80%.At present, the microbiologist it be unclear that the pollution source of Enterobacter sakazakii, but many parts of research reports show that infant formula powder is the main infection channel that current discovery causes baby, premature infant's meningitis, septicemia and necrotic colitis, Enterobacter sakazakii has caused the attention of the multinational relevant departments in the world, has been listed in the category-A pathogenic bacterium of infant formula powder.
The classification position of Enterobacter sakazakii is just set up at present, and the research of its O antigen serotype also becomes focus gradually.Lipopolysaccharides is the main surface composition of the blue formula negative bacteria of leather, comprises lipoid A, core polysaccharide and O antigen.O antigen is the outermost layer structure of gram negative bacterium lipopolysaccharide molecule, and it is by the polysaccharide chain that a plurality of oligosaccharides repeating unit forms, and repeating unit generally is made up of 3～6 monose.In intestinal bacteria, Salmonellas and Shigellae, be responsible for that O antigen synthetic gene is adjacent to be arranged between two housekeeping gene galF and the gnd, on karyomit(e), form a gene cluster.The gene of O antigen gene bunch inside is divided three classes usually: monose synthase gene, glycosyltransferase gene and oligosaccharide unit treatment enzyme gene.The monose synthetic enzyme is responsible for the synthetic of monose precursor, and glycosyltransferase is responsible for monose is connected into oligosaccharides repeating unit, and few pool unit treatment enzyme is responsible for oligosaccharide unit is transported to the inner membrance outside from the inner membrance inboard, and oligosaccharide unit is connected.
Lipopolysaccharides particularly wherein the variation of the antigenic The Nomenclature Composition and Structure of Complexes of O-has determined the diversity of gram negative bacterium cell-surface antigens determinant, such as nearly 2107 of antigenic types identifying salmonella in the world according to the structural performance of lipopolysaccharides.Serotype is a kind of classics and epidemiology survey means commonly used, and is significant for clinical study and vaccine development.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, often has cross reaction between the antiserum(antisera) that different O antigen produces.Therefore, the serum authentication method of setting up based on Protocols in Molecular Biology becomes present developing direction.Generally believe that now this traditional serology detection method will replace for the modern molecular biology method.
In recent years, increasing molecular engineering is used for somatotype, evaluation, detection and the disease screening of pathogenic bacteria, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) analysis, rDNA restriction fragment length polymorphism (RFLP) analysis etc.Molecular biology method not only can be used for the quick serotype examination of Enterobacter sakazakii, and stable qualification result can remedy the deficiency of phenotypic characteristic authentication method.Compare with the traditional detection technology, these are based on the molecular detection technology of polymerase chain reaction (PCR), do not need the processes such as separation, pure culture through pathogenic bacteria, and have fast, advantages such as sensitivity, high specificity.
The sequence homology of different antigenic glycosyltransferases and oligosaccharide unit treatment enzyme is very low, has the serotype specificity of height, can be used as target gene and carries out molecular biology identification.The oligosaccharide unit treatment enzyme gene comprises O antigen transhipment enzyme gene (wzx) and O antigen pol gene (wzy).Utilizing above-mentioned two genes is very good as the specific target gene that the serotype somatotype detects.As intestinal bacteria (Wang Wei, Peng Xia, 2006), Bacillus proteus and Salmonellas the example that utilizes this gene to do special detection is arranged all.
Polymerase chain reaction technology (Polymerase chain reaction, be called for short round pcr) obtaining approval and popularization at present as the microorganism detection technology, this technology has advantages such as high-throughput, detection speed are fast, high specificity, sensitivity height with respect to traditional method, only need sample is increased bacterium simply in advance or increases the bacterium process, prepare the DNA of bacteria template by centrifugal and cracking again, the target sequence that just can increase in the PCR process under the high specific primer mediation reaches the purpose that whether contains invasive organism to be measured in the test sample.The amplification procedure of PCR only needs 1 and a half hours.This has greatly improved working speed undoubtedly and has reduced job costs inspection and quarantine department and Clinical Laboratory.
No matter from internal and international angle, identify serum type quickly and accurately, it is crucial providing the effect technique support for the prevention and control of Enterobacter sakazakii.
Summary of the invention
The object of the present invention is to provide a kind of Nucleotide to the Enterobacter sakazakii antigen-specific, described Nucleotide comprises:
1) at least a in the Nucleotide shown in the SEQ ID NO:1-15;
2) with at least a in the Nucleotide complementary Nucleotide shown in the SEQ ID NO:1-15.
The present invention further discloses nucleotides sequence and be listed in of the application of preparation detection Enterobacter sakazakii with PCR test kit or gene chip aspect.
The invention also discloses a kind of PCR test kit of rapid detection clinical samples, this test kit comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer comprises at least a in the Nucleotide shown in the SEQ ID NO:1-15.
This test kit comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer comprises at least a in the Nucleotide shown in the SEQ IDNO:1-15; Each 0.5 μ l of 10 μ M primers wherein, 10mM dNTP 0.5 μ l, 10 * enzyme spcificity reaction buffer, 2.5 μ l, 5U/ μ l hot resistant DNA polymerase 0.3 μ l, 25mM MgCl
22.5 μ l, remaining volume is used ddH after the amount of removing 5 μ l testing samples
2O complements to 25 μ l.
Disclosed by the invention to the special Nucleotide of individual gene in the Enterobacter sakazakii O antigen gene bunch and use compared with prior art, have following advantage:
(1) practical
The invention provides serotype and detect used special primer, utilize this PCR method to detect clinical samples.Finally can utilize the method for PCR to detect 7 serotypes of discriminating.
Compound methods such as the PCR test kit that the present invention prepared are easy, and sense cycle is short, speed is fast, and is workable, is easy to industrialization production, and it is relatively low to detect cost.
(2) accuracy height
The present invention is by the PCR reaction to special gene wzx of each serotype of Enterobacter sakazakii and wzy, and each sample obtains the band of clauses and subclauses, will obtain the purpose fragment and compare with known length, just can obtain the affiliated serotype of Enterobacter sakazakii.
(3) highly sensitive
Detection primer provided by the invention and Enterobacter sakazakii detection kit etc. have higher susceptibility, and the accuracy of detection height can detect the dna profiling of 1ng/ μ l.
Description of drawings
Fig. 1 represents the screening of O1wzy gene P1 and P2 primer, and the purpose band is 364bp, and remaining serotype is without any band.
Fig. 2 represents the screening of O2wzy gene P3 and P4 primer, and the purpose band is 473bp, and remaining serotype is without any band.
Fig. 3 represents the screening of O3wzy gene P5 and P6 primer, and the purpose band is 704bp, and remaining serotype is without any band.
Fig. 4 represents the screening of O4wzy gene P7 and P8 primer, and the purpose band is 890bp, and remaining serotype is without any band.
Fig. 5 represents the screening of O5wzy gene P9 and P10 primer, and the purpose band is 235bp, and remaining serotype is without any band.
Fig. 6 represents the screening of O6wzx gene P11 and P12 primer, and the purpose band is 615bp, and remaining serotype is without any band.
Fig. 7 represents the screening of O7wzy gene P13 and P14 primer, and the purpose band is 424bp, and remaining serotype is without any band.
Fig. 8 represents species specific evaluation, has wherein detected four strain bacteriums and other kind four strain bacteriums between planting, does not all have any band.Concrete bacterial strain information sees Table 2.
Fig. 9 represents PCR test kit application somatotype result, and the bacterial strain and the non-corresponding O type that can detect corresponding O type do not have band.
Wherein: O1, O2, O3, O4, O5, O6, O7 and the corresponding serotype primer amplification result of negative contrast (-) expression, last swimming lane is 2000bp ladder marker.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989).
Embodiment 1: genomic extraction
37 ℃ of LB liquid nutrient mediums are cultivated Enterobacter sakazakii, collect bacterium, and it is as follows to extract the genome concrete steps:
With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution extracting twice, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2:Sequence is decoded
(1) the O antigen gene by Long-PCR amplification Enterobacter sakazakii bunch.According to the JumpStart design upstream primer between galF gene among the Genbank and the O antigen gene bunch is wl-10324,5 '-GCACTGGTAGCTATTGAGCCAGGGGCGGTAGCAT-3 ' is wl-22115 '-ACTGCCATACCGACGACGCCGATCTGTTGCTTGG-3 ' (being respectively SEQ ID NO:15-16) according to gnd gene design downstream primer again.
The PCR response procedures was as follows: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change are 30 seconds then, annealed 30 seconds for 61 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 8 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 8 pipes, 50 μ l long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.
(2) make up O antigen gene bunch library: make up O antigen gene bunch library with the shot-gun method, reaction system is the 600ngPCR purified product, and the carrier that cuts into 1～3kb fragment, the flat viscosity art end of benefit, pUC18 through the nucleic acid fragment instrument connects the gene library that (adopting fastlink ligase) connection obtains checking order.
(3) to the cloning and sequencing in the library: from the library, select 200 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 8～10 times fraction of coverage, thereby obtains all sequences of O antigen gene bunch.
(4) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Stadenpackage software package of publishing with Britain Camb MRC Molecular Biology Lab and edit all sequences after, orf finder with American National biotechnology information science center finds gene, find open reading frame, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain Sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the sequence of the gene cluster of Enterobacter sakazakii at last.
Embodiment 3: design of primers and screening
Decode situation according to gene cluster, we find that wzy and wzx gene are the special gene of serotype really, so choose this gene specific section design special primer.Because wzy is more special, thus be target gene mainly with the wzy gene, but do not contain this gene in the O6 serotype because of having, so this serotype designs primer with wzx for target gene.
Design of primers is the core of this invention.The design primer designs according to the specific gene of narrating in the document.These two genes of wzx and wzy are more special genes in the Enterobacter sakazakii O antigen gene bunch, can be used as the target gene that serotype is identified.Said gene is imported Primer Premier 5 carry out design of primers, the length of primer is preferably between 18～24bp, and the Tm value is at 50～55 ℃.The a pair of primer of each gene design has the single purpose band.
In Genbank, carry out BLAST after the 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 other nearly edge bacterium or the nearly edge bacterium in the environment of collect specimen.This point is very important for the success or failure of the generation of avoiding non-specific band and experiment.
The primer of designing is as shown in table 1.
Table 1 is used for the primer sequence of PCR
PCR system that the gene identification primer screening is used (25 μ l) and reaction conditions are as follows:
Ultrapure water 15.7 μ l
Damping fluid (10X) 2.5 μ l
dNTP(10mM) 0.5μl
Primer 1 (10 μ M) 0.5 μ l
Primer 2 (10 μ M) 0.5 μ l
Archaeal dna polymerase (rTaq) 0.3 μ l (5U/ μ l)
Template DNA 5 μ l
Reaction cycle parameter on the PCR instrument in this step comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially:
94 ℃, 5 minutes
72 ℃, 5 minutes
Wherein, O1, O3 and O5 use 50 ℃ of amplifications, and other types adopt 55 ℃ of expanding effects best.For for simplicity, also can be all with 50 ℃, but have the non-specific amplification of non-target stripe to exist.
Above-mentioned steps is the electrophoresis amplified production in electrophoresis equipment, and record result's concrete steps are:
1. getting 2～5 μ l amplified productions mixes with 5: 1 volume ratio with 6 * tetrabromophenol sulfonphthalein sample-loading buffer;
2. mixed solution is splined on 1.2% the sepharose;
3. with about 10 minutes of agarose gel electrophoresis 120v voltage stabilizing electrophoresis, contrast with DL2000Marker;
4. observe and write down the result.
The genomic templates that this link of primer screening is used is extracted the bacterium method of boiling that adopts.Work by primer screening after the basic PC R reaction finishes substantially, and necessary length adjustment is little to the W-response condition influence, and the primer sequence of using among the present invention all is summarised in the table 1.
Embodiment 4: sensitivity detects
Behind above-mentioned each O antigenic type of cultural method cultivation Enterobacter sakazakii, use physiological saline according to 10
0～10
-8Power dilutes laggard performing PCR and detects.Final sensitivity is exactly will have at least 10 in the 25 μ l systems
2Individual above bacterium could detect and obtain.
Embodiment 5: specific detection between kind
Choose that the close kind bacterial strain with other of bacterial strain carries out specific detection between the kind of Enterobacter sakazakii, method is the same.Bacterial strain uses therefor sees Table 2, the results are shown in Figure 8.All non-Enterobacter sakazakiis all do not have amplified band, illustrate that the primer specificity of design in the invention is good, are suitable for being suitable for as somatotype in the Enterobacter sakazakii kind.
Table 2 is used for the bacterial strain of specific detection
Embodiment 6: the application of PCR test kit
(1) composition of PCR test kit
Comprise PCR primer, dNTP, damping fluid, archaeal dna polymerase, positive reference substance and negative control product.The amount that test kit uses all is that 20 μ l are a basic consumption.Each test kit can detect 20 samples.
(2) preparation of test experience material requested and equipment
MgCl wherein
2, 10 * damping fluid, dNTP, rTaq polysaccharase give birth to the worker by Shanghai and provide; Primer mixture is synthetic for the sequence that designs voluntarily offers Shanghai Ying Jun biotech company; Positive reference substance, negative control product and ultrapure water are prepared voluntarily by us, wherein positive reference substance is an Enterobacter sakazakii O1 type genome, each reaction consumption is 1 μ l, add reaction system as template, add ultrapure water and supply 25 μ l, the negative control product are ultrapure water, add 5 μ l to reaction system.
Equipment PCR instrument, electrophoresis equipment, gel imaging instrument ,-20 ℃ of refrigerators, supercentrifuge, micropipet and 0.2ml PCR thin-walled tubes of experiment.
(3) the use specific examples of PCR test kit
1. the processing of testing sample.Extract genome if having ready conditions also passable, in order to save time and consumptive material, can directly get 500 μ l bacterium liquid, centrifugal back with 500 μ l ddH
2O is resuspended, and 100 ℃ were boiled 10 minutes, the centrifugal 10min of 12000rpm then, and getting middle layer supernatant is template.The testing sample specifying information sees Table 3.
Table 3 testing sample
2. the reagent 20 μ l that get the PCR test kit add 5 μ l templates in the PCR pipe.
3. the PCR reaction conditions is as follows:
94 ℃, 5 minutes
72 ℃, 5 minutes
4. 1.2% agarose gel electrophoresis detects test-results.
5. result's analysis and record.The results are shown in Figure 9.Corresponding bacterial strain has corresponding single correct band, and irrelevant bacterial strain does not have band.
The above, only be operation of the present invention and implementation method, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Claims (3)
1. Nucleotide to Enterobacter sakazakii O antigen-specific is characterized in that Nucleotide comprises:
1) at least a in the Nucleotide shown in the SEQ ID NO:1-15;
2) with at least a in the Nucleotide complementary Nucleotide shown in the SEQ ID NO:1-15.
2. the described nucleotides sequence of claim 1 is listed in preparation and detects the application of Enterobacter sakazakii with PCR test kit or gene chip aspect.
3. a PCR test kit is characterized in that this test kit comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer comprises at least a in the Nucleotide shown in the SEQ ID NO:1-15; Each 0.5 μ l of 10 μ M primers wherein, 10mM dNTP 0.5 μ l, 10 * enzyme spcificity reaction buffer, 2.5 μ l, 5U/ μ l hot resistant DNA polymerase 0.3 μ l, 25mM MgCl
22.5 μ l, remaining volume is used ddH after the amount of removing 5 μ l testing samples
2O complements to 25 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110004830A CN102154270B (en) | 2011-01-11 | 2011-01-11 | Cronobacter sakazakii O antigen specific nucleotides and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110004830A CN102154270B (en) | 2011-01-11 | 2011-01-11 | Cronobacter sakazakii O antigen specific nucleotides and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102154270A true CN102154270A (en) | 2011-08-17 |
| CN102154270B CN102154270B (en) | 2012-09-05 |
Family
ID=44435944
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110004830A Expired - Fee Related CN102154270B (en) | 2011-01-11 | 2011-01-11 | Cronobacter sakazakii O antigen specific nucleotides and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102154270B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898227A (en) * | 2014-04-16 | 2014-07-02 | 南开大学 | Nucleotide specific to cronobacter O antigen and application of nucleotide |
| CN104593487A (en) * | 2014-12-15 | 2015-05-06 | 合肥工业大学 | PCR-RFLP molecular subtyping method for distinguishing different types of cronobacters |
| CN111676304A (en) * | 2020-07-08 | 2020-09-18 | 南开大学 | Method for real-time fluorescence PCR detection of Enterobacter sakazakii O antigen and application |
| CN112375833A (en) * | 2020-11-23 | 2021-02-19 | 南开大学 | Loop-mediated isothermal amplification detection method for O antigen serotype typing of Enterobacter sakazakii |
-
2011
- 2011-01-11 CN CN201110004830A patent/CN102154270B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 20080630 N. Mullane Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus 3783-3794 1-3 第74卷, 第12期 * |
| 《微生物学报》 20060604 王威 大肠杆菌O11 O-抗原基因簇序列的破译及特异分子标识的鉴定 341-346 1-3 第46卷, 第3期 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898227A (en) * | 2014-04-16 | 2014-07-02 | 南开大学 | Nucleotide specific to cronobacter O antigen and application of nucleotide |
| CN104862393A (en) * | 2014-04-16 | 2015-08-26 | 南开大学 | Nucleotide specific to Cronobacter O antigen and application thereof |
| CN103898227B (en) * | 2014-04-16 | 2015-12-02 | 南开大学 | A kind of Nucleotide to Cronobacter sakazakii O antigen-specific and application thereof |
| CN104593487A (en) * | 2014-12-15 | 2015-05-06 | 合肥工业大学 | PCR-RFLP molecular subtyping method for distinguishing different types of cronobacters |
| CN111676304A (en) * | 2020-07-08 | 2020-09-18 | 南开大学 | Method for real-time fluorescence PCR detection of Enterobacter sakazakii O antigen and application |
| WO2022007326A1 (en) * | 2020-07-08 | 2022-01-13 | 南开大学 | Method and use for real-time fluorescent pcr detection of cronobacter sakazakii o-antigen |
| CN112375833A (en) * | 2020-11-23 | 2021-02-19 | 南开大学 | Loop-mediated isothermal amplification detection method for O antigen serotype typing of Enterobacter sakazakii |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102154270B (en) | 2012-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105112519A (en) | CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method | |
| CN103898227B (en) | A kind of Nucleotide to Cronobacter sakazakii O antigen-specific and application thereof | |
| CN103898108B (en) | The nucleotide special to vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof | |
| CN104059975B (en) | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof | |
| CN102154270B (en) | Cronobacter sakazakii O antigen specific nucleotides and use thereof | |
| CN102876774B (en) | Primer, fluorescence probe and kit for quantitative detection of streptococcus pneumonia nucleic acid | |
| CN103993090B (en) | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof | |
| CN103060447B (en) | Triple real-time fluorescent PCR testing primers of four kinds of bacterium, probe, detection kit and detection method | |
| CN105063228B (en) | The detection kit and detection method of a kind of flavobacterium columnare | |
| CN105154559A (en) | Specific nucleotide for vibrio parahaemolyticus K36, K37 and K68 and application 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 | |
| CN105256041B (en) | The nucleotide special to aeromonas hydrophila O44, O24, O25 and O28 and application | |
| CN106399512A (en) | Detecting kit for aeromonas hydrophila and detecting method of detecting kit | |
| CN111621578A (en) | RU 61-00441 gene-based Salmonella spelt constant-temperature detection kit and method | |
| CN105154438B (en) | To Hafnia alvei G5907, G5908, G5913, nucleotide special G5916 and its application | |
| CN102337261A (en) | Neisseria meningitidis antigen-specific nucleotide and application thereof | |
| 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 | |
| CN105087569B (en) | To vibrio cholerae O 18, O19, O23 and the special nucleotides of O12 and its application | |
| CN102311950A (en) | Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof | |
| CN105177133B (en) | The nucleotide special to comma bacillus O6, O4, O7 and O15 and its application | |
| CN105177144A (en) | Nucleotide for specificity of K4, K32 and K34 of vibrio parahaemolyticus and application thereof | |
| CN105925683A (en) | Detection kit of edwardsiella tarda and application of detection kit | |
| CN105256043B (en) | The nucleotide special to aeromonas hydrophila O29, O30, O33 and O35 and application |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120905 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |