CN104862393B - A kind of nucleotides and its application to Cronobacter sakazakii O antigen-specifics - Google Patents
A kind of nucleotides and its application to Cronobacter sakazakii O antigen-specifics Download PDFInfo
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Abstract
The invention discloses a kind of nucleotides to Cronobacter sakazakii O antigen-specifics and its application.The nucleotides is：SEQ ID NO:At least one of nucleotides shown in 1 12 or at least one of the nucleotides of complementation.The special nucleotides can be used for the PCR kit and/or genetic chip for preparing quick detection Cronobacter sakazakii.Detect that the Cronobacter sakazakii in clinical and food is simple and convenient with the kit, accuracy is high, and sensitivity is good, workable, reproducible, and testing cost is low.
Description
The present patent application is application number：201410150358.2, the applying date：2014.04.16, denomination of invention：It is a kind of to gram
The nucleotides of Luo Nuo bacillus O antigen-specifics and its divisional application of application.
Technical field
The present invention relates to a kind of nucleotides special to Cronobacter sakazakii O antigen gene clusters, more particularly to it is a kind of to Crow
The special nucleotides of individual gene in promise bacillus O antigen gene clusters.
Background technology
Cronobacter（Cronobacter sakazakii）It is a category of most recently newly classification, original claims Enterobactersakazakii
（Enterobacter sakazakii）.2007-2008 rises to category by kind, and one as enterobacteriaceae newly belongs toCronobacter.Wrap at present
7 kinds are included,Cronobacter sakazakii, C.malonaticus, C.muytjensii, C.dublinensis, C.turicensis, C.condimentiWithC.universalis.Now there are some researches show all Cronobacter sakazakii different strains
Between have obvious Difference in Pathogenicity, it neutralizes the related bacterium of infant infection and concentrated onCronobacter sakazakii, C.malonaticusWithC.dublinensisOn.
Cronobacter sakazakii is important food-borne pathogens, widely distributed, in baby milk powder, cheese, butcher's meat, water, vegetable
It is detected in the numerous foods such as dish, rice, bread, tealeaves, herbal medicine, flavoring and bean curd.To some neonate Crows
Find that baby milk powder is main infection channel in the investigation of promise bacillus infection event.With a series of milk powder related to the bacterium
The infection problems of Cronobacter sakazakii in the outburst with great infection event, baby formula milk powder are recalled by global universal
Concern, is defined as causing the essential condition pathogenic bacteria of Infant and child deaths by the World Health Organization and many countries, can cause to appoint
The threat of the disease of what age level crowd, the especially baby light to premature, birth weight or immunocompromised host baby is maximum, sternly
Severe one can cause septicemia, meningitis or necrotizing enterocolitis, and the death rate is up to 40%~80%.The World Health Organization
（WHO）With food and medicine Surveillance Authority of the U.S.（FDA）The meeting that the bacterium is discussed has been held at 2004 and 2006 in succession.
The classification position of current Cronobacter sakazakii is just set up, and the research of its O antigen serotype is increasingly becoming focus.Fat
Polysaccharide is the major surfaces composition of gram-negative bacteria, including lipoid A, core polysaccharide and O antigens.O antigens are gram-negatives
The outermost Rotating fields of property bacteria lipopolysaccharide molecule, the polysaccharide chain that it is made up of multiple oligosaccharides recurring units, recurring unit is general
It is made up of 3~6 monose.In Escherichia coli, salmonella and Shigella, it is responsible for the gene arranged adjacent of O antigens synthesis
Between two housekeeping genes galF and gnd, a gene cluster is formed on chromosome.Gene inside O antigen gene clusters leads to
Often it is divided into three classes：Monose synthase gene, glycosyltransferase gene and oligosaccharide unit treatment enzyme gene.Monose synzyme is responsible for list
The synthesis of sugar precursor, glycosyl transferase is responsible for monose being connected into oligosaccharides recurring unit, and few pool unit treatment enzyme is responsible for widow
Sugared unit is transported on the outside of inner membrance from its inner side, and oligosaccharide unit is connected.
The change that lipopolysaccharides is particularly the Nomenclature Composition and Structure of Complexes of O- antigens therein determines gram negative bacterial cell table
The diversity of face antigenic determinant, such as identified the Antigens of Salmonella according to the architectural characteristic of lipopolysaccharides in the world
Up to 2107, type.Serotype is a kind of classical and conventional epidemiology survey means, is ground for clinical research and vaccine
Fixture is significant.This diagnostic method needs substantial amounts of antiserum, and antiserum general classes are not complete, lazy weight, greatly
The antiserum of amount is difficult there is also some in preparation and storage.On the other hand this method time-consuming, sensitivity is low, loss is high,
Accuracy is poor, and cross reaction is frequently present of between the antiserum that different O antigens are produced.Therefore, set up and be based on molecular biology
The serological diagnosis method of technology turns into current developing direction.Generally believing this traditional Serology test now will be
Modern molecular biology method replaces.
In recent years, increasing molecular engineering is used for parting, identification, detection and the disease screening of pathogen, including turns
Record spacer region (ITS) sequence analysis, random amplification length polymorphism (RAPD) analysis, rDNA Restriction Fragment Length are more
State property (RFLP) analysis etc..Molecular biology method cannot be only used for the rapid serum parting examination of Enterobacter sakazakii, stabilization
Qualification result can make up the deficiency of phenotypic characteristic authentication method.Compared with traditional sensing techniques, these are based on polymerase chain type
React the molecular detection technology of (PCR), it is not necessary to by processes such as separation, the pure cultures of pathogen, and with quick, spirit
The advantages of quick, high specificity.
The glycosyl transferase of synantigen and the sequence homology of oligosaccharide unit treatment enzyme be not very low, the serotype with height
Specificity, can be used as target gene and carries out molecular biology identification.Oligosaccharide unit treatment enzyme gene includes O antigen delivery enzyme genes
And O antigens pol gene (wzy) (wzx).The specific target gene detected by the use of above-mentioned two gene as Serotypes is
Very good.Such as Escherichia coli（Wang Wei, Peng Xia, 2006 year）, proteus and salmonella have and be spy using the gene
The example of different detection.
Polymerase chain reaction technology (Polymerase chain reaction, abbreviation round pcr) is examined as microorganism
Survey technology is currently accepted and promoted, and the technology has high flux, detection speed fast, specific relative to traditional method
By force, the advantages of sensitivity is high, simply pre- increasing bacterium need to be only carried out to sample or increases bacterium process, then is prepared carefully by centrifuging and cracking
Bacterium DNA profiling, it is possible to expand target sequence during the PCR under the mediation of high specific primer, reaching in detection sample is
The no purpose containing invasive organism to be measured.PCR amplification procedure is only needed 1 and a half hours.This to inspection and quarantine department and
Clinical examination undoubtedly greatly improves operating rate and reduces job costs.No matter from the perspective of internal and international, soon
Speed, serum type is identified exactly, for the prevention and control of Cronobacter sakazakii, to provide effective technical support be highly important.
The content of the invention
It is an object of the present invention to provide a kind of nucleotides special to Cronobacter sakazakii O antigen genes, it is special
Levy and be that the nucleotides is：
1）SEQ ID NO:At least one of nucleotides shown in 1-12
2）With SEQ ID NO:At least one of nucleotides of nucleotide complementary shown in 1-12.It is wherein described complementary
Nucleotides is referred to by SEQ ID NO:Nucleotides shown in 1-12 is according to basepairing rule（A=T,G≡C）It is drawing and it
Complementary nucleotides, such as SEQ ID NO1：5'GAAGCGGCAGATTCATTTA3' complementary nucleotide is 5
‘TAAATGAATCTGCCGCTTC 3’。
Another object of the present invention is the provision of a kind of PCR kit for being used to detect Cronobacter sakazakii, including PCR draws
Thing, dNTP, buffer solution and archaeal dna polymerase, the PCR primer such as SEQ ID NO:At least one of nucleotides shown in 1-12.
Such as it is used to detectC.dublinensisO1 primer SEQ ID NO:1 and SEQ ID NO:2, and for detectingC.dublinensisO2 primer SEQ ID NO:3 and SEQ ID NO:4.
This kit, in addition to following reagent：10 mM dNTP 30μL；The μ L of 10 × enzyme spcificity reaction buffer 50；
The μ L of 5 U/ μ L hot resistant DNA polymerases 5；Primer 1（10μM） 10μl；Primer 2（10μM）10μl；The μ L of positive reference substance 10；It is negative
The μ L of reference substance 10；ddH2O 5mL。
Further object of the present invention is the provision of the nucleotides and is preparing the gene core for detecting Cronobacter sakazakii
Application in piece.Described genetic chip is microarray chip.Including solid phase carrier and the few nucleosides being fixed on solid phase carrier
Acid probe, wherein the oligonucleotide probe includes above-mentioned nucleotides；Wherein described nucleotides such as SEQ ID NO:1-12 institutes
At least one of nucleotides shown.
Nucleotides disclosed by the invention for quick detection Cronobacter sakazakii compared with prior art, the present invention have with
Lower advantage：
（1）It is practical
The compound methods such as the PCR kit prepared using the present invention are easy, and detection cycle is short, speed is fast, operability
By force, it is easy to industrialization production, and testing cost is relatively low.
（2）Accuracy is high
The present invention devises the primer special to Cronobacter sakazakii O antigen genes, using the genome of Cronobacter sakazakii as mould
Plate does PCR single, the correct purpose bands of size that can obtain band, and when being PCR by template of the genome of other bacteriums,
Correct purpose band can not be obtained.
Brief description of the drawings：
G2539 in accompanying drawing isC.sakazakiiO1, G2356 areC.sakazakiiO2, G2594 areC.sakazakiiO4, G2732 areC.dublinensisO1, G3983 areC. dublinensisO2, G3882 areC.turicensisO2, G3874 areC.turicensisO3, G3886 areC.muytjensiiO2, G3864 areC.malonaticusO1；
Fig. 1 represents g2732wzmThe screening of gene P1 and P2 primer, purpose band is 336bp, and remaining bacterium is not any
Band；
Fig. 2 represents g 3983wzyThe screening of gene P3 and P4 primer, purpose band is 725bp, and remaining bacterium is not any
Band；
Fig. 3 represents g3882wzyThe screening of gene P5 and P6 primer, purpose band is 266bp, and remaining bacterium is not any
Band；
Fig. 4 represents g3874wzyThe screening of gene P7 and P8 primer, purpose band is 362bp, and remaining bacterium is not any
Band；
Fig. 5 represents g3886wzyThe screening of gene P9 and P10 primer, purpose band is 378bp, and remaining bacterium is not appointed
What band；
Fig. 6 represents g3864wzyThe screening of gene P11 and P12 primer, purpose band is 267bp, and remaining bacterium is not appointed
What band.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning：Laboratory manual（New York: Cold Spring Harbor Laboratory
Press,1989）Described in condition.
Embodiment 1：
The extraction of genome：
37 DEG C of 2YT fluid nutrient mediums（It is formulated and is：Peptone 16g/L, dusty yeast 10g/L, NaCl5g/L）Cultivate Cronobacter
Bacillus, collects bacterium, extracts genome and comprises the following steps that：
Cell is resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, 37 DEG C incubate 20 minutes,
Then the lysozyme for adding 10ul 10mg/ml continues to be incubated 20 minutes.3ul 20mg/ml Proteinase K, 15ul is added afterwards
10%SDS, 50 DEG C incubate 2 hours, add 3 ul 10mg/ml RNase, and 65 DEG C incubate 30 minutes.Plus isometric phenol extracting
Mixture, takes supernatant, then with isometric phenol：Chloroform：Isoamyl alcohol（25：24：1）Solution is extracted twice, takes supernatant, then use
Isometric ether extraction with remove remnants phenol.Supernatant precipitates DNA with 2 times of volume ethanols, rolls out DNA with glass fiber and is used in combination
70% ethanol washes DNA, and finally DNA is resuspended in 30ul TE.Genomic DNA is detected by 0.4% agarose gel electrophoresis.
Embodiment 2：
O antigen gene clusters are decoded：
（1）The O antigen gene clusters of Cronobacter sakazakii are expanded by Long-PCR.According in GenbankgalFGene is set
Sense primer is counted, further according togndGene designs anti-sense primer.
PCR response procedures are as follows：In 94 DEG C of pre-degenerations 5 minutes;Then 94 DEG C are denatured 30 seconds, and 61 DEG C are annealed 30 seconds, 68 DEG C
Extension 15 minutes, so carries out 30 circulations, finally, continues to extend 8 minutes at 68 DEG C, PCR primer is obtained, with 0.8% agar
The size and its specificity of sugared detected through gel electrophoresis PCR primer, merge the μ l long PCR primers of 8 pipe 50, and public with Promega
The Wizard PCR Preps Purification Kit PCR primers of department.
Build O antigen gene clusters library：O antigen gene clusters library is built with shot-gun methods, reaction system is
600ng PCR purified products, by nucleic acid fragment instrument cut into 1~3kb fragments, filling-in cohesive end, pUC18 carrier connect
（Using fastlink ligase）Connect the gene library being sequenced.
To the cloning and sequencing in library：200 clones that Insert Fragment is selected from library in more than 1kb use this reality
Test room ABI3730 types automatic dna sequencer the Insert Fragment in clone is sequenced, sequence reaches 8~10 times of coverage rate,
So as to obtain all sequences of O antigen gene clusters.
The splicing and analysis of nucleotide sequence：The Staden published with Britain Camb MRC Molecular Biology Labs
Pregap4 the and Gap4 softwares of package software kits splice and edited after all sequences, are believed with American National biotechnology
The orf finder at Xi Xue centers have found gene, find open reading frame, with the base in the blast groupwares and GenBank
Because comparing to find the function of these reading frames opened and determine what gene they are, then with Britain Sanger centers
Artemis softwares complete gene annotation, and the precise alignment between DNA and protein sequence is done with Clustral W softwares, finally
To the sequence of the gene cluster of Cronobacter sakazakii.
Embodiment 3：
Design of primers and screening
Situation is decoded according to gene cluster, it has been found thatwzy/wzmThe strictly special gene of Cronobacter sakazakii, so choosing
The gene specific section designs special primer.Said gene is imported into Primer Premier 5 and carries out design of primers, primer
Length is preferably between 18~24bp, and Tm values are at 50~55 DEG C.
Carry out BLAST after design of primers in Genbank, the sequence phase that the primer of design can not be with other nearly edge bacteriums
It is too high like property, so ensure that the primer is only expanded in the precalculated position of oneself, without with other closely edge bacterium or
Nearly edge bacterium in the environment of collect specimen does not produce positive reaction.This point is for avoiding the generation and experiment of non-specific band
Success or failure are particularly significant.The primer designed is as shown in table 1.
Table 1 is used for PCR primer sequence
The PCR system (25 μ l) and reaction condition that identified for genes primer screening is used are as follows：
The μ l of ultra-pure water 15.7
The μ l of buffer solution (10X) 2.5
dNTP(10mM) 0.5μl
Primer 1（10μM） 0.5μl
Primer 2（10μM） 0.5μl
Archaeal dna polymerase（rTaq） 0.3μl(5U/μl)
The μ l of template DNA 5
The reaction cycle parameter in PCR instrument in the step include DNA denaturation, renaturation, the temperature and time of extension,
Cycle-index, be specially：
Above-mentioned steps electrophoresis amplified production in electrophoresis equipment, records concretely comprising the following steps for result：
2~5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer are taken with 5：1 volume ratio mixing；
Mixed liquor is splined on 1.2% Ago-Gel；
By agarose gel electrophoresis 120v voltage stabilizings electrophoresis about 10 minutes, compareed with DL2000 Marker；
Observe and record result.
The genomic templates that this link of primer screening is used, which are extracted to use, boils bacterium method.Draw after being reacted by basic PC R
The work of thing screening terminates substantially, and necessary length adjustment influences little, to be used in present invention primer on W-response condition
Sequence is all summarised in table 1.
Embodiment 4：
The application of PCR detection kit
The composition of PCR kit
Including PCR primer, dNTP, buffer solution, archaeal dna polymerase, positive reference substance and negative controls.What kit was used
Amount is all that 20 μ l are a basic consumption.Each kit can detect 20 samples.
（2）The preparation of test experience material requested and equipment
Wherein 10 × buffer, dNTP, Taq polymerase by Shanghai Sheng Gong companies by providing；Primer mixture is certainly
The sequence of row design is supplied to Beijing Ying Jun biotech companies to synthesize；Positive reference substance, negative controls and ddH2O is by us
Voluntarily prepare.The equipment PCR instrument of experiment, electrophoresis equipment, gel imager, -20 DEG C of refrigerators, supercentrifuge, micropipettors
With 0.2ml PCR light-wall pipes.
The use instantiation of PCR kit
The processing of testing sample.If have ready conditions extraction genome can also, can be with order to save time and consumptive material
500 μ l bacterium solutions directly are taken, with 500 μ l ddH after centrifugation2O is resuspended, and 100 DEG C are boiled 10 minutes, then 12000rpm centrifugations
10min, it is template to take middle layer supernatant.
The μ l of reagent 20 of PCR kit are taken in PCR pipe, 5 μ l templates are added.
PCR reaction conditions are as follows：
1.2% agarose gel electrophoresis detection result of the test.
As a result analysis and record.
Conclusion：Specific detection is carried out with these primer pair Cronobacter sakazakiis and other close kind bacterial strains, primer is found
Specifically.By carrying out specific detection between strain in these primer pair Cronobacter sakazakiis category, accompanying drawing is as a result seen.It is used in experiment
Bacterial strain be shown in Table 2.
The bacterial strain that the specific detection of table 2 is used
SEQUENCE LISTING
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Claims (3)
1. a kind of nucleotides special to Cronobacter sakazakii O antigen genes, it is characterised in that the nucleotides is SEQ ID NO:
3rd, the nucleotides shown in 4.
2. a kind of PCR kit, including
10 mMdNTP 30μL；The μ L of 10 × enzyme spcificity reaction buffer 50；
The μ L of 5 U/ μ L hot resistant DNA polymerases 5；Each 10 μ l of 10 μM of PCR primers；
The μ L of positive reference substance 10, the μ L of negative controls 10；
ddH2O 5mL；
The PCR primer is respectively such as SEQ ID NO in claim 1:3rd, shown in 4.
3. the nucleotides special to Cronobacter sakazakii O antigen genes described in claim 1 is being prepared for detecting Cronobacter sakazakii
PCR kit in application.
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| CN104593487B (en) * | 2014-12-15 | 2017-06-27 | 合肥工业大学 | It is a kind of to distinguish the PCR RFLP molecular typing methods that Cronobacter sakazakii belongs to not of the same race |
| CN105256028B (en) * | 2015-10-20 | 2018-10-26 | 南开大学 | The nucleotide special to citric acid bacillus 017 and O39 and its application |
| CN105219769B (en) * | 2015-10-20 | 2018-05-15 | 南开大学 | The nucleotide special to citric acid bacillus O6 and O9 and its application |
| CN105256042B (en) * | 2015-11-02 | 2018-10-26 | 南开大学 | The nucleotide special to aeromonas hydrophila O13, O36, O16 and O19 and application |
| CN105256041B (en) * | 2015-11-02 | 2018-10-26 | 南开大学 | The nucleotide special to aeromonas hydrophila O44, O24, O25 and O28 and application |
| CN105255871B (en) * | 2015-11-02 | 2018-10-26 | 南开大学 | The nucleotide special to aeromonas hydrophila O7, O8, O9 and O10 and application |
| CN105256043B (en) * | 2015-11-02 | 2018-10-26 | 南开大学 | The nucleotide special to aeromonas hydrophila O29, O30, O33 and O35 and application |
| CN105331693B (en) * | 2015-11-03 | 2019-05-14 | 南开大学 | To the gene liquid chip and detection method of 8 kinds of O antigens genotypings of Plesiomonas shigelloides |
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| CN102154270B (en) * | 2011-01-11 | 2012-09-05 | 天津生物芯片技术有限责任公司 | Cronobacter sakazakii O antigen specific nucleotides and use thereof |
| US20120322676A1 (en) * | 2011-06-17 | 2012-12-20 | Life Technologies Corporation | Compositions and methods for detection of cronobacter spp. and cronobacter species and strains |
| CN103243171A (en) * | 2013-05-29 | 2013-08-14 | 光明乳业股份有限公司 | Method for detecting cronobacter sakazakii as well as kit and primer thereof |
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2014
- 2014-04-16 CN CN201410150358.2A patent/CN103898227B/en active Active
- 2014-04-16 CN CN201510233954.1A patent/CN104862393B/en active Active
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| Publication number | Publication date |
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| CN103898227A (en) | 2014-07-02 |
| CN103898227B (en) | 2015-12-02 |
| CN104862393A (en) | 2015-08-26 |
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