CN104862393A

CN104862393A - Nucleotide specific to Cronobacter O antigen and application thereof

Info

Publication number: CN104862393A
Application number: CN201510233954.1A
Authority: CN
Inventors: 王敏; 王磊; 王来友; 冯露
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2014-04-16
Filing date: 2014-04-16
Publication date: 2015-08-26
Anticipated expiration: 2034-04-16
Also published as: CN104862393B; CN103898227A; CN103898227B

Abstract

The invention discloses a nucleotide specific to Cronobacter O antigen and application thereof. The nucleotide is at least one of nucleotides as shown in SEQ ID NO: 1-12 or at least one of complementary nucleotides. The specific nucleotide can be used for preparing a PCR (Polymerase Chain Reaction) kit and/or a gene chip for quickly detecting the Cronobacters. The kit is simple and convenient, high in accuracy, good in sensitivity, excellent in operability, good in repeatability and low in detection cost when being applied to detecting clinical Cronobacters or Cronobacters in food.

Description

A kind of Nucleotide to Cronobacter sakazakii O antigen-specific and application thereof

The present patent application is application number: 201410150358.2, the applying date: 2014.04.16, denomination of invention: a kind of to the Nucleotide of Cronobacter sakazakii O antigen-specific and the divisional application of application thereof.

Technical field

The present invention relates to a kind of Nucleotide special to Cronobacter sakazakii O antigen gene cluster, particularly relate to a kind of Nucleotide special to individual gene in Cronobacter sakazakii O antigen gene cluster.

Background technology

cronobacter(Cronobacter sakazakii) is a genus of new classification recently, former title Enterobactersakazakii(Enterobacter sakazakii).2007-2008 rises to genus by kind, and one that becomes enterobacteriaceae newly belongs to cronobacter.Comprise 7 kinds at present, cronobacter sakazakii, C.malonaticus, C.muytjensii, C.dublinensis, C.turicensis, C.condimentiwith c.universalis.Now there are some researches show, have obvious Difference in Pathogenicity between all Cronobacter sakazakii different strains, bacterium relevant with infant infection in it concentrates on cronobacter sakazakii, C.malonaticuswith c.dublinensison.

Cronobacter sakazakii is important food-borne pathogens, widely distributed, is detected in the numerous food such as baby milk powder, cheese, butcher's meat, water, vegetables, rice, bread, tealeaves, herbal medicine, food flavouring and bean curd.In the investigation some newborn infant's Cronobacter sakazakii being infected to event, find that baby milk powder is main infection channel.Along with the outburst with great infection event recalled by a series of milk powder relevant to this bacterium, in baby formula milk powder, the infection problems of Cronobacter sakazakii is subject to global common concern, be defined as by the World Health Organization and many countries the essential condition pathogenic bacterium causing Infant and child deaths, the disease of any age level crowd can be caused, especially maximum to the threat of the light baby of premature infant, baby weight or immunocompromised host baby, severe patient can cause septicemia, meningitis or necrotizing enterocolitis, and mortality ratio can reach 40% ~ 80%.The World Health Organization (WHO) and U.S. food Drug Administration (FDA) have held the meeting that this bacterium is discussed in succession 2004 and 2006.

The classification position of current Cronobacter sakazakii is just set up, and the research of its O antigen serotype also becomes focus gradually.Lipopolysaccharides is the major surfaces composition of Gram negative bacteria, comprises lipoid A, core polysaccharide and O antigen.O antigen is the outermost layer structure of gram negative bacterium lipopolysaccharide molecule, the polysaccharide chain that it is made up of multiple oligosaccharides repeating unit, and repeating unit is generally made up of 3 ~ 6 monose.In intestinal bacteria, Salmonellas and Shigellae, the gene arranged adjacent being responsible for the synthesis of O antigen, between two housekeeping gene galF and gnd, forms a gene cluster on chromosome.The gene of O antigen gene cluster inside is divided three classes usually: monose synthase gene, glycosyltransferase gene and oligosaccharide unit treatment enzyme gene.Monose synthetic enzyme is responsible for the synthesis of monose precursor, and glycosyltransferase is responsible for monose to be connected into oligosaccharides repeating unit, and few pool unit treatment enzyme is responsible for oligosaccharide unit to be transported to outside inner membrance from its inner side, and oligosaccharide unit is connected.

The change of the lipopolysaccharides particularly the Nomenclature Composition and Structure of Complexes of O-antigen wherein determines the diversity of gram negative bacterial cell surface antigen determinant, such as identifies that the antigenic type of salmonella reached 2107 according to the structural performance of lipopolysaccharides in the world.Serotype is a kind of classics and conventional epidemiology survey means, for clinical study and vaccine development significant.This diagnostic method needs a large amount of antiserum(antisera)s, and antiserum(antisera) general classes is incomplete, quantity not sufficient, and a large amount of antiserum(antisera)s is in preparation and also there are some difficulties in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, often there is cross reaction between the antiserum(antisera) that different O antigen produces.Therefore, the serological diagnosis method set up based on Protocols in Molecular Biology becomes current developing direction.Generally believe now that this traditional Serology test will replace for modern molecular biology method.

In recent years, increasing molecular engineering is used for the somatotype of pathogenic bacteria, qualification, detection and disease screening, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) is analyzed, rDNA restriction fragment length polymorphism (RFLP) is analyzed.Molecular biology method not only can be used for the rapid serum somatotype examination of Enterobacter sakazakii, and stable qualification result can make up the deficiency of phenotypic characteristic authentication method.Compare with traditional sensing techniques, these are based on the molecular detection technology of polymerase chain reaction (PCR), do not need the process such as separation, pure culture through pathogenic bacteria, and have the advantages such as quick, sensitive, high specificity.

Not the glycosyltransferase of synantigen and the sequence homology of oligosaccharide unit treatment enzyme very low, have height serotype specificity, can be used as target gene and carry out molecular biology identification.Oligosaccharide unit treatment enzyme gene comprises O antigen delivery enzyme gene (wzx) and O antigen pol gene (wzy).Above-mentioned two genes are utilized to be very good as the specific target gene that Serotypes detects.As intestinal bacteria (Wang Wei, Peng Xia, 2006), Bacillus proteus and Salmonellas all have the example utilizing this gene to do specific detection.

Polymerase chain reaction technology (Polymerase chain reaction, be called for short round pcr) admitted at present as microorganism detection technology and promoted, this technology has relative to traditional method that high-throughput, detection speed are fast, high specificity, sensitivity advantages of higher, only need carry out simple pre-increasing bacterium to sample or increase bacterium process, again by the centrifugal and detailed bacterium DNA profiling of cracking, just can to increase target sequence in the PCR process under the mediation of high specific primer, reach the object detected in sample whether containing invasive organism to be measured.The amplification procedure of PCR only needs 1 and a half hours.This greatly improves working speed undoubtedly to inspection and quarantine department and Clinical Laboratory and reduces job costs.No matter from internal and international angle, identify serum type quickly and accurately, it is very important that the prevention and control for Cronobacter sakazakii provide effect technique support.

Summary of the invention

One object of the present invention there are provided a kind of Nucleotide special to Cronobacter sakazakii O antigen gene, it is characterized in that described Nucleotide is:

1) at least one in the Nucleotide shown in SEQ ID NO:1-12

2) with at least one in the Nucleotide of the nucleotide complementary shown in SEQ ID NO:1-12.The Nucleotide of wherein said complementation refers to by the Nucleotide shown in SEQ ID NO:1-12 according to basepairing rule (A=T, G ≡ C) draw with the Nucleotide of their complementations, the complementary nucleotide as SEQ ID NO1:5'GAAGCGGCAGATTCATTTA 3' is 5 ' TAAATGAATCTGCCGCTTC 3 '.

Another object of the present invention there are provided a kind of PCR kit for detecting Cronobacter sakazakii, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, at least one in described PCR primer Nucleotide as shown in SEQ ID NO:1-12.As for detect c.dublinensisthe primer SEQ ID NO:1 of O1 and SEQ ID NO:2, and for detecting c.dublinensiso2 primer SEQ ID NO:3 and SEQ ID NO:4.

This test kit, also comprises following reagent: 10 mM dNTP 30 μ L; 10 × enzyme spcificity reaction buffer 50 μ L; 5 U/ μ L hot resistant DNA polymerase 5 μ L; Primer 1(10 μM) 10 μ l; Primer 2 (10 μMs) 10 μ l; Positive reference substance 10 μ L; Negative controls 10 μ L; ddH ₂o 5mL.

Further object of the present invention there are provided the application of described Nucleotide in the gene chip for the preparation of detection Cronobacter sakazakii.Described gene chip is microarray chip.Comprise solid phase carrier and be fixed on the oligonucleotide probe on solid phase carrier, wherein said oligonucleotide probe comprises above-mentioned Nucleotide; At least one in the Nucleotide of wherein said Nucleotide as shown in SEQ ID NO:1-12.

Compared with prior art, the present invention's Nucleotide for rapid detection Cronobacter sakazakii disclosed by the invention has the following advantages:

(1) practical

The compound methods such as the PCR kit that application the present invention prepares are easy, and sense cycle is short, speed is fast, workable, is easy to industrialization produce, and testing cost is relatively low.

(2) accuracy is high

The present invention devises the primer special to Cronobacter sakazakii O antigen gene, with the genome of Cronobacter sakazakii, for template is PCR, can to obtain band single, the object band that size is correct, and with the genome of other bacterium for template be PCR time, correct object band can not be obtained.

Accompanying drawing illustrates:

G2539 in accompanying drawing is c.sakazakiio1, G2356 are c.sakazakiio2, G2594 are c.sakazakiio4, G2732 are c.dublinensiso1, G3983 are c. dublinensiso2, G3882 are c.turicensiso2, G3874 are c.turicensiso3, G3886 are c.muytjensiio2, G3864 are c.malonaticuso1;

Fig. 1 represents g2732 wzmthe screening of gene P1 and P2 primer, object band is 336bp, and remaining bacterium is without any band;

Fig. 2 represents g 3983 wzythe screening of gene P3 and P4 primer, object band is 725bp, and remaining bacterium is without any band;

Fig. 3 represents g3882 wzythe screening of gene P5 and P6 primer, object band is 266bp, and remaining bacterium is without any band;

Fig. 4 represents g3874 wzythe screening of gene P7 and P8 primer, object band is 362bp, and remaining bacterium is without any band;

Fig. 5 represents g3886 wzythe screening of gene P9 and P10 primer, object band is 378bp, and remaining bacterium is without any band;

Fig. 6 represents g3864 wzythe screening of gene P11 and P12 primer, object band is 267bp, and remaining bacterium is without any band.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.These embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition as people such as Sambrook, molecular cloning: the condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).

Embodiment 1:

Genomic extraction:

37 DEG C of 2YT liquid nutrient mediums (formula is: peptone 16g/L, yeast powder 10g/L, NaCl5g/L) cultivate Cronobacter sakazakii, 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, the N,O-Diacetylmuramidase then adding 10ul 10mg/ml continues insulation 20 minutes.Add the Proteinase K of 3ul 20mg/ml, 15ul 10%SDS afterwards, 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.Supernatant liquor 2 times of volume ethanol precipitation DNA, roll out DNA with glass yarn and wash DNA with 70% ethanol, being finally resuspended in 30ul TE by DNA.Genomic dna is detected by the agarose gel electrophoresis of 0.4%.

Embodiment 2:

O antigen gene cluster is decoded:

(1) the O antigen gene cluster of the Cronobacter sakazakii that increased by Long-PCR.According in Genbank galFgene design upstream primer, then basis gndgene design downstream primer.

PCR response procedures was as follows: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, anneal 30 seconds for 61 DEG C, 68 DEG C extend 15 minutes, carry out 30 circulations like this, finally, continue extension 8 minutes at 68 DEG C, obtain PCR primer, agarose gel electrophoresis with 0.8% detects the size of PCR primer and specificity thereof, merges 8 pipe 50 μ l long PCR primer, and by the Wizard PCR Preps Purification Kit PCR primer of Promega company.

build O antigen gene cluster library: build O antigen gene cluster library by shot-gun method, reaction system is 600ng PCR purified product, cuts into 1 ~ 3kb fragment, fills sticky end, the carrier of pUC18 connects (adopt fastlink ligase) and connect the gene library obtaining checking order through nucleic acid fragment instrument.

cloning and sequencing in library: select Insert Fragment and at 200 clone's this lab A BI3730 type automatic dna sequencers of more than 1kb, the Insert Fragment in clone is checked order from library, sequence reaches the fraction of coverage of 8 ~ 10 times, thus obtains all sequences of O antigen gene cluster.

the splicing of nucleotide sequence and analysis: with Pregap4 and the Gap4 software splicing of the Staden package software package of Britain Camb MRC Molecular Biology Lab publication with after editing all sequences, gene is found with the orf finder at American National Biotechnology Information center, find open reading frame, with the genetic comparison in the blast groupware and GenBank to find the function of these open reading frames and to determine that what gene they are, gene annotation is completed again with the Artemis software at Britain Sanger center, the precise alignment between DNA and protein sequence is done with Clustral W software, finally obtain the sequence of the gene cluster of Cronobacter sakazakii.

Embodiment 3:

Design of primers and screening

Decode situation according to gene cluster, we find wzy/wzmreally be the special gene of Cronobacter sakazakii, so choose this gene specific section design special primer.Said gene is imported 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.

In Genbank, BLAST is carried out after design of primers, the primer of design can not be too high with the sequence similarity of other nearly edge bacterium, so just can guarantee that this primer only increases in the predetermined position of oneself, and not produce positive reaction with the nearly edge bacterium in the environment of other nearly edge bacterium or collect specimen.This point is very important for the success or failure of the generation and experiment of avoiding non-specific band.The primer designed is as shown in table 1.

Table 1 is for the primer sequence of PCR

The PCR system (25 μ l) that gene identification primer screening is used and reaction conditions as follows:

Ultrapure water 15.7 μ l

Damping fluid (10X) 2.5 μ l

dNTP(10mM) 0.5μl

Primer 1(10 μM) 0.5 μ l

Primer 2 (10 μMs) 0.5 μ l

Archaeal dna polymerase (rTaq) 0.3 μ l (5U/ μ l)

Template DNA 5 μ l

Reaction cycle parameter in PCR instrument in this step comprises the sex change of DNA, renaturation, the temperature and time of extension, cycle index, is specially:

Above-mentioned steps is electrophoresis amplified production in electrophoresis equipment, and the concrete steps of record result are:

get 2 ~ 5 μ l amplified productions to mix with the volume ratio of 5:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;

mixed solution is splined on the sepharose of 1.2%;

by agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 10 minutes, contrast with DL2000 Marker;

observe and record result.

The genomic templates that this link of primer screening is used is extracted employing and is boiled bacterium method.Substantially terminated by the work of primer screening after basic PC R reaction, necessary length adjustment is little on the impact of W-response condition, and the primer sequence used in the present invention is all summed up in Table 1.

Embodiment 4 :

The application of PCR detection kit

the composition of PCR kit

Comprise PCR primer, dNTP, damping fluid, archaeal dna polymerase, positive reference substance and negative controls.The amount that test kit uses is all 20 μ l is a basic consumption.Each test kit can detect 20 samples.

(2) preparation of test experience material requested and equipment

Wherein 10 × buffer, dNTP, Taq polysaccharase provide by by Shanghai Sheng Gong company; Primer mixture is that the sequence of designed, designed is supplied to the synthesis of Beijing Ying Jun biotech company; Positive reference substance, negative controls and ddH ₂o is prepared voluntarily by us.The equipment PCR instrument of experiment, electrophoresis equipment, gel imaging instrument ,-20 DEG C of refrigerators, supercentrifuge, micropipet and 0.2ml PCR thin-walled tubes.

the use specific examples of PCR kit

the process of testing sample.Extract genome if had ready conditions also passable, in order to save time and consumptive material, directly can get 500 μ l bacterium liquid, centrifugal rear use 500 μ l ddH ₂o is resuspended, and 100 DEG C are boiled 10 minutes, then the centrifugal 10min of 12000rpm, and getting middle layer supernatant is template.

the reagent 20 μ l getting PCR kit, in PCR pipe, adds 5 μ l templates.

pCR reaction conditions is as follows:

the agarose gel electrophoresis detection experiment result of 1.2%.

the analysis of result and record.

Conclusion: carry out specific detection with these primer pair Cronobacter sakazakii kind bacterial strain close to other, finds primer specific.Carry out specific detection between bacterial classification in being belonged to by these primer pair Cronobacter sakazakii, the results are shown in accompanying drawing.Bacterial strain used in experiment is in table 2.

The bacterial strain that table 2 specific detection is used

SEQUENCE LISTING

<110> Nankai University

<120> Nucleotide to Cronobacter sakazakii O antigen-specific and application thereof

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Claims

1. a Nucleotide special to Cronobacter sakazakii O antigen gene, is characterized in that described Nucleotide is for the Nucleotide shown in SEQ ID NO:3,4.

2. a PCR kit, comprises

10 mMdNTP 30 μ L; 10 × enzyme spcificity reaction buffer 50 μ L;

5 U/ μ L hot resistant DNA polymerase 5 μ L; 10 μMs of each 10 μ l of PCR primer;

Positive reference substance 10 μ L, negative controls 10 μ L;

ddH ₂O 5mL；

Described PCR primer is respectively as shown in SEQ ID NO:3,4 in claim 1.

3. the application of Nucleotide in the PCR kit for the preparation of detection Cronobacter sakazakii special to Cronobacter sakazakii O antigen gene described in claim 1.

4. application according to claim 3, is characterized in that primer in described PCR kit is respectively as shown in SEQ ID NO:3,4; The method that wherein said PCR kit detects Cronobacter sakazakii is further comprising the steps of:

1) get 2 ~ 5 μ l amplified productions to mix with the volume ratio of 5:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;

2) mixed solution is splined on the sepharose of 1.2%;

3) by agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 10 minutes, contrast with DL2000 Marker;

4) observe and record result;

5) analytical results judging.