CN1234858C - Nucleotide peculiar to 0-antigen of -56 type bacillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O56. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O56 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 13074 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the oligonucleotides of oligosaccharide unit processing genes such as wzx genes or genes with the similar functions of wzx and wzy genes or genes with the similar functions of wzy stemmed from the O-antigen gene clusters of Escherichia coli O56. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O56 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Escherichia coli O56 in human bodies and environment.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O56

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster among the intestinal bacteria O56 (Escherichia coii O56), particularly relate among the intestinal bacteria O56 oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O56 in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Intestinal bacteria O56 is important pathogenic bacterium, it belongs to STEC (Shiga toxin-producingEscherichia coli), promptly produce the intestinal bacteria of Shiga toxin, it is important pathogenic bacteria [Steven P et al (2001) " Virulence Properties and Serotypesof Shiga Toxin-Producing Escherichia coli from Healthy AustralianSlaughter-Age Sheep " the .Journal of Clinical Microbiology that causes livestock disease, Vol.39, No.5,2017-2021]; This external Middle East and North Africa finds that also intestinal bacteria O56 can cause the diarrhoea [Leonard F.Perusk I et al (1999) " Phenotypic Diversity ofEnterotoxigenic Escherichia coli Strains from a Community-Based Studyof Pediatric Diarrhea in Periurban Egypt " JOURNAL OF CLINICALMICROBIOLOGY.2974-2978 Vol.37, No.9] of children below three years old.Therefore the detection to intestinal bacteria O56 is important, needs the method that can detect intestinal bacteria O56 quickly and accurately.

The lipopolysaccharides that is positioned at the intestinal bacteria surface is the morbific inducements of intestinal bacteria, and O-antigen is lipopolysaccharides outermost layer structure, is the target of immune system recognition and the site of phage absorption.The antigenic disappearance of O-can cause the serum sensitivity of many pathogenic agent, perhaps seriously undermines the virulence [Frank etal (1987) " The function of antibody and complement in the lysis ofbacteria " .Rev Infect Dis 177:1750-1753.Pluschke G et al " Role of thecapsule and the O-antigen in resistance of O18:K1 Escherichia coli tocomplement-mediated king " .J Bacteriol 42:907-913] of pathogenic agent.Intestinal bacteria are kinds, and the bacterial strain in planting is generally identified by O-antigen and H-antigen (sometimes by K-antigen).Wherein O-antigen has the height diversity, intestinal bacteria have 166 kinds of different O-antigens, the antigenic variation of O-may be colibacillary origin and keep its multifarious major cause [Reeves, P.R (1992) " Variation in antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516].

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by O-antigen transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In intestinal bacteria, Shigellae and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichiacoli O111 and Salmonella enterica O35 gene clusters:gene clusters encodingthe same colitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises o-antigen transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, it is one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of O one antigenic specificity thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.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, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of S.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome eaused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) " Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111 " .Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet.The antigenic structure of the O-of intestinal bacteria O56 is known, and is as follows.The repeating unit that it is made up of 4 sugar, its very similar [A Gamian et al (1994) " Structure of the Escherichia coli O24 andO56 O-specific sialic-acid-containing polysaccharides and linkage ofthese structures to the core region in lipopolysaccharides " .EuropeanJournal of Biochemistry to the antigenic structure of the O-of intestinal bacteria O24, Vol 225,1211-1220].

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O56.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O56, is the special Nucleotide that comes from o-antigen transhipment enzyme gene, pol gene and glycosyltransferase gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O56.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O56: transhipment enzyme gene is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf5, orf6, orf7 gene; Sugar synthesis path gene comprises wckD, nnaB, nnaC, nnaA.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.

Another purpose of the present invention has provided oligonucleotide, and they come from the gene of coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O56 respectively, comprises the wzx gene or with wzx the gene of identity function is arranged; Come from the gene of coding polysaccharase, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from the gene of encoding glycosyl transferring enzyme, comprise orf5, orf6, orf7 gene.They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O56; The oligonucleotide of the gene of especially listing in the table 1 that comes from coding transhipment enzyme and the gene of polysaccharase, they are high specials to the O-antigen of intestinal bacteria O56, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O56.

The above-mentioned oligonucleotide that another object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect O-antigen and detection and identification of escherichia coli O56 with identification of escherichia coli O56 by these methods.

A further object of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O56.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

The objective of the invention is to realize by following technical scheme.

The present invention is characterized in that to the Nucleotide of the O-antigen-specific of intestinal bacteria O56 it is the isolating Nucleotide shown in SEQID NO:1,13074 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is characterized in that it comprises called after wckD, nnaB, and nnaC, nnaA, orf5, orf6, orf7, wzy, 9 genomic constitutions of wzx are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is characterized in that the gene that has high degree of specificity in the described gene comprises: transhipment enzyme gene comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf5, orf6, orf7 gene.Wherein said transhipment enzyme gene is the Nucleotide of 10552 to 11751 bases among the SEQ IDNO:1; Described pol gene is the Nucleotide of 9488 to 10537 bases among the SEQ ID NO:1; Described orf5 gene is the Nucleotide of 6642 to 7397 bases among the SEQ ID NO:1; The orf6 gene is the Nucleotide of 7390 to 8478 bases among the SEQ ID NO:1; The orf7 gene is the Nucleotide of 8468 to 9475 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is characterized in that it also comprises the oligonucleotide that comes from described wzx gene or wzy gene or glycosyltransferase gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to intestinal bacteria O56, it is characterized in that, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 10645 to 10662 bases among the SEQ ID NO:1 and the Nucleotide of 11320 to 11337 bases, the Nucleotide of 10920 to 10937 bases among the SEQ ID NO:1 and the Nucleotide of 11255 to 11272 bases; The oligonucleotide of the described wzy of coming from gene is to being: the Nucleotide of 9612 to 9629 bases among the SEQ ID NO:1 and the Nucleotide of 9850 to 9867 bases, the Nucleotide of 9847 to 9864 bases among the SEQ ID NO:1 and the Nucleotide of 10408 to 10425 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is in the application that detects other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is providing the O-antigen of expressing intestinal bacteria O56 by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is characterized in that, comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O56 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O56 type bunch: with the genome of intestinal bacteria O56 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;

(4) to the cloning and sequencing in the library: from the library, select the clone of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with laboratory automatic dna sequencer commonly used, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O56 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O56 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O56 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O56, after the bacterial count respectively with 5 * 10 ³, 5 * 10 ², 5 * 10 ¹5 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O56.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O56 is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O56 in the LB of 5mL substratum, centrifugal collecting cell.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 and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene among the pcr amplification intestinal bacteria O56 bunch: with the genome of intestinal bacteria O56 is that template is passed through its O-antigen gene of Long pcr amplification bunch; At first according to the galf gene design upstream primer (5 '-ATT GTG GCT GCA GGG ATC AAAGAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCGCGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the ExpandLong Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of sex change were 10 seconds then, 60 ℃ of annealing 30 seconds, 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product; Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature; Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction; Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water; In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ were reacted 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and this mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged; Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 c mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds-6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O56;

(4) to the cloning and sequencing in the library: from the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage, carry out backward sequencing by the sequence that will interrelate again and survey logically obtaining remaining 20% sequence, thereby obtain all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical ResearchCouncil) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O56, the quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O56 is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base; Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O56 bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 9 openings, 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 structure of the O-antigen gene bunch of intestinal bacteria O56 at last;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O56 bunch; Respectively designed two pairs of primers in each gene, the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity; Is that template is carried out PCR with these primers with the intestinal bacteria of 166 kinds of serotypes and the genome of 43 strain Shigellaes, all primers all obtain positive findings in intestinal bacteria O56, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though obtain PCR product band in the minority group, its size does not meet the expection size, so wzx, wzy gene pairs intestinal bacteria O56 and O-antigen thereof all are high specials.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O56 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 10645 to 10662 bases among the SEQ ID NO:1 and the Nucleotide of 11320 to 11337 bases, the Nucleotide of 10920 to 10937 bases among the SEQ ID NO:1 and the Nucleotide of 11255 to 11272 bases, the Nucleotide of 9612 to 9629 bases among the SEQ ID NO:1 and the Nucleotide of 9850 to 9867 bases, the Nucleotide of 9847 to 9864 bases among the SEQ ID NO:1 and the Nucleotide of 10408 to 10425 bases, carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O56 in the pork filling when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O56, its complete sequence shown in SEQ ID NO:1,13074 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of intestinal bacteria O56 by method of the present invention, as described in Table 3, it it comprise called after wckD, nnaB, nnaC, nnaA, orf5, orf6, orf7, wzy, 9 genome Chengdu of wzx are between galf gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O56, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf5, orf6, orf7 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises wckD, nnaB, nnaC, nnaA gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.The invention particularly relates to o-antigen transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not special, and the o-antigen that the present invention relates to transhipment enzyme gene, pol gene and glycosyltransferase gene are special to the O-antigen of intestinal bacteria O56.The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O56 is provided or the gene of identity function and wzx gene is arranged or with wzx the oligonucleotide of gene of identity function and the oligonucleotide that glycosyltransferase gene comprises orf5, orf6, orf7 gene are arranged with wzy, they are any one section oligonucleotide in these genes.But, be to list in the wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O56 in the table 1 or the gene, wzx gene of identity function arranged or have the oligonucleotide of gene of identity function right preferentially with wzx with wzy by usefulness.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O56 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, so, can determine these primers promptly the listed oligonucleotide of table 1 be high special to intestinal bacteria O56 and their O-antigen.

The separation and the authentication method of the Nucleotide of described O-antigen-specific to intestinal bacteria O56 comprise the steps: 1) genomic extraction; 2) the O-antigen gene among the pcr amplification intestinal bacteria O56 bunch; 3) structure in O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis finally obtain the structure of O-antigen gene bunch; 6) screening of specific gene; 7) detection of primer sensitivity.

Other aspects of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

As described herein, " oligonucleotide " mainly is meant gene, the gene of coding polysaccharase and one section nucleic acid molecule in the encoding glycosyl transferase gene of the coding transhipment enzyme that derives from the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes.Especially come from wzx gene (nucleotide position is 10552 to 11751 bases from SEQ ID NO:1), the oligonucleotide in the wzy gene (nucleotide position is 9488 to 10537 bases from SEQ ID NO:1) all is a high special to intestinal bacteria O56.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function are arranged; Come from pol gene, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from glycosyltransferase gene, comprise orf5, orf6, orf7 gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from transhipment enzyme gene, come from pol gene and come from the combination of the oligonucleotide in the glycosyltransferase gene.

On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) coding transhipment enzyme gene, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged, comprise the wzy gene or the gene of identity function is arranged with wzy with wzx.(iii) the encoding glycosyl transferase gene comprises orf5, orf6, orf7 gene.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O56.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

The present inventor considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant and comes from that coding transhipment enzyme gene comprises the wzx gene or have the gene of gene, the coding polysaccharase of identity function to comprise the wzy gene with wzx or with wzy the oligonucleotide of gene of identity function and the oligonucleotide that the encoding glycosyl transferase gene comprises orf5, orf6, orf7 gene are arranged.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O56.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf5, orf6, orf7 gene.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O56.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

General a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of and be used to detect the many to oligonucleotide of the inventive method, many here is that the gene that comes from coding transhipment enzyme comprises the wzx gene or with wzx the gene of identity function arranged to oligonucleotide; The gene that comes from the coding polysaccharase comprises the wzy gene or with wzy the gene of identity function is arranged; The gene that comes from the encoding glycosyl transferring enzyme comprises orf5, orf6, orf7 gene.This cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf5, orf6, orf7 gene.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O56.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from the wzx gene or the gene of identity function and wzy gene are arranged or have the gene of identity function and glycosyltransferase gene to comprise on the sequence of orf5, orf6, orf7 gene with wzy with wzx that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.

The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.Because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, method of the present invention and molecule also are applied to these other polysaccharide antigen.

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O56 first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O56 by inserting to express, and become useful vaccine.

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 (NewYork:Cold Spring Harbor Laboratory Press, 1989).

Embodiment 1: genomic extraction.

37 ℃ of incubated overnight intestinal bacteria O56 in the LB of 5mL substratum, centrifugal collecting cell.With 500ul50mM 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: by the O-antigen gene among the pcr amplification intestinal bacteria O56 bunch

With the genome of intestinal bacteria O56 is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF gene design upstream primer (5 '-ATTGTG GCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 61 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

Embodiment 3: make up O-antigen gene bunch library.

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ were reacted 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, get in the LB substratum that the 2ml culture is transferred to 200ml, 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant liquor, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant liquor, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O56 with the EcoRI enzyme.

Embodiment 4: to the cloning and sequencing in the library.

From the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is surveyed logical obtaining by backward sequencing and with some sequence again, obtains all sequences of O-antigen gene bunch at last.

Embodiment 5: the splicing of nucleotide sequence and analysis.

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O56 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O56 is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O56 bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 openings, 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 structure of the O-antigen gene bunch of intestinal bacteria O56 at last, as shown in table 3.

By retrieving and relatively, finding that the WckD albumen of orf1 and Escherichia coli has 74% homogeny, 87% similarity in 206 amino acid; WckD albumen is the biosynthetic transferring enzyme of polysaccharide, and the homogeny of height shows that orf1 also is a transferring enzyme, so with orf1 called after WckD.The NnaB albumen of the Prehemataminic acid of Orf2 and Escherichia coli has 79% homogeny in 346 amino acid, 89% similarity, NnaB is a synthetic enzyme in the N-acetylneuram biosynthetic pathway, the homogeny of height shows the orf2 NnaB albumen of also encoding, so orf2 is named as nnaB.The NnaC albumen of orf3 and Escherichia coli has 57% homogeny in 419 amino acid, 74% similarity, NnaC is the CMP-N-acetylneuram synthetic enzyme in the N-acetylneuram biosynthetic pathway, higher homogeny shows the orf3 NnaC that also encodes, therefore with orf3 called after nnaC.The NnaA albumen of Orf4 and Escherichia coli has 56% homogeny in 387 amino acid, 72% similarity, NnaA is the UDP-N-acetylglucosamine-2-mutase in the N-acetylneuram biosynthetic pathway, higher homology shows the orf4 NnaA that also encodes, therefore with orf4 called after nnaA.The glycosyltransferase of Orf5 and Oceanobacillus iheyensis HTE831 has 37% homogeny in 352 amino acid, 56% similarity is sought conservative functional domain in genbank, find that the Evalue of orf5 and PF00535 is 3.4 * e ^-35, therefore infer that orf5 also is a glycosyltransferase, with the temporary called after orf5 of orf5.The glycosyltransferase of Orf6 and Bacteroides thetaiotaomicron VPI-5482 has 40% homogeny in 222 amino acid, 61% similarity, in genbank, seek conservative functional domain, find that the E value of the PF00534 of orf6 and glycosyltransferase family 1 is 5 * e ^-55, therefore infer that orf6 also is a glycosyltransferase, temporarily called after orf6.The glycosyltransferase WbwA of Orf7 and Escherichia coli has 30% homogeny in 324 amino acid, 50% similarity is sought conservative functional domain in genbank, find that the E value of the transferring enzyme PF05855 of orf7 and lipopolysaccharides is 5.8 * e ^-39, therefore infer that orf7 also is a glycosyltransferase, temporarily called after orf7.The O-antigen polysaccharase of Orf8 and Shigella boydii has 24% homogeny, 41% similarity in 341 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf8 has 10 potential transmembrane domains, it has similar secondary structure to many Wzy albumen, a big loop is arranged, feature with typical O-antigen polysaccharase, so determine that orf8 is the wzy gene, called after wzy.Blast comparison shows that the O-antigen transhipment enzyme Wzx of orf9 and Streptococcus thermophilus has 24% homogeny, 46% similarity in 473 amino acid whose sequences.And find that by people's such as Eisenberg algorithm orf9 has 12 potential transmembrane domains, the proteic aminoterminal of Wzx has about 40 amino acid whose conservative motifs, is the wzx gene so can determine orf9, called after wzx.

Embodiment 6: the screening of specific gene

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O56 bunch, the position of these genes in nucleotide sequence sees Table 1.

Transhipment enzyme gene, pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O56 in table 1, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer (5 '-TTC ATC CTA AACTCC TTA TT-3 ') and (5 '-TAA TCG CAG GGG AAA GCA GG-3 '), extract genome then from the intestinal bacteria of 166 kinds of serotypes, method as previously mentioned.With this to primer from the colibacillary genome of 166 kinds of serotypes PCR with identification of escherichia coli and detect its genomic quality.

Table 2 is intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen 166 kinds of serotypes of specific gene, and for the convenience that detects, we are divided into one group, 13 groups altogether with their every 12-19 bacterium.All list in the table in their source.

In the 3rd group, contain the genomic dna of intestinal bacteria O56 as positive control.In the 13rd group is the genomic dna that does not contain intestinal bacteria O56, as negative control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 2 minutes, 95 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get 10ul PCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, and every pair of primer has obtained except be PCR in the 3rd group after the correct band of expection size, and the correct band of any size does not all increase in other groups.So wzx, wzy gene pairs intestinal bacteria O56 and O-antigen thereof all are high specials.

At last, from intestinal bacteria O56, screen gene by PCR: wzx, wzy gene to the O-antigen high special of intestinal bacteria O56.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O56, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O56.These all oligonucleotide all can be used for the intestinal bacteria O56 in the human body and environment rapidly and accurately, and can identify their O-antigen.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O56 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 10645 to 10662 bases among the SEQ IDNO:1 and the Nucleotide of 11320 to 11337 bases, the Nucleotide of 10920 to 10937 bases among the SEQID NO:1 and the Nucleotide of 11255 to 11272 bases, the Nucleotide of 9612 to 9629 bases among the SEQ ID NO:1 and the Nucleotide of 9850 to 9867 bases, the Nucleotide of 9847 to 9864 bases among the SEQ ID NO:1 and the Nucleotide of 10408 to 10425 bases, carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O56 in the pork filling when using aforesaid method.

By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O antigen gene bunch.O antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O antigen-specific gene order of O56 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ ID NO:1) according to the O-antigen-specific to intestinal bacteria O56 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O56, has listed the structure of the O-antigen gene bunch of intestinal bacteria O56 in table, altogether by 9 genomic constitutions, and each gene box indicating, and in square frame, write the title of gene.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O56, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O56, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in bacterium: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O56

<160>1

<170>PatentIn version 3.1

<210>1

<211>13074

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ttggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaactgcg cgtgaagcgt 120

caactgctgg cggaagtaca gtccatctgt ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttatgtg cacgacctgc cattggtgac 240

aatccgtttg tcgtggtact gccggacgtt gtgatcgacg acgccagcgc cgatccgctg 300

cgctacaacc ttgcagccat gattgcgcgc ttcaacgaaa cgggccgcag tcaggtgctg 360

gcaaaacgta tgccgggcga tctctctgaa tactccgtta ttcagaccaa agaaccgctg 420

gatcgtgaag gtaaagtcag tcgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagatatcat ggcagtaggt cgttatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcagcc tggtgcatgg gggcgtattc agctgactga tgccatcgct 600

gaactggcga aaaaacagtc cgttgatgcc attctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaggcgttt ttgaagtatg ggctacgcaa ccacaaagaa 720

ggggcgaagt tccgtaaata tattgaagag ttattgagta aaagttattg actggataat 780

tgaaaatatt aaataattgt ttaactttta taatctacta attatgaaaa ctggttaaag 840

tatttaggga tctagtaact attaagctag gggcggtagc gttgtctacg gttcgtatta 900

tttacatatt taaagcactc cagagagatg ttaactcctc ctgtaagaca atatctgaag 960

aaagtcatgt ctaatatatg aattcactca gatgagtaca aagatgagaa acagatacga 1020

gaagggaaag aacatcagag ataccttttt atactaaata aatcacattg aatattcaat 1080

aattgagata ttccttatta ggtaatgatt gttttttaat gagctgtata tgacacaaat 1140

attatttgta taaataaaga agagtaaaaa gaaaattaaa aattgataaa atatatttct 1200

attatccccg gttacagacc gtcttataaa gtagaacatc tacttttgac tatttgcgct 1260

attattcatg gtgcagatgg ttggggtata taaatgtttt ttgggcaacg cttctagatt 1320

tttgaagtaa tatggtgatc ttaaaagtgg tattcctgtt cataatacca ttgtcagagt 1380

ggtatcatgc ttcattcctg caaataatta ctggtgcttt attaactaag taagtgacta 1440

ttattcttca aatgataaag atgtcgttgc aattgatgga gaatgtactg gctcttttat 1500

gataagagtc gcagcaaggg tacgatttat gttattaatg tgttttaaca atgcacaatc 1560

tggtcctagg ataacggatg agtaatccaa tcaaagctat tcctaatatt cttaacatat 1620

tagatattag aggataatta tcatgattgg cgcggcggtg ggcgagaaat atcttgtggt 1680

gaagatacaa aagcaaggaa gtgattattt attcgctgta ataatcagag gctgcttaat 1740

aaagtatttg accactgaaa gaattgaata attcaaagca atgactgtta cgtaatcagt 1800

gaaaggcgtc atgacaaaaa agaaaaccgt cttcatatta tttgcgatac ccctgataaa 1860

attatttatt tcactgttga atgaaatgaa tgatgcgtga cagctatctt tagctaaata 1920

atagtagagc aaatgaaaga actagaaatg atggtcagat attatatcag ttctgatgat 1980

aaaacagcgt agaggttcgc tacaattatc cgaaatcact ggcgagtgga gaataagtgt 2040

actggaagca gaaagtggtg atgaatgaat acgactgcga aattacctcg cgtgagagct 2100

tgcggggggg aggctaaatt tggtcctgtg tgcatcttgt attgagataa acatcaaaaa 2160

tttagatttc aatataataa tgatgtacct gtaagtcaca gcataatcaa accttgtgat 2220

cgatttttca tcttagaagg aatttatgga aaattttttt gtatgtgtgt taccaattgt 2280

tggtacataa gcgtagacta agtaattatt agattagata taaaacgcta agaaaacata 2340

gaacaaacct atgaacaaaa atggagttgc tgcgtttaat ataaatatgg aatataaccc 2400

tcgcctcttt ctaggaggta ttctgataaa caaccattct aggaaatgaa ctttattatt 2460

tttggttgat ttttggactc tggataataa gacatcgaat ggcatatata tgaagaaaag 2520

attaatcatc attggagctg gcggttttgc aaaagcagtc attgatagct tagaccatga 2580

acagtatgaa gtttatggtt tcatcgacag ctttaagagt ggggaacatc aaggctataa 2640

aatactttca aaaacaatag atggtttaaa tgaacctaat caatactgtt attttattgc 2700

gattggcgaa ccctgttgta gggctatatg gctagaaact attaataata tgaggcttga 2760

aacgatcaat gtcattgata aaacagcaat agtctcaaaa cgagcaattc ttgggacatg 2820

tatctatgtc ggtaagatgg caatcgttaa ttgtgattca catcttgagg atggcgttgt 2880

tattaataca agagctcttg ttgagcatgg taattatata tcatattgct ccaatatatc 2940

caccaacgtc gtattgaatg gagatgtttt tgttggtaga aaatgtttta taggtagttg 3000

tactgtagtt aatggtcaac tgaaaatagg cgattcatct atagttggct ctggctcagt 3060

tgttattaga aatatagata aaaatattgt cgtcgctggt acaccaacaa gacttattag 3120

gaaaaggtaa taaaatgtca tctgtatata tagtcgcgga aattggatgt aatcataatg 3180

gagactttaa tttagctaaa aggatgatag aggaggctta taatgctggg gtcgatgctg 3240

ttaaatttca gacatttaaa gccgatagtt taatatcaaa atatgctcct aaagcagatt 3300

atcagattaa aacgacaggt aatagtgagt cacagttgga aatgactcgg aaactagagc 3360

tttcctataa agaatatatc aagttagaag aatattgtaa aagtttaggg ttggatgttt 3420

tttcaacccc ctttgatttt gagtcaatag aatttttata tacgagaaaa caaaccacat 3480

ggaagatccc atctggtgag atactgaacc taccatattt ggagaaaatt gcattgttgc 3540

ctattaataa gaaaattatt ttatctacag gcatggcaac tattgatgaa ataaagcagg 3600

ctctgaaagt atttactgac aatgggatta attctgaaga tatcacgata ttacattgta 3660

acactgagta tcctacgcct tatgaagacg ttaatctcaa tgctctcgcg ggtttaaaaa 3720

aaacattcca aggatataac attggttttt cggatcattc tccagggttc tttgctggta 3780

tagcagctgt tccttatgga ataactttca ttgaaaagca ttttacatta aataaaaact 3840

tcgaaggacc agatcataaa gcatctgtta ctcctgatga attaagattg ttatgtgaag 3900

gtattagagc gattgaaaaa tcattgggaa gttttgataa gttagttact gattcggaga 3960

gaaaaaataa aatagtggcg aggaagtcca ttgtagccaa acgtgatatc aaaaaagggg 4020

aagtttttac tgtcgataat attactacta aacgcccagg aaacggaatc agtcctatgt 4080

attggtatga ggttttgggt aaaaaagctg agcaagattt cttagaagat cagctaataa 4140

aaatgtctgg tgttgacgaa caagagattt aaaaatgaga aaaaataaaa ttgcaattat 4200

tcctgctcgt tcaggatcaa aagggctact caataaaaat atattaatgc tttgtgataa 4260

acccttaatt gcttatacaa tagaagctgc tattgaaagt aaagtatttg acaaagttat 4320

tgtttcgacg gattctactg aatataagga tattgcttta cgttatgggg ctgaagttat 4380

tatgagggat aaagagctgg cctctgataa agctacatca tttgtggttg tgaaagatgt 4440

gttagaaaaa aataatggct atgattattt tgctttatta cagccaacat caccatttag 4500

aaactataaa catattaggg aagctattaa tctttttgaa aatacagtag aaattgattt 4560

tttggtctca atggtcaaaa cgaataaaag tgcagagttg attaatcaat tgacacccaa 4620

caatactttg aaatatttta atgcggactt ttctaattat agaaggcaaa accaagataa 4680

ttactgtcca aatggtgcga tatttatagg caatgtggac gcatatatta aaaaaaagag 4740

tttctttggt gaaagaagta ttgcctatat aatggataga atcgattcaa tagatattga 4800

tgacaaacta gatttcgagt tcgcaataac aataatgcat aaaaaaaata gatctagaga 4860

acttcaaaag aaaatactaa ataggatatg cgaaaaaaaa aattacttta accaaaaaaa 4920

agatataacg ttaatggggc attcaatttt cgactattgg gatataactc agttaaaaaa 4980

acgtgatgtg aataatttag gaatcgctgg tattaatagt aaacaatatt atgattatat 5040

tataaaaaaa aatatagcaa aggttggtca agatgtcata ttaatgcttg gtacgaatga 5100

tattgttact aatgattgga atttagactt cacaactaat tggattgaaa aaataattaa 5160

tcgcttaaaa agaataaacc caaatgttaa tatttatatt ttatccgtac cccctgtagt 5220

tggacgaatt gataggaata acggagtaat aaaaaaattg aattgcaatc tgttaaaata 5280

ttttgataat tgtcaaaatg tgtttttttt acccctgtca gaagcttttt atgataaata 5340

tggaaatcta aaggaagagt tcacatatga tggattgcat tttactcagc tagcttataa 5400

tcaactgaag aaagagatcg aggaaaaatt aaaatgaaaa aattattata tataactggt 5460

tcaagagcgg agtatgggat catgaaacgg ttgttaagaa aacttcagga tcatgagcgt 5520

attgaacttt ctattattgc aacaggaatg cattgtgatg ttaagtatgg ttatacatat 5580

caaaatatta ttgacgatgg atttgaaata aaagaattat ttgatataaa aattgaaaca 5640

tctacaaata gtaatattat ttctacgatg agtcgtgcac aaagtttgtt tggtgctcat 5700

tttcaggaca acaaatatga tgcaataatt attcttggtg atagatatga aatgctttct 5760

gtagctatag ctgcatctat acatggaatt ccaattatcc atttacatgg aggagaacaa 5820

acattaggga attatgatga attcattcgt cactgtatta caaaaatgag tcacctacat 5880

ttagttgcaa cagataaata tagagaaaga gtaatacaac taggagaacc acctgcttct 5940

gttgtaaata tcggagcgat gggggctgag aatgtaatat atttacctat accagaacat 6000

aacgaactac agtcaaaata tagtaatgtt ttaagtaatt attttatggt tctttttcat 6060

cccgagacat taactagaaa atctgtgcat gaccaaataa atgaactaat taaagcattg 6120

gaaattgcgc ataaaaaaaa tcattgtaac tttatcttca taggatctaa ctcggacacg 6180

agttcagaaa tcattactga gcaaatttta accttctgta agtcatacaa tttcccatat 6240

ttagtatcgt taccgactca agattattta ggattatgta aatattcttt agggcttatt 6300

ggtaattcat cttctggcct aatagaaatt ccatcactcg gagtccctac tattaatatt 6360

ggacatcgac aggctgggcg agaacgtgga aaatcagtga ttgatgttga atgctgccat 6420

gttgatatac ttaataagat acatgatatt ttatcgaatc gaaatatcag tgctgatgat 6480

aatgttaatc cttattataa ggagaacagt gctgacttag ctatgtctag tataattagt 6540

tttctagaca ggattgggga caataaaagt catatcaaaa agttttatga tattcctaat 6600

attgataatc tgattaactc ataataagta aacatggtta tatggaacta gttagcatca 6660

ttatagcagc ttataactgt aaagatacca tctatgctac agttgaatca gcgctatctc 6720

agacatacaa aaacattgaa ataataattt gcgatgactc atctacagat gatacatggg 6780

atataattaa taaaattaaa gactcacgaa ttatttgcat aaaaaacaat tattgtaagg 6840

gggcggcggg agcaagaaat tgtgcattaa aaatagccaa aggcagatac atagcatttc 6900

tggatagtga tgattactgg gttacaacta agatatcaaa tcagatccat ttcatggaaa 6960

cagaaaaggt tttcttttca tacagcaatt attatattga aaaagatttt gttattacgg 7020

gcgtctttag ttctccgcct gaaatcaatt atggtgccat gcttaaatat tgtaatattg 7080

catgttcaac agtcatcctc gataggacgg gggtaaaaaa tatatctttc ccttacatag 7140

ataaagaaga ttatgcatta tggcttaaca tcctttcaaa aggaataaag gctaggaaca 7200

ctaatttagt agatacatat tatagagttc atgctggttc agtttcggca aataagttta 7260

aagagttaat acgacaaagt aatgttttaa agtctatcgg gatcaaagca catcacagaa 7320

taatatgttt attttattat gctataaatg gtttgattaa acactgtttt agttatagag 7380

ataaaagaaa tgcttagagt ttgttttatt attgcagatg ttacatggac aggcggaata 7440

gaacgagtgc tatctaggtt agcgttatcg attccagatg ataatataga cttagaaata 7500

ttaagcttat ataggactta taaagataca tattatacat ttccagataa tgttaaaatc 7560

aaatatctta atgataacta tcaatataaa ggtcgaccag gatcgttcac tcggttagtt 7620

cagcatctaa gaacatctat taaactaagg gaatttctaa attcgtgtaa ctatgatatt 7680

attgtggttc atacatttcc gcttgcattc atcaattttt ttactgaaaa aagatcaaag 7740

tgggtcgttg ttgaacatgt aaaatattat tattataata ttttattaag acgtatacgc 7800

tcatttatat atgataaata tgatgctatt gtgttattaa cagataaaga tgttacccat 7860

ttcaaaaatg cgaataataa tgtatatgta atacctaatc ctttaagttt ttcgcaaact 7920

aaaaaagcga atctgaatag cgttagaata atatctgttg gaagattaga aaaacagaag 7980

ggattcgata tgctcttaaa ggcattctca tatatatcat ataaatatcc agaatggcaa 8040

ttggatattt ttgggaaagg taatgaagaa gaaaatctga ggaaacttat atctaaatta 8100

aatttaaaaa atgtgaatct tatgggaacg tcaaaaaata ttcatcaaga atatcttagt 8160

tctgcatttt atgtaatgtc ttcacggtat gaaggttttc ctatggtact cttggaggcg 8220

atggctagcg gtttaccatg tatatcattt aattgtgaga ctggccctgc agatattatt 8280

attgacaatg aaaatggttt tcttatagaa cactttgatg ttgttcaatt atcaaaagca 8340

atggagaaac tgatcaaaga tgaatatttg agaacaaaaa tgggtaatgc cgcatttgag 8400

cgagcagatg aattttcatt agaaaaaatc acattgaaat ggaaggaatt atttaaaggg 8460

cttactcatg aaaagtaatg tttatatttg taaaaccaca tgggaaattt ttgtttctat 8520

tctattggct agcgatgttt actatagtaa aaatataaaa tcagtttttg ttattgaagt 8580

tgctgaggaa aatattaatt atattcctag gctgtctgat ttttgcttta tattaaaggt 8640

tgtttctgtt aaattttctt ctaataaata cattaaagct ctgtgttttt tatatagagt 8700

taaatatttt ttacctaaag tacttggaga gtatctaaaa aatgatgtaa gggttaatct 8760

gtttatagat caaggtgtat ttggacagtt tttcctgaaa aatcacaaag acgtttatct 8820

atatgagcat gggaatggaa attattgtgt gggaccatat cctaattata aactaataaa 8880

aaagatttta ggtataactg aaggttatgg tagacacagt agagtgaaga gggtatacct 8940

acagcatccg gaaaaagctc ctcaagatat tcgtaataag gtaattggtt tttctattaa 9000

acatttatat tgtaaattat gctcgagaga gcaacaagcc ttattgagtt tttttggagt 9060

gaaagatatt aataaatcaa atgatattat tattttaaca cagccaatca gtgaggatgg 9120

tttttacagt gaaaaagaga agatagaaat ttacaaggag attattaata gccgcttttc 9180

ccatgataaa tctagagttg ttataaagcc gcatccccgc gataaaacag attatagtaa 9240

atacatacca gatgtaatgc tattgccaag aaattttcct atggaaatat taaattttac 9300

agacgtgaga tttgttaaag ctataacaat ttgttccagt gctgtatata attttggata 9360

tgatatttca gttgattata taggtacctt aaaatatcca aaattaatta aagcgttggg 9420

gcgattacct gcacaatatg tacataaaga taactataaa aaaatcaagt attaattgga 9480

gtctgatgtg ataccttata ttatatttta ttctttatca ttaactttgg ttgtattatc 9540

tttggtgtta aagaaatatg attggttttg gttgcttttt cttttgttta tttcaattat 9600

ttttattggc cttagggtga atgtaggcgc tgattatact gaatatgtca gaatttacaa 9660

tcaatcttac gatactaatt ttgaattagg gtttgatata atatttaatc tctttaagag 9720

gttcggttat gattatgtat ttgtatcttt gtttgtgttt atattgacat catttttttt 9780

tctatattca attaagtcat tacaacacaa aactttaatt tatttctgtt ttttgctttt 9840

tatgtttgtc cctctaacat caactattag acaagggtta gctattcctt tttttataat 9900

ttgcctttta aacgctgata gacctaaagt ctactttatg agtatcgcta tagggtgttt 9960

tttccattat tcgattttat ttatgctatt tttctttttt gttagatact ttaaacaatc 10020

gtattgcaga gcttatttta taatcctgtt ttttttaggt ttaagtgttt ttaatatcgt 10080

tgagtatatg attatactat tacattatat tcctagtctg ggaacgttaa gctctaaact 10140

ccttatgtat acacagcgat ataatgttac gctttcatta acttcagttg cttataaatt 10200

agctggttta ctattggtta cgagttttat gtcttttatc aaaataaata aaaagttact 10260

gtgtagttat aacttatatt atatatcatt gtgtttgttt atcttattca aagataattc 10320

tgtttttaca aatcgtttaa tgtatgccac taatatatcc ttggtgactt ttttttcatt 10380

tttgttatcg atgatgaatg cgttttatag gaaagccatt gttacttttt tgtttgtaat 10440

ctatttttcc ataaattatt ttaaatttac agcaacagat ttaagacact ctattgagtc 10500

tgcttatatg ccttataaat cggtacttat aaattaagta aaatataaat catgaaatta 10560

atgaaagata gattttttta tttttttatc gaactaataa ataaaggcgt cccgtttttc 10620

ttattacctt atgtttcttc tgctgtcggt gttgaagtct atggtaaaat cgagcttttt 10680

actactaatt atataatatt atctttcctt tttgccttgg ggtttgaagg ttggatgaat 10740

tctcattatt ttaaaatatc tgaaaataat tttattacta tattgtcttc attttatttt 10800

ttgagttttc ttattttttt tattagtgtg ataacaacgt tgctatattc atctgactgg 10860

actgctcttg ttattgttgg atatattcag tcgcttctta atattacaac tttgcattta 10920

agactcaagg gaaaatatat aagatcaggc gcattgttat tagcagcgtc aattgttaat 10980

gcagtgatgg tatatgtgtg ctttgagtta tttggtaaag attataatgc gcgcattgaa 11040

gcttttttat tttcttctgt gttattggta atatttatta ttctctgtaa cattaatata 11100

tggggggggg gggtaaaaat aaaaattaat tttagctttt taaataaaga cattttgtta 11160

ttttgtttac ccctgtgttt aacaatggtt attaactggg ctaagggtaa tatagataag 11220

tattttattg ctaatctact tgatttgaag tctcttggta tctatgctgt tgcatatcag 11280

ttagcttcag ttattaatgt tgttggcatt attttgaata ggacattaca ggctgatttg 11340

cttaaaagca tggctgatgg tttttatcaa accaaaaaga taatactcat ttttttatcc 11400

gttttggcta tattctttct cctatatgtt atagctgtgt gttatggttt taattatgtt 11460

ttttctgccg attatgcccc ctctcgtaat atggcgttac tgttaatatt ttgtttttta 11520

ttcttttcca cctcatcgtt tttaattaat tttgtattgt tttacggtag acctaaattg 11580

attttgcttc aaagtttatt aatcacaacc atacatgttg cactatcgtt tcttttgatt 11640

cagaaatacg ctctgtttgg ggctttattt acacaggtta ttacttcgat attaacattt 11700

atttccactt tattattatg ctatatatta atgaaagcat ctcacaaata acacattctt 11760

acatgttgta agatatttat ttaatattat cttaaattat gaactcattt ctcaggagta 11820

aacaatgtca aagcaacaga tcggcgtcgt cggtatggct gtgatggggc gcaaccttgc 11880

gctcaatatc gaaagccgtg gttataccgt ctctattttc aaccgttccc gtgagaagac 11940

ggaagaagtg attgccgaaa atccaggcaa gaagctggtt ccttactata cggtgaaaga 12000

gtttgttgaa tccctggaaa cgcctcgtcg gatcctgtta atggtgaaag caggtgcagg 12060

cacggatgct gctattgatt ctctcaagcc atacctcgat aaaggtgaca tcatcattga 12120

tggcggaaat accttcttcc tggacaccat tcgtcgtaat cgtgagcttt ctgcagaagg 12180

ctttaacttt attggtaccg gtgtctccgg tggtgaagaa ggcgcgctga aaggtccttc 12240

cattatgcct ggtgggcaga aagaagccta tgaactggtt gctccgatcc tgaccaaaat 12300

cgccgcagtg gctgaagacg gcgagccatg cgttacctat attggtgccg atggtgcagg 12360

tcactatgtg aagatggttc acaacggtat tgaatacggc gatatgcagc tgattgctga 12420

agcctattct ctgcttaaag gtggcctgaa tctttccaac gaagaactgg cacagacctt 12480

taccgaatgg aataacggtg aactgagcag ctacctgatc gacatcacca aagatatctt 12540

caccaaaaaa gatgaagacg gtaactacct ggttgatgtg attctggatg aagcagcaaa 12600

caaaggtacg ggcaaatgga ccagccagag tgcgctggat ctcggcgaac cgctgtcgct 12660

gattaccgag tctgtgtttg cacgttatat ctcttctctg aaagatcagc gcgttgccgc 12720

gtctaaagtt ctcactggcc cgaaagcgca gccagcaggc gataaggctg agttcatcga 12780

aaaagttcgt cgtgcgctgt atctgggcaa aatcgtttct tacgcccagg gcttctctca 12840

gctacgcgca gcgtctgaag agtacaactg ggatctgaac tatggcgaaa tcgcgaagat 12900

ttttcgtgct ggctgcatca tccgagcgca gttcctgcag aaaatcaccg atgcctatgc 12960

cgaaaatcca cagatcgcta acctgctgct ggctccgtac ttcaagcaaa ttgccgatga 13020

ctaccagcag gcgctgcgtg atgtcgttgc ttatgcggta cagaacggta tccc 13074

Wzx gene, wzy gene and wherein primer and PCR data in the O antigen gene of table 1 intestinal bacteria O56 bunch

Gene	Function	The base position of gene	The forward primer position	The reverse primer position	PCR product length	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzy wzx	O-antigen polysaccharase O-antigen transhipment enzyme	9488-10537 10552-11751	9612-9629 9847-9864 10645-10662 10920-10937	9850-9867 10408-10425 11320-11337 11255-11272	394bp 579bp 693bp 353bp	0 0 0 0	58 60 61 62

The intestinal bacteria of 166 kinds of serotypes of table 2 and 43 strain Shigellaes and their source

The bacterium source that contains in this group of group number

1, wild-type e. coli O1, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, IMVSa

O19ab，O20，O21，O22，O23，O24

2, wild-type e. coli O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, IMVSa

O36，O37，O38，O40，O41，O42，O43

3, wild-type e. coli O6, O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, IMVSa

O57，O58，O60，O61，O62，O53

4, wild-type e. coli O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, IMVSa

O79，O80，O81，O82，O83，O68

5, wild-type e. coli O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, IMVSa

O101，O102，O103，O104，O105，O106，O97，

6, wild-type e. coli O107, O108, O109, O110, O111, O112ab, O112ac, O113, IMVSa

O115，O116，O118，O120，O123，O125，O126，O128，O117

7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O56, O137, IMVSa

O138，O139，O141，O142，O143，O144，O145，O140

8, wild-type e. coli O146, O147, O148, O150, O152, O154, O156, O157, O158, IMVSa

O159，O160，O161，O163，O164，O165，O166，O153 b

9, wild-type e. coli O168, O169, O170, O171, O172, O173, c

Shigella dysenteriae D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 d

10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B12, B12, B13, B14, B15, d

B16，B17，B18

11, shigella flexneri F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, d

DS，DR

12, wild-type e. coli O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVSa

O124，O167，O162，O121，O127，O149，O119

13, wild-type e. coli is removed the 3rd group of bacterium of intestinal bacteria O56

For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group as negative control

a.Institude of Medical and Veterinary Science，Anelaide，Australia

b.Statens Serum Institut，Copenhagen，Denmark

C.O172 and O173 come from Statens Serum Institut, Copenhagen, and Denmark, all the other come from IMVS

D. China Preventive Medicial Science Institute's epidemiological study institute

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O56

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O56

ATTGTGGCTG CAGGGATCAA AGAAATCCTC TTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAACTGCG CGTGAAGCGT 120

CAACTGCTGG CGGAAGTACA GTCCATCTGT CCGCCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT AGGCCACTCC ATTTTATGTG CACGACCTGC CATTGGTGAC 240

AATCCGTTTG TCGTGGTACT GCCGGACGTT GTGATCGACG ACGCCAGCGC CGATCCGCTG 300

CGCTACAACC TTGCAGCCAT GATTGCGCGC TTCAACGAAA CGGGCCGCAG TCAGGTGCTG 360

GCAAAACGTA TGCCGGGCGA TCTCTCTGAA TACTCCGTTA TTCAGACCAA AGAACCGCTG 420

GATCGTGAAG GTAAAGTCAG TCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGCTGGACT CAGATATCAT GGCAGTAGGT CGTTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACTCAGCC TGGTGCATGG GGGCGTATTC AGCTGACTGA TGCCATCGCT 600

GAACTGGCGA AAAAACAGTC CGTTGATGCC ATTCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGCTATAT GCAGGCGTTT TTGAAGTATG GGCTACGCAA CCACAAAGAA 720

GGGGCGAAGT TCCGTAAATA TATTGAAGAG TTATTGAGTA AAAGTTATTG ACTGGATAAT 780

TGAAAATATT AAATAATTGT TTAACTTTTA TAATCTACTA ATTATGAAAA CTGGTTAAAG 840

TATTTAGGGA TCTAGTAACT ATTAAGCTAG GGGCGGTAGC GTTGTCTACG GTTCGTATTA 900

TTTACATATT TAAAGCACTC CAGAGAGATG TTAACTCCTC CTGTAAGACA ATATCTGAAG 960

AAAGTCATGT CTAATATATG AATTCACTCA GATGAGTACA AAGATGAGAA ACAGATACGA 1020

GAAGGGAAAG AACATCAGAG ATACCTTTTT ATACTAAATA AATCACATTG AATATTCAAT 1080

AATTGAGATA TTCCTTATTA GGTAATGATT GTTTTTTAAT GAGCTGTATA TGACACAAAT 1140

ATTATTTGTA TAAATAAAGA AGAGTAAAAA GAAAATTAAA AATTGATAAA ATATATTTCT 1200

ATTATCCCCG GTTACAGACC GTCTTATAAA GTAGAACATC TACTTTTGAC TATTTGCGCT 1260

ATTATTCATG GTGCAGATGG TTGGGGTATA TAAATGTTTT TTGGGCAACG CTTCTAGATT 1320

TTTGAAGTAA TATGGTGATC TTAAAAGTGG TATTCCTGTT CATAATACCA TTGTCAGAGT 1380

GGTATCATGC TTCATTCCTG CAAATAATTA CTGGTGCTTT ATTAACTAAG TAAGTGACTA 1440

TTATTCTTCA AATGATAAAG ATGTCGTTGC AATTGATGGA GAATGTACTG GCTCTTTTAT 1500

GATAAGAGTC GCAGCAAGGG TACGATTTAT GTTATTAATG TGTTTTAACA ATGCACAATC 1560

TGGTCCTAGG ATAACGGATG AGTAATCCAA TCAAAGCTAT TCCTAATATT CTTAACATAT 1620

TAGATATTAG AGGATAATTA TCATGATTGG CGCGGCGGTG GGCGAGAAAT ATCTTGTGGT 1680

GAAGATACAA AAGCAAGGAA GTGATTATTT ATTCGCTGTA ATAATCAGAG GCTGCTTAAT 1740

AAAGTATTTG ACCACTGAAA GAATTGAATA ATTCAAAGCA ATGACTGTTA CGTAATCAGT 1800

GAAAGGCGTC ATGACAAAAA AGAAAACCGT CTTCATATTA TTTGCGATAC CCCTGATAAA 1860

ATTATTTATT TCACTGTTGA ATGAAATGAA TGATGCGTGA CAGCTATCTT TAGCTAAATA 1920

ATAGTAGAGC AAATGAAAGA ACTAGAAATG ATGGTCAGAT ATTATATCAG TTCTGATGAT 1980

AAAACAGCGT AGAGGTTCGC TACAATTATC CGAAATCACT GGCGAGTGGA GAATAAGTGT 2040

ACTGGAAGCA GAAAGTGGTG ATGAATGAAT ACGACTGCGA AATTACCTCG CGTGAGAGCT 2100

TGCGGGGGGG AGGCTAAATT TGGTCCTGTG TGCATCTTGT ATTGAGATAA ACATCAAAAA 2160

TTTAGATTTC AATATAATAA TGATGTACCT GTAAGTCACA GCATAATCAA ACCTTGTGAT 2220

CGATTTTTCA TCTTAGAAGG AATTTATGGA AAATTTTTTT GTATGTGTGT TACCAATTGT 2280

TGGTACATAA GCGTAGACTA AGTAATTATT AGATTAGATA TAAAACGCTA AGAAAACATA 2340

GAACAAACCT ATGAACAAAA ATGGAGTTGC TGCGTTTAAT ATAAATATGG AATATAACCC 2400

TCGCCTCTTT CTAGGAGGTA TTCTGATAAA CAACCATTCT AGGAAATGAA CTTTATTATT 2460

Orf1's is initial

TTTGGTTGAT TTTTGGACTC TGGATAATAA GACATCGAAT GGCATATAT A TGAAGAAAAG 2520

ATTAATCATC ATTGGAGCTG GCGGTTTTGC AAAAGCAGTC ATTGATAGCT TAGACCATGA 2580

ACAGTATGAA GTTTATGGTT TCATCGACAG CTTTAAGAGT GGGGAACATC AAGGCTATAA 2640

AATACTTTCA AAAACAATAG ATGGTTTAAA TGAACCTAAT CAATACTGTT ATTTTATTGC 2700

GATTGGCGAA CCCTGTTGTA GGGCTATATG GCTAGAAACT ATTAATAATA TGAGGCTTGA 2760

AACGATCAAT GTCATTGATA AAACAGCAAT AGTCTCAAAA CGAGCAATTC TTGGGACATG 2820

TATCTATGTC GGTAAGATGG CAATCGTTAA TTGTGATTCA CATCTTGAGG ATGGCGTTGT 2880

TATTAATACA AGAGCTCTTG TTGAGCATGG TAATTATATA TCATATTGCT CCAATATATC 2940

CACCAACGTC GTATTGAATG GAGATGTTTT TGTTGGTAGA AAATGTTTTA TAGGTAGTTG 3000

TACTGTAGTT AATGGTCAAC TGAAAATAGG CGATTCATCT ATAGTTGGCT CTGGCTCAGT 3060

TGTTATTAGA AATATAGATA AAAATATTGT CGTCGCTGGT ACACCAACAA GACTTATTAG 3120

The termination orf2's of orf1 is initial

GAAAAGG TAA TAAA ATGTCA TCTGTATATA TAGTCGCGGA AATTGGATGT AATCATAATG 3180

GAGACTTTAA TTTAGCTAAA AGGATGATAG AGGAGGCTTA TAATGCTGGG GTCGATGCTG 3240

TTAAATTTCA GACATTTAAA GCCGATAGTT TAATATCAAA ATATGCTCCT AAAGCAGATT 3300

ATCAGATTAA AACGACAGGT AATAGTGAGT CACAGTTGGA AATGACTCGG AAACTAGAGC 3360

TTTCCTATAA AGAATATATC AAGTTAGAAG AATATTGTAA AAGTTTAGGG TTGGATGTTT 3420

TTTCAACCCC CTTTGATTTT GAGTCAATAG AATTTTTATA TACGAGAAAA CAAACCACAT 3480

GGAAGATCCC ATCTGGTGAG ATACTGAACC TACCATATTT GGAGAAAATT GCATTGTTGC 3540

CTATTAATAA GAAAATTATT TTATCTACAG GCATGGCAAC TATTGATGAA ATAAAGCAGG 3600

CTCTGAAAGT ATTTACTGAC AATGGGATTA ATTCTGAAGA TATCACGATA TTACATTGTA 3660

ACACTGAGTA TCCTACGCCT TATGAAGACG TTAATCTCAA TGCTCTCGCG GGTTTAAAAA 3720

AAACATTCCA AGGATATAAC ATTGGTTTTT CGGATCATTC TCCAGGGTTC TTTGCTGGTA 3780

TAGCAGCTGT TCCTTATGGA ATAACTTTCA TTGAAAAGCA TTTTACATTA AATAAAAACT 3840

TCGAAGGACC AGATCATAAA GCATCTGTTA CTCCTGATGA ATTAAGATTG TTATGTGAAG 3900

GTATTAGAGC GATTGAAAAA TCATTGGGAA GTTTTGATAA GTTAGTTACT GATTCGGAGA 3960

GAAAAAATAA AATAGTGGCG AGGAAGTCCA TTGTAGCCAA ACGTGATATC AAAAAAGGGG 4020

AAGTTTTTAC TGTCGATAAT ATTACTACTA AACGCCCAGG AAACGGAATC AGTCCTATGT 4080

ATTGGTATGA GGTTTTGGGT AAAAAAGCTG AGCAAGATTT CTTAGAAGAT CAGCTAATAA 4140

The termination orf3's of Orf2 is initial

AAATGTCTGG TGTTGACGAA CAAGAGATT T AAAA ATGAGA AAAAATAAAA TTGCAATTAT 4200

TCCTGCTCGT TCAGGATCAA AAGGGCTACT CAATAAAAAT ATATTAATGC TTTGTGATAA 4260

ACCCTTAATT GCTTATACAA TAGAAGCTGC TATTGAAAGT AAAGTATTTG ACAAAGTTAT 4320

TGTTTCGACG GATTCTACTG AATATAAGGA TATTGCTTTA CGTTATGGGG CTGAAGTTAT 4380

TATGAGGGAT AAAGAGCTGG CCTCTGATAA AGCTACATCA TTTGTGGTTG TGAAAGATGT 4440

GTTAGAAAAA AATAATGGCT ATGATTATTT TGCTTTATTA CAGCCAACAT CACCATTTAG 4500

AAACTATAAA CATATTAGGG AAGCTATTAA TCTTTTTGAA AATACAGTAG AAATTGATTT 4560

TTTGGTCTCA ATGGTCAAAA CGAATAAAAG TGCAGAGTTG ATTAATCAAT TGACACCCAA 4620

CAATACTTTG AAATATTTTA ATGCGGACTT TTCTAATTAT AGAAGGCAAA ACCAAGATAA 4680

TTACTGTCCA AATGGTGCGA TATTTATAGG CAATGTGGAC GCATATATTA AAAAAAAGAG 4740

TTTCTTTGGT GAAAGAAGTA TTGCCTATAT AATGGATAGA ATCGATTCAA TAGATATTGA 4800

TGACAAACTA GATTTCGAGT TCGCAATAAC AATAATGCAT AAAAAAAATA GATCTAGAGA 4860

ACTTCAAAAG AAAATACTAA ATAGGATATG CGAAAAAAAA AATTACTTTA ACCAAAAAAA 4920

AGATATAACG TTAATGGGGC ATTCAATTTT CGACTATTGG GATATAACTC AGTTAAAAAA 4980

ACGTGATGTG AATAATTTAG GAATCGCTGG TATTAATAGT AAACAATATT ATGATTATAT 5040

TATAAAAAAA AATATAGCAA AGGTTGGTCA AGATGTCATA TTAATGCTTG GTACGAATGA 5100

TATTGTTACT AATGATTGGA ATTTAGACTT CACAACTAAT TGGATTGAAA AAATAATTAA 5160

TCGCTTAAAA AGAATAAACC CAAATGTTAA TATTTATATT TTATCCGTAC CCCCTGTAGT 5220

TGGACGAATT GATAGGAATA ACGGAGTAAT AAAAAAATTG AATTGCAATC TGTTAAAATA 5280

TTTTGATAAT TGTCAAAATG TGTTTTTTTT ACCCCTGTCA GAAGCTTTTT ATGATAAATA 5340

TGGAAATCTA AAGGAAGAGT TCACATATGA TGGATTGCAT TTTACTCAGC TAGCTTATAA 5400

The termination of the initial Orf3 of Orf4

TCAACTGAAG AAAGAGATCG AGGAAAAATT AAA ATGAAAA AATTATTATA TATAACTGGT 5460

TCAAGAGCGG AGTATGGGAT CATGAAACGG TTGTTAAGAA AACTTCAGGA TCATGAGCGT 5520

ATTGAACTTT CTATTATTGC AACAGGAATG CATTGTGATG TTAAGTATGG TTATACATAT 5580

CAAAATATTA TTGACGATGG ATTTGAAATA AAAGAATTAT TTGATATAAA AATTGAAACA 5640

TCTACAAATA GTAATATTAT TTCTACGATG AGTCGTGCAC AAAGTTTGTT TGGTGCTCAT 5700

TTTCAGGACA ACAAATATGA TGCAATAATT ATTCTTGGTG ATAGATATGA AATGCTTTCT 5760

GTAGCTATAG CTGCATCTAT ACATGGAATT CCAATTATCC ATTTACATGG AGGAGAACAA 5820

ACATTAGGGA ATTATGATGA ATTCATTCGT CACTGTATTA CAAAAATGAG TCACCTACAT 5880

TTAGTTGCAA CAGATAAATA TAGAGAAAGA GTAATACAAC TAGGAGAACC ACCTGCTTCT 5940

GTTGTAAATA TCGGAGCGAT GGGGGCTGAG AATGTAATAT ATTTACCTAT ACCAGAACAT 6000

AACGAACTAC AGTCAAAATA TAGTAATGTT TTAAGTAATT ATTTTATGGT TCTTTTTCAT 6060

CCCGAGACAT TAACTAGAAA ATCTGTGCAT GACCAAATAA ATGAACTAAT TAAAGCATTG 6120

GAAATTGCGC ATAAAAAAAA TCATTGTAAC TTTATCTTCA TAGGATCTAA CTCGGACACG 6180

AGTTCAGAAA TCATTACTGA GCAAATTTTA ACCTTCTGTA AGTCATACAA TTTCCCATAT 6240

TTAGTATCGT TACCGACTCA AGATTATTTA GGATTATGTA AATATTCTTT AGGGCTTATT 6300

GGTAATTCAT CTTCTGGCCT AATAGAAATT CCATCACTCG GAGTCCCTAC TATTAATATT 6360

GGACATCGAC AGGCTGGGCG AGAACGTGGA AAATCAGTGA TTGATGTTGA ATGCTGCCAT 6420

GTTGATATAC TTAATAAGAT ACATGATATT TTATCGAATC GAAATATCAG TGCTGATGAT 6480

AATGTTAATC CTTATTATAA GGAGAACAGT GCTGACTTAG CTATGTCTAG TATAATTAGT 6540

TTTCTAGACA GGATTGGGGA CAATAAAAGT CATATCAAAA AGTTTTATGA TATTCCTAAT 6600

The termination orf5's of Orf4 is initial

ATTGATAATC TGATTAACTC A TAATAAGTA AACATGGTTA T ATGGAACTA GTTAGCATCA 6660

TTATAGCAGC TTATAACTGT AAAGATACCA TCTATGCTAC AGTTGAATCA GCGCTATCTC 6720

AGACATACAA AAACATTGAA ATAATAATTT GCGATGACTC ATCTACAGAT GATACATGGG 6780

ATATAATTAA TAAAATTAAA GACTCACGAA TTATTTGCAT AAAAAACAAT TATTGTAAGG 6840

GGGCGGCGGG AGCAAGAAAT TGTGCATTAA AAATAGCCAA AGGCAGATAC ATAGCATTTC 6900

TGGATAGTGA TGATTACTGG GTTACAACTA AGATATCAAA TCAGATCCAT TTCATGGAAA 6960

CAGAAAAGGT TTTCTTTTCA TACAGCAATT ATTATATTGA AAAAGATTTT GTTATTACGG 7020

GCGTCTTTAG TTCTCCGCCT GAAATCAATT ATGGTGCCAT GCTTAAATAT TGTAATATTG 7080

CATGTTCAAC AGTCATCCTC GATAGGACGG GGGTAAAAAA TATATCTTTC CCTTACATAG 7140

ATAAAGAAGA TTATGCATTA TGGCTTAACA TCCTTTCAAA AGGAATAAAG GCTAGGAACA 7200

CTAATTTAGT AGATACATAT TATAGAGTTC ATGCTGGTTC AGTTTCGGCA AATAAGTTTA 7260

AAGAGTTAAT ACGACAAAGT AATGTTTTAA AGTCTATCGG GATCAAAGCA CATCACAGAA 7320

TAATATGTTT ATTTTATTAT GCTATAAATG GTTTGATTAA ACACTGTTTT AGTTATAGAG 7380

The termination of the initial Orf5 of Orf6

ATAAAAGAA A TGCT TAGAGT TTGTTTTATT ATTGCAGATG TTACATGGAC AGGCGGAATA 7440

GAACGAGTGC TATCTAGGTT AGCGTTATCG ATTCCAGATG ATAATATAGA CTTAGAAATA 7500

TTAAGCTTAT ATAGGACTTA TAAAGATACA TATTATACAT TTCCAGATAA TGTTAAAATC 7560

AAATATCTTA ATGATAACTA TCAATATAAA GGTCGACCAG GATCGTTCAC TCGGTTAGTT 7620

CAGCATCTAA GAACATCTAT TAAACTAAGG GAATTTCTAA ATTCGTGTAA CTATGATATT 7680

ATTGTGGTTC ATACATTTCC GCTTGCATTC ATCAATTTTT TTACTGAAAA AAGATCAAAG 7740

TGGGTCGTTG TTGAACATGT AAAATATTAT TATTATAATA TTTTATTAAG ACGTATACGC 7800

TCATTTATAT ATGATAAATA TGATGCTATT GTGTTATTAA CAGATAAAGA TGTTACCCAT 7860

TTCAAAAATG CGAATAATAA TGTATATGTA ATACCTAATC CTTTAAGTTT TTCGCAAACT 7920

AAAAAAGCGA ATCTGAATAG CGTTAGAATA ATATCTGTTG GAAGATTAGA AAAACAGAAG 7980

GGATTCGATA TGCTCTTAAA GGCATTCTCA TATATATCAT ATAAATATCC AGAATGGCAA 8040

TTGGATATTT TTGGGAAAGG TAATGAAGAA GAAAATCTGA GGAAACTTAT ATCTAAATTA 8100

AATTTAAAAA ATGTGAATCT TATGGGAACG TCAAAAAATA TTCATCAAGA ATATCTTAGT 8160

TCTGCATTTT ATGTAATGTC TTCACGGTAT GAAGGTTTTC CTATGGTACT CTTGGAGGCG 8220

ATGGCTAGCG GTTTACCATG TATATCATTT AATTGTGAGA CTGGCCCTGC AGATATTATT 8280

ATTGACAATG AAAATGGTTT TCTTATAGAA CACTTTGATG TTGTTCAATT ATCAAAAGCA 8340

ATGGAGAAAC TGATCAAAGA TGAATATTTG AGAACAAAAA TGGGTAATGC CGCATTTGAG 8400

CGAGCAGATG AATTTTCATT AGAAAAAATC ACATTGAAAT GGAAGGAATT ATTTAAAGGG 8460

The termination of the initial Orf6 of Orf7

CTTACTC ATG AAAAG TAATG TTTATATTTG TAAAACCACA TGGGAAATTT TTGTTTCTAT 8520

TCTATTGGCT AGCGATGTTT ACTATAGTAA AAATATAAAA TCAGTTTTTG TTATTGAAGT 8580

TGCTGAGGAA AATATTAATT ATATTCCTAG GCTGTCTGAT TTTTGCTTTA TATTAAAGGT 8640

TGTTTCTGTT AAATTTTCTT CTAATAAATA CATTAAAGCT CTGTGTTTTT TATATAGAGT 8700

TAAATATTTT TTACCTAAAG TACTTGGAGA GTATCTAAAA AATGATGTAA GGGTTAATCT 8760

GTTTATAGAT CAAGGTGTAT TTGGACAGTT TTTCCTGAAA AATCACAAAG ACGTTTATCT 8820

ATATGAGCAT GGGAATGGAA ATTATTGTGT GGGACCATAT CCTAATTATA AACTAATAAA 8880

AAAGATTTTA GGTATAACTG AAGGTTATGG TAGACACAGT AGAGTGAAGA GGGTATACCT 8940

ACAGCATCCG GAAAAAGCTC CTCAAGATAT TCGTAATAAG GTAATTGGTT TTTCTATTAA 9000

ACATTTArAT TGTAAATTAT GCTCGAGAGA GCAACAAGCC TTATTGAGTT TTTTTGGAGT 9060

GAAAGATATT AATAAATCAA ATGATATTAT TATTTTAACA CAGCCAATCA GTGAGGATGG 9120

TTTTTACAGT GAAAAAGAGA AGATAGAAAT TTACAAGGAG ATTATTAATA GCCGCTTTTC 9180

CCATGATAAA TCTAGAGTTG TTATAAAGCC GCATCCCCGC GATAAAACAG ATTATAGTAA 9240

ATACATACCA GATGTAATGC TATTGCCAAG AAATTTTCCT ATGGAAATAT TAAATTTTAC 9300

AGACGTGAGA TTTGTTAAAG CTATAACAAT TTGTTCCAGT GCTGTATATA ATTTTGGATA 9360

TGATATTTCA GTTGATTATA TAGGTACCTT AAAATATCCA AAATTAATTA AAGCGTTGGG 9420

The termination of Orf7

GCGATTACCT GCACAATATG TACATAAAGA TAACTATAAA AAAATCAAGT AT TAATTGGA 9480

Orf8's is initial

GTCTGATGTG ATACCTTATA TTATATTTTA TTCTTTATCA TTAACTTTGG TTGTATTATC 9540

TTTGGTGTTA AAGAAATATG ATTGGTTTTG GTTGCTTTTT CTTTTGTTTA TTTCAATTAT 9600

TTTTATTGGC CTTAGGGTGA ATGTAGGCGC TGATTATACT GAATATGTCA GAATTTACAA 9660

TCAATCTTAC GATACTAATT TTGAATTAGG GTTTGATATA ATATTTAATC TCTTTAAGAG 9720

GTTCGGTTAT GATTATGTAT TTGTATCTTT GTTTGTGTTT ATATTGACAT CATTTTTTTT 9780

TCTATATTCA ATTAAGTCAT TACAACACAA AACTTTAATT TATTTCTGTT TTTTGCTTTT 9840

TATGTTTGTC CCTCTAACAT CAACTATTAG ACAAGGGTTA GCTATTCCTT TTTTTATAAT 9900

TTGCCTTTTA AACGCTGATA GACCTAAAGT CTACTTTATG AGTATCGCTA TAGGGTGTTT 9960

TTTCCATTAT TCGATTTTAT TTATGCTATT TTTCTTTTTT GTTAGATACT TTAAACAATC 10020

GTATTGCAGA GCTTATTTTA TAATCCTGTT TTTTTTAGGT TTAAGTGTTT TTAATATCGT 10080

TGAGTATATG ATTATACTAT TACATTATAT TCCTAGTCTG GGAACGTTAA GCTCTAAACT 10140

CCTTATGTAT ACACAGCGAT ATAATGTTAC GCTTTCATTA ACTTCAGTTG CTTATAAATT 10200

AGCTGGTTTA CTATTGGTTA CGAGTTTTAT GTCTTTTATC AAAATAAATA AAAAGTTACT 10260

GTGTAGTTAT AACTTATATT ATATATCATT GTGTTTGTTT ATCTTATTCA AAGATAATTC 10320

TGTTTTTACA AATCGTTTAA TGTATGCCAC TAATATATCC TTGGTGACTT TTTTTTCATT 10380

TTTGTTATCG ATGATGAATG CGTTTTATAG GAAAGCCATT GTTACTTTTT TGTTTGTAAT 10440

CTATTTTTCC ATAAATTATT TTAAATTTAC AGCAACAGAT TTAAGACACT CTATTGAGTC 10500

The termination Orf9's of Orf8 is initial

TGCTTATATG CCTTATAAAT CGGTACTTAT AAAT TAAGTA AAATATAAAT C ATGAAATTA 10560

ATGAAAGATA GATTTTTTTA TTTTTTTATC GAACTAATAA ATAAAGGCGT CCCGTTTTTC 10620

TTATTACCTT ATGTTTCTTC TGCTGTCGGT GTTGAAGTCT ATGGTAAAAT CGAGCTTTTT 10680

ACTACTAATT ATATAATATT ATCTTTCCTT TTTGCCTTGG GGTTTGAAGG TTGGATGAAT 10740

TCTCATTATT TTAAAATATC TGAAAATAAT TTTATTACTA TATTGTCTTC ATTTTATTTT 10800

TTGAGTTTTC TTATTTTTTT TATTAGTGTG ATAACAACGT TGCTATATTC ATCTGACTGG 10860

ACTGCTCTTG TTATTGTTGG ATATATTCAG TCGCTTCTTA ATATTACAAC TTTGCATTTA 10920

AGACTCAAGG GAAAATATAT AAGATCAGGC GCATTGTTAT TAGCAGCGTC AATTGTTAAT 10980

GCAGTGATGG TATATGTGTG CTTTGAGTTA TTTGGTAAAG ATTATAATGC GCGCATTGAA 11040

GCTTTTTTAT TTTCTTCTGT GTTATTGGTA ATATTTATTA TTCTCTGTAA CATTAATATA 11100

TGGGGGGGGG GGGTAAAAAT AAAAATTAAT TTTAGCTTTT TAAATAAAGA CATTTTGTTA 11160

TTTTGTTTAC CCCTGTGTTT AACAATGGTT ATTAACTGGG CTAAGGGTAA TATAGATAAG 11220

TATTTTATTG CTAATCTACT TGATTTGAAG TCTCTTGGTA TCTATGCTGT TGCATATCAG 11280

TTAGCTTCAG TTATTAATGT TGTTGGCATT ATTTTGAATA GGACATTACA GGCTGATTTG 11340

CTTAAAAGCA TGGCTGATGG TTTTTATCAA ACCAAAAAGA TAATACTCAT TTTTTTATCC 11400

GTTTTGGCTA TATTCTTTCT CCTATATGTT ATAGCTGTGT GTTATGGTTT TAATTATGTT 11460

TTTTCTGCCG ATTATGCCCC CTCTCGTAAT ATGGCGTTAC TGTTAATATT TTGTTTTTTA 11520

TTCTTTTCCA CCTCATCGTT TTTAATTAAT TTTGTATTGT TTTACGGTAG ACCTAAATTG 11580

ATTTTGCTTC AAAGTTTATT AATCACAACC ATACATGTTG CACTATCGTT TCTTTTGATT 11640

CAGAAATACG CTCTGTTTGG GGCTTTATTT ACACAGGTTA TTACTTCGAT ATTAACATTT 11700

The termination of Orf9

ATTTCCACTT TATTATTATG CTATATATTA ATGAAAGCAT CTCACAAA TA ACACATTCTT 11760

ACATGTTGTA AGATATTTAT TTAATATTAT CTTAAATTAT GAACTCATTT CTCAGGAGTA 11820

AACAATGTCA AAGCAACAGA TCGGCGTCGT CGGTATGGCT GTGATGGGGC GCAACCTTGC 11880

GCTCAATATC GAAAGCCGTG GTTATACCGT CTCTATTTTC AACCGTTCCC GTGAGAAGAC 11940

GGAAGAAGTG ATTGCCGAAA ATCCAGGCAA GAAGCTGGTT CCTTACTATA CGGTGAAAGA 12000

GTTTGTTGAA TCCCTGGAAA CGCCTCGTCG GATCCTGTTA ATGGTGAAAG CAGGTGCAGG 12060

CACGGATGCT GCTATTGATT CTCTCAAGCC ATACCTCGAT AAAGGTGACA TCATCATTGA 12120

TGGCGGAAAT ACCTTCTTCC TGGACACCAT TCGTCGTAAT CGTGAGCTTT CTGCAGAAGG 12180

CTTTAACTTT ATTGGTACCG GTGTCTCCGG TGGTGAAGAA GGCGCGCTGA AAGGTCCTTC 12240

CATTATGCCT GGTGGGCAGA AAGAAGCCTA TGAACTGGTT GCTCCGATCC TGACCAAAAT 12300

CGCCGCAGTG GCTGAAGACG GCGAGCCATG CGTTACCTAT ATTGGTGCCG ATGGTGCAGG 12360

TCACTATGTG AAGATGGTTC ACAACGGTAT TGAATACGGC GATATGCAGC TGATTGCTGA 12420

AGCCTATTCT CTGCTTAAAG GTGGCCTGAA TCTTTCCAAC GAAGAACTGG CACAGACCTT 12480

TACCGAATGG AATAACGGTG AACTGAGCAG CTACCTGATC GACATCACCA AAGATATCTT 12540

CACCAAAAAA GATGAAGACG GTAACTACCT GGTTGATGTG ATTCTGGATG AAGCAGCAAA 12600

CAAAGGTACG GGCAAATGGA CCAGCCAGAG TGCGCTGGAT CTCGGCGAAC CGCTGTCGCT 12660

GATTACCGAG TCTGTGTTTG CACGTTATAT CTCTTCTCTG AAAGATCAGC GCGTTGCCGC 12720

GTCTAAAGTT CTCACTGGCC CGAAAGCGCA GCCAGCAGGC GATAAGGCTG AGTTCATCGA 12780

AAAAGTTCGT CGTGCGCTGT ATCTGGGCAA AATCGTTTCT TACGCCCAGG GCTTCTCTCA 12840

GCTACGCGCA GCGTCTGAAG AGTACAACTG GGATCTGAAC TATGGCGAAA TCGCGAAGAT 12900

TTTTCGTGCT GGCTGCATCA TCCGAGCGCA GTTCCTGCAG AAAATCACCG ATGCCTATGC 12960

CGAAAATCCA CAGATCGCTA ACCTGCTGCT GGCTCCGTAC TTCAAGCAAA TTGCCGATGA 13020

CTACCAGCAG CCGCTGCGTG ATGTCGTTGC TTATGCGGTA CAGAACGGTA TCCC 13074

Only being preferred embodiment of the present invention below, is not that the present invention is imposed any restrictions, all according to the technology of the present invention essence to above embodiment make an amendment, equivalent variations and modification, all belong in the scope of technical solution of the present invention.

Claims (3)

1, a kind of oligonucleotide is characterized in that, is selected from the Nucleotide of 10645 to 10662 bases among the SEQ ID NO:1; The Nucleotide of 11320 to 11337 bases among the SEQ ID NO:1; The Nucleotide of 10920 to 10937 bases among the SEQ ID NO:1; The Nucleotide of 11255 to 11272 bases among the SEQ ID NO:1; The Nucleotide of 9612 to 9629 bases among the SEQ ID NO:1; The Nucleotide of 9850 to 9867 bases among the SEQ ID NO:1; The Nucleotide of 9847 to 9864 bases among the SEQ ID NO:1; The Nucleotide of 10408 to 10425 bases among the SEQ ID NO:1.

2, a kind of oligonucleotide is right, it is characterized in that, is selected from the Nucleotide of 10645 to 10662 bases among the SEQ ID NO:1 and the Nucleotide of 11320 to 11337 bases among the SEQ ID NO:1; The Nucleotide of 11255 to 11272 bases among the Nucleotide of 10920 to 10937 bases among the SEQ ID NO:1 and the SEQ ID NO:1; The Nucleotide of 9850 to 9867 bases among the Nucleotide of 9612 to 9629 bases among the SEQ ID NO:1 and the SEQ ID NO:1; The Nucleotide of 10408 to 10425 bases among the Nucleotide of 9847 to 9864 bases among the SEQ ID NO:1 and the SEQ ID NO:1.

3, the application of the nucleotide pair of the Nucleotide of claim 1 or claim 2 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray as probe, for detecting intestinal bacteria O56 type.