CN1316025C - Nucleotide against O-antigen of bacillus coli-086 - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O86. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O86 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 14156 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 specific nucleotide also comprises the oligonucleotides of glycosyl transferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O86. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O86 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O86 by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O86 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O86 type (Escherichia coli O86), particularly relate in the intestinal bacteria O86 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O86 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

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 the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 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) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-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 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, be 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 the antigenic specificity of O-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 of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.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 [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused 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.and Reeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichiacoli O111.Gene 164:17-23], and in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards andEwing ' s identification of the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152 and and Shigella O antigens " J.clin Microbiol, 17 (4): 681-684].

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O86 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O86 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O86 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O86 type: the gene of transhipment enzyme 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 orf2, orf9, orf11, orf12 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O86 type respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-25nt; They are high specials to the O-antigen of intestinal bacteria O86 type; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O86 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, or be used for hybridization as probe, or be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O86 type of these methods detections and identification of escherichia coli O86 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O86 type.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 the Nucleotide of the O-antigen-specific of intestinal bacteria O86 type: it is the isolating Nucleotide shown in SEQ ID NO:1,14156 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O86 type is comprising called after gne, orf2, gmd, fcl, gmm, manC, manB, wzx, orf9, wzy, orf11,12 genomic constitutions of orf12 are all between JUMPStart sequence and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O86 type, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf2, orf9, orf11, orf12 gene; Wherein said gene: wzx is the Nucleotide of 8601 among the SEQIDNO:1 to 9803 bases; Wzy is the Nucleotide of 10491 to 11831 bases among the SEQID NO:1; Orf2 is the Nucleotide of 2113 to 3129 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9806 to 10510 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 11843 to 12586 bases among the SEQ ID NO:1; Orf12 is the Nucleotide of 12598 to 13506 bases among the SEQ IDNO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O86 type wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O86 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 8987 to 9008 bases among the SEQ ID NO:1 and the Nucleotide of 9399 to 9419 bases; The Nucleotide of 9108 to 9130 bases among the SEQ ID NO:1 and the Nucleotide of 9532 to 9556 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 11055 to 11075 bases among the SEQ IDNO:1 and the Nucleotide of 11766 to 11786 bases; The Nucleotide of 10674 to 10692 bases among the SEQID NO:1 and the Nucleotide of 11319 to 11339 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O86 type in detecting 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 O86 type is providing the O-antigen of expressing intestinal bacteria O86 type 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 O86 type 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 O86 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O86 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 O86 type bunch: with the genome of intestinal bacteria O86 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 O86 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O86 type bunch: in each gene, respectively designed two pairs of primers, 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 O86 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O86, 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 O86.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O86 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O86 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O86 type bunch: with the genome of intestinal bacteria O86 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JUMPStart sequences Design upstream primer (#wl-1098-ATTGGT AGC TGT AAG CCA AGG GCG GTA GCG T) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGTTCG C) in O-antigen gene bunch downstream: with the Expand Long Template PCR method amplification O-antigen gene of Boehringer Mannheim company bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, Annealed 15 seconds, 68 ℃ were extended 15 minutes, carry 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 5 pipe longPCR products, and with the Wizard PCRPreps 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, 0.9 μ l 0.1MMnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut 10 minute hands concentrates between the 1.5kb-3kb dna fragmentation size, then adds 2 μ l 0.1M EDTA termination reactions.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 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends 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 80 μ l, 70 ℃ of reactions 20 minutes make 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 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, 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 to 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, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, 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, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O86 type;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains 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 Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O86 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O86 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O86 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) Orffinder finds gene, find the reading frame of 12 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 O86 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O86 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, except that a band that in containing intestinal bacteria O86 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O86 type all is high special.

(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 O86 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 444 to 459 bases among the SEQ ID NO:1 and the Nucleotide of 936 to 951 bases; The Nucleotide of 285 to 304 bases among the SEQ ID NO:1 and the Nucleotide of 1079 to 1094 bases: the Nucleotide of 3898 to 3913 bases among the SEQ ID NO:1 and the Nucleotide of 4205 to 4220 bases; The Nucleotide of 3807 to 3823 bases among the SEQ ID NO:1 and the Nucleotide of 4258 to 4273 bases carry out the PCR reaction, and 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 ¹, every part of pork filling of 0 and 5 viable bacteria 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 O86 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 O86 type, its complete sequence shown in SEQIDNO:1,14156 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 O86 type by method of the present invention, as shown in table 3, it comprises called after gne, orf2, gmd, fcl, gmm, manC, manB, wzx, orf9, wzy, orf11,12 genomic constitutions of orf12 are all between JUMPStart sequence and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O86 type, 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 (orf, orf3, orf6, orf11 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 glycosyltransferase gene, 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 very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria O86 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O86 type is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.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 except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O86 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O86 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O86 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 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 used herein, " oligonucleotide " mainly refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 8601 to 9803 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 10491 to 11831 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O86 type.

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 and pol gene, comprise the wzx gene or the gene, wzy gene of identity function are arranged or with wzy the gene of identity function is arranged with wzx.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 the wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.

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) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.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 O86 types.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.

Planner of the present invention 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 the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O86 type.

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 the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.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 O86 types.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 is used to detect the many to oligonucleotide of the inventive method, many here is that the gene of the gene that comes from the encoding glycosyl transferring enzyme, coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, 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 the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.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 O86 types.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 with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy 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-.And 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 O86 type 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 O86 type 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 O86 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l50mM Tris-HCl (pH8.0) and 10 μ l0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30 μ l TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O86 type bunch:

With the genome of intestinal bacteria O86 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JUMPStart sequences Design upstream primer (#wl-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNGCCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand LongTemplate 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, Annealed 15 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 5 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, 0.9 μ l 0.1M MnCl2, and 1 μ l1: the DNaseI of the 1mg/mL of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.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 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, lmMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 μ l 100mM DTT and 5 units, 11 ℃ 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 80 μ l, 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 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l 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 in the 30 μ l water and obtain connecting product.

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, got in the LB substratum that the 2mL culture is transferred to 200mL, and 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, 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, precipitate 10% glycerine suspension cell with the precooling of 1mL ice at last, are competent cell.The competent cell that makes is packed as 50 μ l, one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, 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 ammonia benzyl mould rope, 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 O86 type with the EcoRI enzyme.

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

From the library, select insert 96 clones of fragment more than 1kb with this lab A BI3730 type automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 100% fraction of coverage, thus all sequences of acquisition O-antigen gene bunch.

Embodiment 5: the splicing of nucleotide sequence and analysis:

The Pregap4 and the splicing of Gap4 software of the Stadenpackage software package of publishing with Britain Camb MRC (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 O86 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O86 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O86 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffinder finds gene, find the reading frame of 12 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 ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O86 type at last, as shown in table 3.

By retrieving and comparing, the albumen of finding the gne genes encoding among orf1 encoded protein and the Yersinia enterocolitica (type 0:8) has 57% consistence and 72% similarity, the antigenic structure of the O-of intestinal bacteria O86 is known, from structure, really need existence with the functionally similar gene of gne, therefore, we to name orf1 be gne.

The albumen of gmd, fcl, gmm, manC, manB genes encoding has very high consensus amino acid sequence (85-99%) in orf3,4,5,6,7 encoded protein and the Escherichia coli O-antigen gene bunch.These five genes were identified it is to be used for trehalose synthesis, and had this sugar of trehalose in the O-antigenic structure of Escherichia coli O86 really, and we name orf3,4,5,6,7 to be gmd, fcl, gmm, manC, manB.

Orf8 and orf10 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O86 kind.The O-antigen transferring enzyme of Orf8 encoded protein and Staphylococcus aureus has 23% sequence identity, and it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf8 is wzx.The O-antigen polysaccharase of Orf10 encoded protein and Shigella boydii has 21% consistence, 42% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf10 is wzy.

Orf2, the albumen of 9,11,12 4 genes encodings and other known glycosyltransferases have the sequence identity of 24-47% and the sequence similarity of 44-68%.Therefore we infer this four genes encoding glycosyltransferases.Because the definite function of these four genes can't be determined, so we are with these four genes temporary called after orf2, orf9, orf11 and orf12.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O86 type bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local 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, except that a band that in containing intestinal bacteria O86 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O86 type all is high special; The position of these genes in nucleotide sequence sees Table 1.

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 O86 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 the 200mLLB 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 8987 to 9008 bases among the SEQ ID NO:1 and the Nucleotide of 9399 to 9419 bases; The Nucleotide of 9108 to 9130 bases among the SEQ ID NO:1 and the Nucleotide of 9532 to 9556 bases; The Nucleotide of 11055 to 11075 bases among the SEQ ID NO:1 and the Nucleotide of 11766 to 11786 bases; The Nucleotide of 10674 to 10692 bases among the SEQ IDNO:1 and the Nucleotide of 11319 to 11339 bases carry out the PCR reaction, and 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 ", 55 ℃: 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 ¹, every part of pork filling of 0 and 5 viable bacteria 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 O86 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 intestinal bacteria O86 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O86 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 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O86 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O86 type in table, 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.

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

The genomic dna that contains intestinal bacteria O86 type in the 5th group is as positive 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 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25 μ l.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get 10 μ lPCR products 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, every pair of primer has obtained except be PCR in the 5th group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O86 type and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-25nt is special to the O-antigen of intestinal bacteria O86 type, 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 O86 type.These all oligonucleotide all can be used for the intestinal bacteria O86 type in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O86 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O86 type, altogether by 12 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are JUMPStart sequence 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 site plan of the gene in the O-antigen gene bunch of intestinal bacteria O86 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O86 type in the drawings, 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 intestinal bacteria: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

SEQUENCE LISTING

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O86 type

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O86 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>14156

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc atgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaacaacg tgtgaagcgt 120

caactgttgg cggaagtaca gtcgatctgc ccgccgggcg taaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttatgtg cacgacccgc cattggtgac 240

aacccatttg tcgtggtact gccagacgtt gtgatcgacg acgccagcgc tgacccgctg 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgtac ccaggtgctg 360

gcaaaacgta tgccgggcga cctctcggaa tactccgtta ttcagaccaa agagccgctg 420

gatcgcgaag gcaaagtcag ccgcattgtt gaattcatcg aaaagccgga tcagccgcag 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcagcc tggtgcatgg gggcgtattc aactgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tggggtatat gcaggcattt gtgaagtatg gactacgcaa cctcaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataaaaatt aatcatccat 780

atatggtgga ttgaataaaa atagaacggc agtgaacatt caaaagcaga gatgcttaca 840

ttataagaat gttcctgtct tttatttaaa gtatttaata acaataagtt accgcaagta 900

tcgctcgggc atagattttc cttgtatcta acttcgtttg gttagacaat tggaaaatag 960

tgaaaagtat taaatgctta gtacctatga ttcattttca gtgagttggt aactgttaag 1020

ccaagggcgg tagcgtgtgt ttttctctca atcctcatgg taaaggaggt ttatatggtg 1080

attttcgtaa caggcggtgc aggatatatt ggatcccata ccatacttga gttacttaat 1140

aatggtcatg atgtcgtttc gatagataat tttgtcaatt cctctataga atcattaaaa 1200

agagtagagc aaataactaa taagaaaatt atttcttatc aaggtgatat ccgtgataaa 1260

aatctacttg atgagatttt ttcaagacac catatcgatg ctgtaattca ctttgcatcg 1320

ttaaaatctg taggtgagtc taagttaaag cccttagagt attattctaa taatgttggt 1380

ggaactttag tattacttga atgcatgaag agatataaca ttaataaaat gatatttagc 1440

tcttctgcta ctgtttatgg gagtaacagt atccctcccc atacggaaga tagacgaatt 1500

ggtgaaacta caaacccata tgggacatcg aaatttataa tagaaataat tttgagtgat 1560

tattgtgata gtgataataa taaatcagta attgcactgc gttactttaa tccaatcgga 1620

gcacataagt ccgggatgat tggtgaaaat cctaacggga tccctaataa tctggttcct 1680

tatatatcta aagttgcaca aaatcaactt cctgtattaa atatttatgg caacgattat 1740

ccaactaaag atggtacagg agtaagagac tatatacatg tctgtgattt ggctaaaggg 1800

catgttaaag cattagaata tatgttttta aatgatgtca attatgaagc ttttaattta 1860

ggtactggtc aaggttattc tgttttagag attgtaaaaa tgtttgagat agtcactaaa 1920

aagagtatac ctgttgctat ttgtaataga cgtgaggggg atgttgcgga gtcatgggcg 1980

tctgctgatt tggcacataa aaagctttcc tggaaagcgg aaaaaaattt gaaagaaatg 2040

atcgaagatg tatggcgttg gcaaacaaac aatccaaatg gatataaaaa ataatttttg 2100

gaattttgat atatgaaaaa tgttggtttt attgttacaa aatcagaaat tggtggtgca 2160

caaacatggg taaatgaaat atctaacctt attaaagagg aatgtaatat atttcttatt 2220

acatctgaag aaggatggct cacacataaa gatgtctttg ccggagtttt tgtcatacca 2280

ggtattaaaa aatattttga cttccttaca ttgtttaaat tgagaaaaat tttaaaagaa 2340

aataacattt caacgttaat agcaagttct gctaatgccg gagtttatgc caggttagtt 2400

cgattactag tcgactttaa atgtatttat gtttcgcatg gatggtcttg tttatataat 2460

ggtggtcgcc taaaatcaat tttttgcatt gttgaaaaat acctttcttt attaactgat 2520

gttatatggt gtgtttccaa aagtgatgaa aaaaaggcaa ttgagaatat tggtataaaa 2580

gaaccaaaga taatcacagt atcgaattca gtgcctcaga tgccgagatg taataataaa 2640

caactccagt ataaggttct gtttgttggt aggttaacac accctaagcg ccccgaattg 2700

ttagcgaatg taatatcgaa aaagccccag tatagcctcc atatcgtagg agggggggaa 2760

aggttagaat cattgaagaa acaattcagt gaatgtgaaa atattcattt tttgggtgag 2820

gtcaataatt tttataacta tcatgagtat gatttatttt cactgatatc cgatagtgaa 2880

ggtttgccta tgtcaggcct tgaggctcac acagctgcaa taccactcct gttaagtgat 2940

gtgggcggat gttttgaatt aattgagggt aatgggttac ttgtggaaaa tactgaagac 3000

gacattggat ataaattgga taaaatattc gatgactatg aaaattatcg ggaacaggca 3060

attcgtgcct ccgggaaatt tgttatcgag aactatgctt cagcatataa aagcattatt 3120

ttaggttgag tatttttaag aacaggaaat gtaaaatggc taaagtcgct ctcattactg 3180

gcataaccgg acaagacggt tcttacctgg cagagtttct gctggaaaaa ggttacgagg 3240

tgcatggtat caagcgtcgt gcatcgtcat tcaacaccga gcgcgtggat catatctatc 3300

aggatccgca cacctgcaac ccgaaattcc atctgcatta tggcgatctg acagatacct 3360

ccaacctgac gcgcattttg tgtgaagtgc agccggatga agtgtacaac ctgggcgcaa 3420

tgagtcacgt tgcggtttct tttgagtcac cggaatatac cgctgacgtc gatgcgatgg 3480

gtacgctgcg cctgctggag gcgatccgct tcctcggtct ggaaaagaaa acccgttttt 3540

atcaggcttc cacttctgaa ctgtatggtc tggtgcagga aattccccag aaagagacca 3600

caccgttcta cccgcgatct ccgtatgcag tcgccaaact gtacgcctac tggatcaccg 3660

ttaactaccg cgaatcctac ggcatgtacg cctgcaacgg tattctcttt aaccatgaat 3720

ccccgcgccg cggcgaaact ttcgttaccc gcaaaatcac ccgcgcaatc gccaacatcg 3780

cccaggggct ggaatcgtgc ctgtacctcg gcaatatgga ttccctgcgt gactggggcc 3840

atgccaaaga ctacgtaaaa atgcagtgga tgatgctgca acaggaacag ccggaagatt 3900

tcgtcattgc taccggcgtt cagtactccg tgcgtcagtt cgtggaaatg gcggcggagc 3960

gagtcggtat caaactgcgt tttgaaggca caggtgtaga tgagaagggg atagtggttt 4020

ctgttaatgg tgaggatgct ccagcagtta aacctggaga tgtaatcgtt aaggttgatc 4080

cgcgttactt ccgtccggct gaagttgaaa cgctgctcgg tgacccgacc aaagcgcacg 4140

aaaaactggg ctggaaacca gaaatcaccc tcagagagat ggtgtctgaa atggtggcta 4200

atgacctcga agcggcgaaa aaacactctc tgctgaaatc tcacggctac gacgtggcga 4260

tcgcgctgga gtcataagca tgagtaaaca acgcattttt atcgctggcc atcgcgggat 4320

ggtcggttct gccatcaggc ggcagctcga acagcgcggt gatgtggaac tggtattacg 4380

cacccgcgac gagctgaacc tgctggacag ccgcgccgtg catgatttct ttgccagcga 4440

acgtattgac caggtctatc tggcggcggc gaaagtgggc ggcattgttg ccaacaacac 4500

gtatccggcg gatttcatct atcagaacat gatgattgag agcaacatca ttcacgccgc 4560

gcatcagaac gacgtgaaca aactgctgtt tctcggatcg tcctgtatct acccgaaact 4620

ggcaaaacag ccaatggcag aaagtgagtt attgcagggc acgctggagc cgaccaacga 4680

accttatgcc attgccaaaa tcgccgggat caaactgtgc gaatcttaca accgtcagta 4740

cggacgcgat taccgctcgg tcatgccgac caacctgtac gggccgcacg acaacttcca 4800

tccgagtaac tcgcatgtga tcccagcatt gctgcgtcgc ttccacgagg cgacggcaca 4860

gaatgcgccg gacgtggtgg tgtggggcag cggtacgccg atgcgtgaat ttctgcacgt 4920

cgatgatatg gcggcggcga gcattcatgt gatggagctg gcgcacgaag tctggctgga 4980

gaacacccag ccgatgttgt cgcacattaa cgtcggcacg ggcgttgact gcactatccg 5040

cgagctggcg caaaccatcg ccaaagtggt gggttacaaa ggtcgggtgg tttttgatgo 5100

cagcaaaccg gatggcacac cgcgcaaact gctggatgtg acgcgcctgc atcagcttgg 5160

ctggtatcac gaaatctcac tggaagcggg gcttgccagc acttaccagt ggttccttga 5220

gaatcaagac cgctttcggg ggtaatgatg tttttacgtc aggaagactt tgccacggta 5280

gtgcgctcca ctccgcttgt ctctctcgac tttattgttg agaacagtcg cggcgagttt 5340

ctgcttggca aaagaaccaa ccgcccggcg cagggttact ggtttgtgcc gggagggcgc 5400

gtgcagaaag acgaaacgct ggaagccgca tttgagcggc tgacgatggc ggaactgggt 5460

ctgcgtctac cgataacagc aggccagttt tacggtgtct ggcagcactt ttatgacgat 5520

aacttctccg gcacggattt caccacgcac tatgtggtgc tcggttttcg cttcagagtc 5580

gcggaagaag agctgttact gccggatgag cagcatgtcg attaccgctg gctgacgccg 5640

gacgcgctgc tcgccagtaa caatgttcac gctaacagcc gagcttattt taatcatgat 5700

ccaagtgctg tttttggttt agataaaaag gatgtcaaaa atgtctgatg taccgttgat 5760

tgctgttgta atggctggtg gtacgggtag ccgtctttgg cccctctcaa gagagcacta 5820

tccgaagcaa tttctgcaat tatcaggtga gaatacttta ttacagtcaa ctttattacg 5880

actatccccg ctttcttgcg aaactccatt agtaataact aacgagcagc atcgttttgt 5940

ggtcgcagaa caattgcgtc aaataaacca attaagcgat aacattattc ttgaaccatg 6000

tggaagaaat actgcaccag ctatagcact ttcagcattt acagccttaa agcgtaatga 6060

acaacaagat cctatacttt tagttcttgc agctgaccat gttataaaca aaacagatgt 6120

tttttgtaat gctattaaaa attccatttc aattgttgaa caaggaaata ttttaacatt 6180

tggtattatt cctgattatg cagaaaccgg ttacggttat atagaaaaag ggagtatagt 6240

taaagaatca caacgtggag ttggtaatac attctatcac gtggaacaat ttgttgaaaa 6300

acctaatcgc tcaagggctg aagagtatat ctcctcaggt aaatatctct ggaatagtgg 6360

gatgtttatg ttcaaagcat cagtatattt agaggagtta aaaaaataca gacctgatat 6420

atacgatata tgtgaaaaaa caatttcttc gtcctaccat gatcttgatt ttatacgact 6480

ttcgaaagat gtttttcaaa attgtccatc ggaatcaatt gattttgcag taatggaaaa 6540

aacaaagcgt tgtatagtct atcccgttga tattggttgg aacgatgttg gatcttggca 6600

atcattgtgg gacgttagtg ataaaacccc tacaggggac gtttgcaaag gtgatatcct 6660

gacctataat acaaaaaata attatattca ttcggagtca gccttagtag ccgctgtagg 6720

tgtggaagat attgttattg ttcagacgaa agatgcaatt ctagtttcaa aaaaatccga 6780

agttcaggat gttaagaaaa ttgttcaaat gcttaaaatg caagaacgtt ctgaatatct 6840

atcacatcgc gaggagtttc ggccttgggg aaagtttgat gctatagaac agggagatcg 6900

atataaggtc aaaaagattg tcgttaaacc aggggaaggg ctatccttac gtatgcatca 6960

tcatcgttca gagcattgga ttgtcctttc aggtacagca aaagtaacgt taaataacaa 7020

aactttctta gttactgcta atgaatccgt ttatattcct ctgggtgcta cttatagcct 7080

tgaaaatcca ggtataattc ctctcaatct tattgaagtt agttcaggag attatttagg 7140

tgaggatgat attgttcgtc aaaaagaacg ttacaaaata gatgactaat aaatgaaaaa 7200

attaacctgc tttaaagcct acgatattcg cgggaaatta ggcgaagaat tgaatgaaga 7260

tattgcctgg cgcattggtc gcgcttatgg cgaatttctc aaaccgaaaa ccattgtgtt 7320

aggcggtgat gtccgcctca ccagcgaaac cttaaaactg gcgctggcga aaggtttaca 7380

ggatgcgggc gtcgatgtgc tggatattgg tatgtccggc accgaagaga tctatttcgc 7440

cacgttccat ctcggcgtgg atggcggcat tgaagttacc gccagccata atccgatgga 7500

ttacaacggc atgaagctgg tgcgcgaagg ggctcgcccg atcagcggcg ataccggact 7560

gcgcgatgtc cagcgcctgg cagaagccaa cgactttcct cccgttgatg aaaccaaacg 7620

cggtcgctat cagcaaatca acctgcgtga tgcatacgtt gatcacctgt tcggttatat 7680

caacgtcaaa aacctcacgc cgctcaagct ggtgatcaac tccgggaacg gcgcagcggg 7740

tccggtagtt gacgccatcg aagcccgctt taaagccctc ggcgcacccg tggaattgat 7800

caaagtgcac aacacgccgg acggaaattt ccccaacggt attcctaacc cgctgctgcc 7860

ggaatgccgc gacgacaccc gcaatgcggt catcaaacat ggcgcggata tgggcattgc 7920

ttttgatggc gattttgacc gctgtttcct gtttgacgaa aaagggcagt ttatcgaggg 7980

ctactacatt gtcggcctgc tggcagaagc gttcctcgaa aaaaatcccg gcgcgaagat 8040

catccacgat ccgcgtctaa tttggaacac cgttgatgtg gtgagtgccg caggtggcac 8100

cccggtaatg tcgaaaaccg gacacgcctt tattaaagaa cgtatgcgca aggaagacgc 8160

catctacggt ggcgaaatga gcgcccacca ttacttccgt gatttcgctt actgcgacag 8220

cggcatgatc ccgtggctgc tggtagccga actggtgtgt ctgaaaggaa aaacgctggg 8280

cgaacttgtg cgtgaccgga tggcagcttt tccagcaagt ggggaaatta atagcacgtt 8340

ggaagatcca ttatgtgcta tcgcacgggt tgaaagttat tatgcaaata aggcaattga 8400

gatcgatcgt acagatggta taagtatgac ttttaatgga tggcgcttta acttacgttc 8460

ttctaatacg gaacccgtag tccggttaaa cgttgaatct agaggagatg tggccgcaac 8520

taaaaaatat acaggcttca tattagactt attaaattat aaaaagtcaa tatcatctta 8580

acaagtaaat gtgagttatt atgataaagt tgctttttag ttcatcttta ttaaagatgg 8640

gtgttgtttt attaaatttg cttattcctg caataataat acgattctat tcagagtatg 8700

attttggaat ttattcttat ttactaacat acgcaatcgt tatttctgtt ttacaaaacg 8760

gcatcactgc aagcttccgt aatctgatat caaatttaga aaaaaatgat ttgcttgatt 8820

atttattata ttcagtgagg aaaataacgt atccattctt attaataata ttttgtttat 8880

tattggctac gctttatgta gttgggtttg atacgttata tggtaaaata tgttttttta 8940

ttgctgtatc gttaataaat atatattatc cagtggttgc ttcatattat gatgctacag 9000

gtaaaacgct caattttgta ctacttgaaa taatattttt actcgtattc attgcaggcg 9060

tgggcatttt aatttactac aaagtgagta tttacattgt ttgcatttta actgctaact 9120

acagaggggt ttatgcaata ttattaataa tgaagaagtg tgttattttt aaacaaataa 9180

ggcttaagaa aaaggggggg tataaaattt tttgttctga tgatatgatt tttattataa 9240

ttcaattaat aaatgttagt aatacattat tatttatgaa cgtgtttgca aaattttacg 9300

gtgtagtggc gtttggggta ttttcagtat attacagatt tgttgctttt ccccaacaag 9360

tgataagctt ttcctcgtcg ttaatatgga ttaactttcg tcaagtccat gttaataaca 9420

gggttgctgc catgcaacta ttaaagagat tattttttat ttttactcta ttgcttatta 9480

tatggggagg gtgcgttcat ttttttatag acaaagtcgt atatctttat tcatccaaac 9540

ctttagaata cccaggtgtg ttgtgttttc taaatattat ggtttgcttg atgttgctaa 9600

aagacttttc aagtataata ctcaatgcat taactttata taaagaacaa atgataatga 9660

atgctttact ttgtttacta aacgttattt ttttcttttt ttataatgaa acgaattttg 9720

ataccatata tctaatcttc gtttcactgt taacattgat gtttgtattt gtaaatttat 9780

cactaattaa aagcaggctc tgaagatggt tattaatata ttttatatat gcacaggcga 9840

atataaaagg ttttttgata aattttattt atcttgtgag gataaattta taccagagtt 9900

tgaaaaaaaa tattatgtgt ttaccgattc tgataggatt tattttagta aatatctgaa 9960

tgttgaagta attaatgttg aaaaaaattg ctggccgctt aatacgttat tacgtttttc 10020

atattttttg aaggttattg ataagttaca aactaactca tatacttttt tctttaatgc 10080

aaatgcagtt attgtcaaag agattccttt tagtacattt atggagagtg atttaatcgg 10140

tgttattcat ccagggtata agaatcgtat atcgattctt tacccttggg aaagaagaaa 10200

aaatgcgacc tgctatcttg ggtatttaaa gaaaggtatt tattatcaag gttgtttcaa 10260

tggaggtaaa acagcgtcat ttaagagatt aatacaaatt tgtaatatga tgacaatggc 10320

tgatttgaaa aaaaacctga ttgctaaagt acatgatgag tcatatttga attattatta 10380

ttactacaat aagccactat tactttctga attgtactca tggcctgaaa aatatggtga 10440

aaataaagac gccaagatta ttatgcgtga taaggaaaga gaaagttggt atggtaatat 10500

caagaagtaa ttatagaaat atatgttcat atactttttt tatggtgaat ttatttatct 10560

taattttatc cgtaatcaat gaaggttttt gtgagattgc ctatgtaata ataagtgttt 10620

cttctgttct tttttgtgta atcataatct gccttgaaag gcaagggggc tttctcaacc 10680

caatgacttt ctgtattata tctgtctttt tctttatctt aatcagacct gtcttttttt 10740

cgcaaaatat aacagagaat ttaaacgaag taataactgc tggtttagaa attgatgaga 10800

tatatgtttt ttattcgtta gctgttgtaa acattccctt ggcttttact gtattactgt 10860

attcagtaca aaaaggtact gtttccaagt tagtagggca gttacctgat ttgttttttt 10920

ataataaaca attgtcaatg attttattat ggggggggct tttctctgct attttcttaa 10980

ttaaatcata caaaaaattt attatattag gtcaggtttc agtatttgag gctgacgcgt 11040

atggactata tgatgagtta ttttggttca ccctttcaaa atattgttat atattgtctt 11100

tgcttttttc taaaaataaa aatttcatac tatattcttt actgatattt ataacatcga 11160

taggttacat tttagtggga ttaagaggct atacaatcgc atatggtttc ttattgttat 11220

tttttctaga tataagatat aggcttaaga tcaaatggtt gttgttagta gctatattgg 11280

taacgaccat ttcttcactg ttccttaact atagaatagg tatagaagta aatagcggat 11340

tattaggaat tatttttaat ccattattac agcaaggtgc ttcatttgaa accgtttatg 11400

gtgctttgaa atacaatgaa aaaatattaa gttgcatatc ttactatgat tattttttta 11460

caaataaaga tatcggttct tgtattgaca tcgctcgtgg tgtttatttt aaagaaggag 11520

gaagtttcgc atcaagtttt tacagtgagc taatatattt ggggtggatc attggaagtg 11580

ttgctttatt actatttgca ttttctctag cttttgttca atcttgttat gagaaaataa 11640

taaaaaattc aatgaataat aaattggcat atacatatcg tttgatcatt tttttagcgc 11700

taccaaatct tatatatttt gcccgctcat cattatttga ttttattact aaggttttat 11760

ttatagctct attcataggt gggctaagca tagtgagaca tattgcttta aatataaaaa 11820

aatgtcattg agaatattag atatgatttc agtaataatg gctgtacacc gatatgataa 11880

atatgttgat atttcaattg atagtatctt aaatcagaca tactctgact ttgagttaat 11940

aataattgca aatggagggg attgtttcga gatagcaaaa cagctgaagc attatacaga 12000

gctggataac agagttaaaa tttatacatt agaaataggg cagttatcgt ttgcattaaa 12060

ttacgcagta actaagtgta aatactctat tattgccaga atggattccg acgatgtttc 12120

actgccgtta cgtctagaaa aacaatatat gtatatgttg cagaatgatt tagaaatggt 12180

ggggactggg atcagactta tcaatgaaaa cggtgagttt attaaagaat taaaatatcc 12240

aaatcataat aagataaata agatacttcc ttttaaaaat tgttttgcgc atcctacttt 12300

gatgttcaag aaagatgtta tactaaagca gcgaggttat tgtggtggtt ttaattcaga 12360

agattatgat ctatggctca gaatcttaaa tgaatgtccg aatatacgct gggataatct 12420

aagtgagtgt ttgctaaatt atcgaattca taacaaatct acgcaaaaat cagcactcgc 12480

atattatgaa tgtgctagtt attctctgcg agaattctta aaaaaaagaa ctattacgaa 12540

ttttctttct tgcctctatc atttttgtaa agcactaata aaataaaggt tgacgctatg 12600

tatagttgtt tgtctggtgg attaggtaat caaatgtttc agtatgctgc ggcatatatc 12660

ttacagagaa agcttaaaca aagatcatta gttttagacg atagctattt tttagattgc 12720

tcaaatcgtg atacacgtag aagatttgaa ttgaatcaat ttaacatatg ttatgatcgt 12780

ctgactacaa gtaaggaaaa aaaagagata tccataatac gacatgtaaa tagatatcgt 12840

ttgcccttat ttgttacaaa ttctatattt ggagttctac taaaaaaaaa ctatttgcct 12900

gaagcaaaat tttatgaatt tttgaacaac tgtaaattac aggttaaaaa tggttattgt 12960

ctattttctt atttccagga tgctacattg atagatagtc atcgtgatat gattctccca 13020

ttattccaga ttaatgaaga tttgctcaat ttatgtaatg acttgcatat ttacaaaaaa 13080

gtgatatgtg agaatgctaa cacaacttca ctacatatca ggcgtggaga ctacatcacc 13140

aaccctcacg cctctaaatt tcatggggtg ttgcccatgg attactatga aaaggctatt 13200

cgttatattg aggatgttca aggagaacag gtgattatcg tattttcaga tgatgtgaaa 13260

tgggctgaga atacatttgc taatcaacct aattattacg ttgttaataa ttctgaatgc 13320

gagtacagtg cgattgatat gtttttaatg tcaaagtgta aaaacaatat aatagccaat 13380

agtacatata gttggtgggg ggcatggtta aatactttcg aagataaaat agttgtttcc 13440

cctcgtaagt ggtttgctgg aaataataaa tctaagttga ccatggatag ttggattaat 13500

ctttgattat gctgtgtgtt ttttttaaaa tatgatatcc attcataatt tgtaattttg 13560

atatattatt tttagactaa aatgtcaatc tgcaacattc cgtagtaatc cctgacagga 13620

gtaaacaatg tcaaagcaac agatcggcgt agtcggtatg gcagtgatgg ggcgtaacct 13680

tgcgctcaac atcgaaagcc gtggttatac cgtctctatt ttcaaccgct cccgtgagaa 13740

aacggaagaa gtaattgccg aaaacccggg caagaaactg gttccttact atacggtgaa 13800

agagtttgtt gaatctctgg aaacgcctcg tcgcatcctg ttaatggtga aagcaggtgc 13860

aggcacggat gctgctattg attccctcaa gccatacctc gataaaggtg acatcatcat 13920

tgatggcggt aacaccttct tccaggacac cattcgtcgt aaccgtgagc tttctgccga 13980

aggctttaac ttcattggta ccggtgtttc cggtggtgaa gagggcgcgc tgaaaggtcc 14040

ttccattatg cctggtggcc agaaagaagc ctatgaactg gttgctccga tcctgaccaa 14100

aatcgccgca gtagctgaag acggtgagcc atgcgttacc tatattggtg ccgatg 14156

Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O86 type bunch and primer and PCR data wherein

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzx wzy	Transhipment enzymatic polymerization enzyme	8601-9803 10491-11831	8987-9008 9108-9130 11055-11075 10674-10692	9399-9419 9532-9556 11766-11786 11319-11339	433 449 732 666	0 * 0 * 0 * 0 *	60 60 60 54

^*Only in intestinal bacteria O86 type, obtain a correct band

Table 2166 strain intestinal bacteria and 43 strain Shigellaes and their source

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli	O1，O2，O5，O7，O8，O9，O12，O13，O14，O15，O16，O17，O18， O19ab，O20，O21，O22，O23，O24	IMVS a

2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli shigella dysenteriae 10, Shigella bogdii 11, shigella flexneri 12, wild-type e. coli 13, the 6th group of bacterial strain adds large intestine bar enterobacteria reference culture

O4，O10，O25，O26，O27，O28，O29，O30，O32，O33，O34，O35， O36，O37，O38，O40，O41，O42，O43 O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O53 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O68 O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99， O101，O102，O103，O104，O105，O106，O97 O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O126，O128，O117 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O141，O142，O143，O144，O145，O140 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166，O153 O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，19，D10，D11，Dl2，D13 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v:4)，F5(v:7)，F6， DS，DR O3，O11，O39，O59，O64，O73，O96，O95，O100，O114，O151，O155， O124，O167，O162，O121，O127，O149，O119 O86

IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a IMVS a

For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group as positive 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 intestinal bacteria O86 type O antigen gene structure iron

orf# galF gne orf2 gmd fcl gmm manC manB wzx orf9 wzy orf11 orf12 gnd

G+C％ 52.2 33.2 34.2 52.5 56.2 52.2 36.4 50.5 27.8 28.2 28.6 31.3 32.3 50.5

content

Table 4 intestinal bacteria O86 type O-antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ATGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTTC TTGAACAACG TGTGAAGCGT 120

CAACTGTTGG CGGAAGTACA GTCGATCTGC CCGCCGGGCG TAACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT GGGCCACTCC ATTTTATGTG CACGACCCGC CATTGGTGAC 240

AACCCATTTG TCGTGGTACT GCCAGACGTT GTGATCGACG ACGCCAGCGC TGACCCGCTG 300

CGTTACAACC TTGCTGCCAT GATTGCGCGC TTCAACGAAA CGGGCCGTAC CCAGGTGCTG 360

GCAAAACGTA TGCCGGGCGA CCTCTCGGAA TACTCCGTTA TTCAGACCAA AGAGCCGCTG 420

GATCGCGAAG GCAAAGTCAG CCGCATTGTT GAATTCATCG AAAAGCCGGA TCAGCCGCAG 480

ACGCTGGACT CAGACATCAT GGCCGTTGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACTCAGCC TGGTGCATGG GGGCGTATTC AACTGACTGA TGCCATTGCT 600

GAACTGGCGA AAAAACAGTC CGTTGATGCC ATGCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGGTATAT GCAGGCATTT GTGAAGTATG GACTACGCAA CCTCAAAGAA 720

GGGGCGAAGT TCCGTAAAGG GATTGAGAAG CTGTTAAGCG AATAAAAATT AATCATCCAT 780

ATATGGTGGA TTGAATAAAA ATAGAACGGC AGTGAACATT CAAAAGCAGA GATGCTTACA 840

TTATAAGAAT GTTCCTGTCT TTTATTTAAA GTATTTAATA ACAATAAGTT ACCGCAAGTA 900

TCGCTCGGGC ATAGATTTTC CTTGTATCTA ACTTCGTTTG GTTAGACAAT TGGAAAATAG 960

TGAAAAGTAT TAAATGCTTA GTACCTATGA TTCATTTTCA GTGAGTTGGT AACTGTTAAG 1020

Gne is initial

CCAAGGGCGG TAGCGTGTGT TTTTCTCTCA ATCCTCATGG TAAAGGAGGT TTAT ATGGTG 1080

ATTTTCGTAA CAGGCGGTGC AGGATATATT GGATCCCATA CCATACTTGA GTTACTTAAT 1140

AATGGTCATG ATGTCGTTTC GATAGATAAT TTTGTCAATT CCTCTATAGA ATCATTAAAA 1200

AGAGTAGAGC AAATAACTAA TAAGAAAATT ATTTCTTATC AAGGTGATAT CCGTGATAAA 1260

AATCTACTTG ATGAGATTTT TTCAAGACAC CATATCGATG CTGTAATTCA CTTTGCATCG 1320

TTAAAATCTG TAGGTGAGTC TAAGTTAAAG CCCTTAGAGT ATTATTCTAA TAATGTTGGT 1380

GGAACTTTAG TATTACTTGA ATGCATGAAG AGATATAACA TTAATAAAAT GATATTTAGC 1440

TCTTCTGCTA CTGTTTATGG GAGTAACAGT ATCCCTCCCC ATACGGAAGA TAGACGAATT 1500

GGTGAAACTA CAAACCCATA TGGGACATCG AAATTTATAA TAGAAATAAT TTTGAGTGAT 1560

TATTGTGATA GTGATAATAA TAAATCAGTA ATTGCACTGC GTTACTTTAA TCCAATCGGA 1620

GCACATAAGT CCGGGATGAT TGGTGAAAAT CCTAACGGGA TCCCTAATAA TCTGGTTCCT 1680

TATATATCTA AAGTTGCACA AAATCAACTT CCTGTATTAA ATATTTATGG CAACGATTAT 1740

CCAACTAAAG ATGGTACAGG AGTAAGAGAC TATATACATG TCTGTGATTT GGCTAAAGGG 1800

CATGTTAAAG CATTAGAATA TATGTTTTTA AATGATGTCA ATTATGAAGC TTTTAATTTA 1860

GGTACTGGTC AAGGTTATTC TGTTTTAGAG ATTGTAAAAA TGTTTGAGAT AGTCACTAAA 1920

AAGAGTATAC CTGTTGCTAT TTGTAATAGA CGTGAGGGGG ATGTTGCGGA GTCATGGGCG 1980

TCTGCTGATT TGGCACATAA AAAGCTTTCC TGGAAAGCGG AAAAAAATTT GAAAGAAATG 2040

Gne stops

ATCGAAGATG TATGGCGTTG GCAAACAAAC AATCCAAATG GATATAAAAA A TAATTTTTG 2100

Orf2 is initial

GAATTTTGAT AT ATGAAAAA TGTTGGTTTT ATTGTTACAA AATCAGAAAT TGGTGGTGCA 2160

CAAACATGGG TAAATGAAAT ATCTAACCTT ATTAAAGAGG AATGTAATAT ATTTCTTATT 2220

ACATCTGAAG AAGGATGGCT CACACATAAA GATGTCTTTG CCGGAGTTTT TGTCATACCA 2280

GGTATTAAAA AATATTTTGA CTTCCTTACA TTGTTTAAAT TGAGAAAAAT TTTAAAAGAA 2340

AATAACATTT CAACGTTAAT AGCAAGTTCT GCTAATGCCG GAGTTTATGC CAGGTTAGTT 2400

CGATTACTAG TCGACTTTAA ATGTATTTAT GTTTCGCATG GATGGTCTTG TTTATATAAT 2460

GGTGGTCGCC TAAAATCAAT TTTTTGCATT GTTGAAAAAT ACCTTTCTTT ATTAACTGAT 2520

GTTATATGGT GTGTTTCCAA AAGTGATGAA AAAAAGGCAA TTGAGAATAT TGGTATAAAA 2580

GAACCAAAGA TAATCACAGT ATCGAATTCA GTGCCTCAGA TGCCGAGATG TAATAATAAA 2640

CAACTCCAGT ATAAGGTTCT GTTTGTTGGT AGGTTAACAC ACCCTAAGCG CCCCGAATTG 2700

TTAGCGAATG TAATATCGAA AAAGCCCCAG TATAGCCTCC ATATCGTAGG AGGGGGGGAA 2760

AGGTTAGAAT CATTGAAGAA ACAATTCAGT GAATGTGAAA ATATTCATTT TTTGGGTGAG 2820

GTCAATAATT TTTATAACTA TCATGAGTAT GATTTATTTT CACTGATATC CGATAGTGAA 2880

GGTTTGCCTA TGTCAGGCCT TGAGGCTCAC ACAGCTGCAA TACCACTCCT GTTAAGTGAT 2940

GTGGGCGGAT GTTTTGAATT AATTGAGGGT AATGGGTTAC TTGTGGAAAA TACTGAAGAC 3000

GACATTGGAT ATAAATTGGA TAAAATATTC GATGACTATG AAAATTATCG GGAACAGGCA 3060

ATTCGTGCCT CCGGGAAATT TGTTATCGAG AACTATGCTT CAGCATATAA AAGCATTATT 3120

It is initial that orf2 stops gmd

TTAGGT TGAG TATTTTTAAG AACAGGAAAT GTAAA ATGGC TAAAGTCGCT CTCATTACTG 3180

GCATAACCGG ACAAGACGGT TCTTACCTGG CAGAGTTTCT GCTGGAAAAA GGTTACGAGG 3240

TGCATGGTAT CAAGCGTCGT GCATCGTCAT TCAACACCGA GCGCGTGGAT CATATCTATC 3300

AGGATCCGCA CACCTGCAAC CCGAAATTCC ATCTGCATTA TGGCGATCTG ACAGATACCT 3360

CCAACCTGAC GCGCATTTTG TGTGAAGTGC AGCCGGATGA AGTGTACAAC CTGGGCGCAA 3420

TGAGTCACGT TGCGGTTTCT TTTGAGTCAC CGGAATATAC CGCTGACGTC GATGCGATGG 3480

GTACGCTGCG CCTGCTGGAG GCGATCCGCT TCCTCGGTCT GGAAAAGAAA ACCCGTTTTT 3540

ATCAGGCTTC CACTTCTGAA CTGTATGGTC TGGTGCAGGA AATTCCCCAG AAAGAGACCA 3600

CACCGTTCTA CCCGCGATCT CCGTATGCAG TCGCCAAACT GTACGCCTAC TGGATCACCG 3660

TTAACTACCG CGAATCCTAC GGCATGTACG CCTGCAACGG TATTCTCTTT AACCATGAAT 3720

CCCCGCGCCG CGGCGAAACT TTCGTTACCC GCAAAATCAC CCGCGCAATC GCCAACATCG 3780

CCCAGGGGCT GGAATCGTGC CTGTACCTCG GCAATATGGA TTCCCTGCGT GACTGGGGCC 3840

ATGCCAAAGA CTACGTAAAA ATGCAGTGGA TGATGCTGCA ACAGGAACAG CCGGAAGATT 3900

TCGTCATTGC TACCGGCGTT CAGTACTCCG TGCGTCAGTT CGTGGAAATG GCGGCGGAGG 3960

GAGTCGGTAT CAAACTGCGT TTTGAAGGCA CAGGTGTAGA TGAGAAGGGG ATAGTGGTTT 4020

CTGTTAATGG TGAGGATGCT CCAGCAGTTA AACCTGGAGA TGTAATCGTT AAGGTTGATC 4080

CGCGTTACTT CCGTCCGGCT GAAGTTGAAA CGCTGCTCGG TGACCCGACC AAAGCGCACG 4140

AAAAACTGGG CTGGAAACCA GAAATCACCC TCAGAGAGAT GGTGTCTGAA ATGGTGGCTA 4200

ATGACCTCGA AGCGGCGAAA AAACACTCTC TGCTGAAATC TCACGGCTAC GACGTGGCGA 4260

It is initial that gmd stops fcl

TCGCGCTGGA GTCA TAAGC A TGAGTAAACA ACGCATTTTT ATCGCTGGCC ATCGCGGGAT 4320

GGTCGGTTCT GCCATCAGGC GGCAGCTCGA ACAGCGCGGT GATGTGGAAC TGGTATTACG 4380

CACCCGCGAC GAGCTGAACC TGCTGGACAG CCGCGCCGTG CATGATTTCT TTGCCAGCGA 4440

ACGTATTGAC CAGGTCTATC TGGCGGCGGC GAAAGTGGGC GGCATTGTTG CCAACAACAC 4500

GTATCCGGCG GATTTCATCT ATCAGAACAT GATGATTGAG AGCAACATCA TTCACGCCGC 4560

GCATCAGAAC GACGTGAACA AACTGCTGTT TCTCGGATCG TCCTGTATCT ACCCGAAACT 4620

GGCAAAACAG CCAATGGCAG AAAGTGAGTT ATTGCAGGGC ACGCTGGAGC CGACCAACGA 4680

ACCTTATGCC ATTGCCAAAA TCGCCGGGAT CAAACTGTGC GAATCTTACA ACCGTCAGTA 4740

CGGACGCGAT TACCGCTCGG TCATGCCGAC CAACCTGTAC GGGCCGCACG ACAACTTCCA 4800

TCCGAGTAAC TCGCATGTGA TCCCAGCATT GCTGCGTCGC TTCCACGAGG CGACGGCACA 4860

GAATGCGCCG GACGTGGTGG TGTGGGGCAG CGGTACGCCG ATGCGTGAAT TTCTGCACGT 4920

CGATGATATG GCGGCGGCGA GCATTCATGT GATGGAGCTG GCGCACGAAG TCTGGCTGGA 4980

GAACACCCAG CCGATGTTGT CGCACATTAA CGTCGGCACG GGCGTTGACT GCACTATCCG 5040

CGAGCTGGCG CAAACCATCG CCAAAGTGGT GGGTTACAAA GGTCGGGTGG TTTTTGATGC 5100

CAGCAAACCG GATGGCACAC CGCGCAAACT GCTGGATGTG ACGCGCCTGC ATCAGCTTGG 5160

CTGGTATCAC GAAATCTCAC TGGAAGCGGG GCTTGCCAGC ACTTACCAGT GGTTCCTTGA 5220

It is initial that fc1 stops gmm

GAATCAAGAC CGCTTTCGGG GG TAATGATG TTTTTACGTC AGGAAGACTT TGCCACGGTA 5280

GTGCGCTCCA CTCCGCTTGT CTCTCTCGAC TTTATTGTTG AGAACAGTCG CGGCGAGTTT 5340

CTGCTTGGCA AAAGAACCAA CCGCCCGGCG CAGGGTTACT GGTTTGTGCC GGGAGGGCGC 5400

GTGCAGAAAG ACGAAACGCT GGAAGCCGCA TTTGAGCGGC TGACGATGGC GGAACTGGGT 5460

CTGCGTCTAC CGATAACAGC AGGCCAGTTT TACGGTGTCT GGCAGCACTT TTATGACGAT 5520

AACTTCTCCG GCACGGATTT CACCACGCAC TATGTGGTGC TCGGTTTTCG CTTCAGAGTC 5580

GCGGAAGAAG AGCTGTTACT GCCGGATGAG CAGCATGTCG ATTACCGCTG GCTGACGCCG 5640

GACGCGCTGC TCGCCAGTAA CAATGTTCAC GCTAACAGCC GAGCTTATTT TAATCATGAT 5700

The initial gmm of manC stops

CCAAGTGCTG TTTTTGGTTT AGATAAAAAG GATGTCAAAA ATGTC TGATG TACCGTTGAT 5760

TGCTGTTGTA ATGGCTGGTG GTACGGGTAG CCGTCTTTGG CCCCTCTCAA GAGAGCACTA 5820

TCCGAAGCAA TTTCTGCAAT TATCAGGTGA GAATACTTTA TTACAGTCAA CTTTATTACG 5880

ACTATCCCCG CTTTCTTGCG AAACTCCATT AGTAATAACT AACGAGCAGC ATCGTTTTGT 5940

GGTCGCAGAA CAATTGCGTC AAATAAACCA ATTAAGCGAT AACATTATTC TTGAACCATG 6000

TGGAAGAAAT ACTGCACCAG CTATAGCACT TTCAGCATTT ACAGCCTTAA AGCGTAATGA 6060

ACAACAAGAT CCTATACTTT TAGTTCTTGC AGCTGACCAT GTTATAAACA AAACAGATGT 6120

TTTTTGTAAT GCTATTAAAA ATTCCATTTC AATTGTTGAA CAAGGAAATA TTTTAACATT 6180

TGGTATTATT CCTGATTATG CAGAAACCGG TTACGGTTAT ATAGAAAAAG GGAGTATAGT 6240

TAAAGAATCA CAACGTGGAG TTGGTAATAC ATTCTATCAC GTGGAACAAT TTGTTGAAAA 6300

ACCTAATCGC TCAAGGGCTG AAGAGTATAT CTCCTCAGGT AAATATCTCT GGAATAGTGG 6360

GATGTTTATG TTCAAAGCAT CAGTATATTT AGAGGAGTTA AAAAAATACA GACCTGATAT 6420

ATACGATATA TGTGAAAAAA CAATTTCTTC GTCCTACCAT GATCTTGATT TTATACGACT 6480

TTCGAAAGAT GTTTTTCAAA ATTGTCCATC GGAATCAATT GATTTTGCAG TAATGGAAAA 6540

AACAAAGCGT TGTATAGTCT ATCCCGTTGA TATTGGTTGG AACGATGTTG GATCTTGGCA 6600

ATCATTGTGG GACGTTAGTG ATAAAACCCC TACAGGGGAC GTTTGCAAAG GTGATATCCT 6660

GACCTATAAT ACAAAAAATA ATTATATTCA TTCGGAGTCA GCCTTAGTAG CCGCTGTAGG 6720

TGTGGAAGAT ATTGTTATTG TTCAGACGAA AGATGCAATT CTAGTTTCAA AAAAATCCGA 6780

AGTTCAGGAT GTTAAGAAAA TTGTTCAAAT GCTTAAAATG CAAGAACGTT CTGAATATCT 6840

ATCACATCGC GAGGAGTTTC GGCCTTGGGG AAAGTTTGAT GCTATAGAAC AGGGAGATCG 6900

ATATAAGGTC AAAAAGATTG TCGTTAAACC AGGGGAAGGG CTATCCTTAC GTATGCATCA 6960

TCATCGTTCA GAGCATTGGA TTGTCCTTTC AGGTACAGCA AAAGTAACGT TAAATAACAA 7020

AACTTTCTTA GTTACTGCTA ATGAATCCGT TTATATTCCT CTGGGTGCTA CTTATAGCCT 7080

TGAAAATCCA GGTATAATTC CTCTCAATCT TATTGAAGTT AGTTCAGGAG ATTATTTAGG 7140

It is initial that manC stops manB

TGAGGATGAT ATTGTTCGTC AAAAAGAACG TTACAAAATA GATGAC TAAT AA ATGAAAAA 7200

ATTAACCTGC TTTAAAGCCT ACGATATTCG CGGGAAATTA GGCGAAGAAT TGAATGAAGA 7260

TATTGCCTGG CGCATTGGTC GCGCTTATGG CGAATTTCTC AAACCGAAAA CCATTGTGTT 7320

AGGCGGTGAT GTCCGCCTCA CCAGCGAAAC CTTAAAACTG GCGCTGGCGA AAGGTTTACA 7380

GGATGCGGGC GTCGATGTGC TGGATATTGG TATGTCCGGC ACCGAAGAGA TCTATTTCGC 7440

CACGTTCCAT CTCGGCGTGG ATGGCGGCAT TGAAGTTACC GCCAGCCATA ATCCGATGGA 7500

TTACAACGGC ATGAAGCTGG TGCGCGAAGG GGCTCGCCCG ATCAGCGGCG ATACCGGACT 7560

GCGCGATGTC CAGCGCCTGG CAGAAGCCAA CGACTTTCCT CCCGTTGATG AAACCAAACG 7620

CGGTCGCTAT CAGCAAATCA ACCTGCGTGA TGCATACGTT GATCACCTGT TCGGTTATAT 7680

CAACGTCAAA AACCTCACGC CGCTCAAGCT GGTGATCAAC TCCGGGAACG GCGCAGCGGG 7740

TCCGGTAGTT GACGCCATCG AAGCCCGCTT TAAAGCCCTC GGCGCACCCG TGGAATTGAT 7800

CAAAGTGCAC AACACGCCGG ACGGAAATTT CCCCAACGGT ATTCCTAACC CGCTGCTGCC 7860

GGAATGCCGC GACGACACCC GCAATGCGGT CATCAAACAT GGCGCGGATA TGGGCATTGC 7920

TTTTGATGGC GATTTTGACC GCTGTTTCCT GTTTGACGAA AAAGGGCAGT TTATCGAGGG 7980

CTACTACATT GTCGGCCTGC TGGCAGAAGC GTTCCTCGAA AAAAATCCCG GCGCGAAGAT 8040

CATCCACGAT CCGCGTCTAA TTTGGAACAC CGTTGATGTG GTGAGTGCCG CAGGTGGCAC 8100

CCCGGTAATG TCGAAAACCG GACACGCCTT TATTAAAGAA CGTATGCGCA AGGAAGACGC 8160

CATCTACGGT GGCGAAATGA GCGCCCACCA TTACTTCCGT GATTTCGCTT ACTGCGACAG 8220

CGGCATGATC CCGTGGCTGC TGGTAGCCGA ACTGGTGTGT CTGAAAGGAA AAACGCTGGG 8280

CGAACTTGTG CGTGACCGGA TGGCAGCTTT TCCAGCAAGT GGGGAAATTA ATAGCACGTT 8340

GGAAGATCCA TTATGTGCTA TCGCACGGGT TGAAAGTTAT TATGCAAATA AGGCAATTGA 8400

GATCGATCGT ACAGATGGTA TAAGTATGAC TTTTAATGGA TGGCGCTTTA ACTTACGTTC 8460

TTCTAATACG GAACCCGTAG TCCGGTTAAA CGTTGAATCT AGAGGAGATG TGGCCGCAAC 8520

ManB stops

TAAAAAATAT ACAGGCTTCA TATTAGACTT ATTAAATTAT AAAAAGTCAA TATCATCT TA 8580

Wzx is initial

ACAAGTAAAT GTGAGTTATT ATGATAAAGT TGCTTTTTAG TTCATCTTTA TTAAAGATGG 8640

GTGTTGTTTT ATTAAATTTG CTTATTCCTG CAATAATAAT ACGATTCTAT TCAGAGTATG 8700

ATTTTGGAAT TTATTCTTAT TTACTAACAT ACGCAATCGT TATTTCTGTT TTACAAAACG 8760

GCATCACTGC AAGCTTCCGT AATCTGATAT CAAATTTAGA AAAAAATGAT TTGCTTGATT 8820

ATTTATTATA TTCAGTGAGG AAAATAACGT ATCCATTCTT ATTAATAATA TTTTGTTTAT 8880

TATTGGCTAC GCTTTATGTA GTTGGGTTTG ATACGTTATA TGGTAAAATA TGTTTTTTTA 8940

TTGCTGTATC GTTAATAAAT ATATATTATC CAGTGGTTGC TTCATATTAT GATGCTACAG 9000

GTAAAACGCT CAATTTTGTA CTACTTGAAA TAATATTTTT ACTCGTATTC ATTGCAGGCG 9060

TGGGCATTTT AATTTACTAC AAAGTGAGTA TTTACATTGT TTGCATTTTA ACTGCTAACT 9120

ACAGAGGGGT TTATGCAATA TTATTAATAA TGAAGAAGTG TGTTATTTTT AAACAAATAA 9180

GGCTTAAGAA AAAGGGGGGG TATAAAATTT TTTGTTCTGA TGATATGATT TTTATTATAA 9240

TTCAATTAAT AAATGTTAGT AATACATTAT TATTTATGAA CGTGTTTGCA AAATTTTACG 9300

GTGTAGTGGC GTTTGGGGTA TTTTCAGTAT ATTACAGATT TGTTGCTTTT CCCCAACAAG 9360

TGATAAGCTT TTCCTCGTCG TTAATATGGA TTAACTTTCG TCAAGTCCAT GTTAATAACA 9420

GGGTTGCTGC CATGCAACTA TTAAAGAGAT TATTTTTTAT TTTTACTCTA TTGCTTATTA 9480

TATGGGGAGG GTGCGTTCAT TTTTTTATAG ACAAAGTCGT ATATCTTTAT TCATCCAAAC 9540

CTTTAGAATA CCCAGGTGTG TTGTGTTTTC TAAATATTAT GGTTTGCTTG ATGTTGCTAA 9600

AAGACTTTTC AAGTATAATA CTCAATGCAT TAACTTTATA TAAAGAACAA ATGATAATGA 9660

ATGCTTTACT TTGTTTACTA AACGTTATTT TTTTCTTTTT TTATAATGAA ACGAATTTTG 9720

ATACCATATA TCTAATCTTC GTTTCACTGT TAACATTGAT GTTTGTATTT GTAAATTTAT 9780

It is initial that wzx stops orf9

CACTAATTAA AAGCAGGCTC TGAAG ATGGT TATTAATATA TTTTATATAT GCACAGGCGA 9840

ATATAAAAGG TTTTTTGATA AATTTTATTT ATCTTGTGAG GATAAATTTA TACCAGAGTT 9900

TGAAAAAAAA TATTATGTGT TTACCGATTC TGATAGGATT TATTTTAGTA AATATCTGAA 9960

TGTTGAAGTA ATTAATGTTG AAAAAAATTG CTGGCCGCTT AATACGTTAT TACGTTTTTC 10020

ATATTTTTTG AAGGTTATTG ATAAGTTACA AACTAACTCA TATACTTTTT TCTTTAATGC 10080

AAATGCAGTT ATTGTCAAAG AGATTCCTTT TAGTACATTT ATGGAGAGTG ATTTAATCGG 10140

TGTTATTCAT CCAGGGTATA AGAATCGTAT ATCGATTCTT TACCCTTGGG AAAGAAGAAA 10200

AAATGCGACC TGCTATCTTG GGTATTTAAA GAAAGGTATT TATTATCAAG GTTGTTTCAA 10260

TGGAGGTAAA ACAGCGTCAT TTAAGAGATT AATACAAATT TGTAATATGA TGACAATGGC 10320

TGATTTGAAA AAAAACCTGA TTGCTAAAGT ACATGATGAG TCATATTTGA ATTATTATTA 10380

TTACTACAAT AAGCCACTAT TACTTTCTGA ATTGTACTCA TGGCCTGAAA AATATGGTGA 10440

Wxy is initial

AAATAAAGAC GCCAAGATTA TTATGCGTGA TAAGGAAAGA GAAAGTTGGT ATGGTAATAT 10500

Orf9 stops

CAAGAAG TAA TTATAGAAAT ATATGTTCAT ATACTTTTTT TATGGTGAAT TTATTTATCT 10560

TAATTTTATC CGTAATCAAT GAAGGTTTTT GTGAGATTGC CTATGTAATA ATAAGTGTTT 10620

CTTCTGTTCT TTTTTGTGTA ATCATAATCT GCCTTGAAAG GCAAGGGGGC TTTCTCAACC 10680

CAATGACTTT CTGTATTATA TCTGTCTTTT TCTTTATCTT AATCAGACCT GTCTTTTTTT 10740

CGCAAAATAT AACAGAGAAT TTAAACGAAG TAATAACTGC TGGTTTAGAA ATTGATGAGA 10800

TATATGTTTT TTATTCGTTA GCTGTTGTAA ACATTCCCTT GGCTTTTACT GTATTACTGT 10860

ATTCAGTACA AAAAGGTACT GTTTCCAAGT TAGTAGGGCA GTTACCTGAT TTGTTTTTTT 10920

ATAATAAACA ATTGTCAATG ATTTTATTAT GGGGGGGGCT TTTCTCTGCT ATTTTCTTAA 10980

TTAAATCATA CAAAAAATTT ATTATATTAG GTCAGGTTTC AGTATTTGAG GCTGACGCGT 11040

ATGGACTATA TGATGAGTTA TTTTGGTTCA CCCTTTCAAA ATATTGTTAT ATATTGTCTT 11100

TGCTTTTTTC TAAAAATAAA AATTTCATAC TATATTCTTT ACTGATATTT ATAACATCGA 11160

TAGGTTACAT TTTAGTGGGA TTAAGAGGCT ATACAATCGC ATATGGTTTC TTATTGTTAT 11220

TTTTTCTAGA TATAAGATAT AGGCTTAAGA TCAAATGGTT GTTGTTAGTA GCTATATTGG 11280

TAACGACCAT TTCTTCACTG TTCCTTAACT ATAGAATAGG TATAGAAGTA AATAGCGGAT 11340

TATTAGGAAT TATTTTTAAT CCATTATTAC AGCAAGGTGC TTCATTTGAA ACCGTTTATG 11400

GTGCTTTGAA ATACAATGAA AAAATATTAA GTTGCATATC TTACTATGAT TATTTTTTTA 11460

CAAATAAAGA TATCGGTTCT TGTATTGACA TCGCTCGTGG TGTTTATTTT AAAGAAGGAG 11520

GAAGTTTCGC ATCAAGTTTT TACAGTGAGC TAATATATTT GGGGTGGATC ATTGGAAGTG 11580

TTGCTTTATT ACTATTTGCA TTTTCTCTAG CTTTTGTTCA ATCTTGTTAT GAGAAAATAA 11640

TAAAAAATTC AATGAATAAT AAATTGGCAT ATACATATCG TTTGATCATT TTTTTAGCGC 11700

TACCAAATCT TATATATTTT GCCCGCTCAT CATTATTTGA TTTTATTACT AAGGTTTTAT 11760

TTATAGCTCT ATTCATAGGT GGGCTAAGCA TAGTGAGACA TATTGCTTTA AATATAAAAA 11820

It is initial that wzy stops orf11

AATGTCAT TG AGAATATTAG AT ATGATTTC AGTAATAATG GCTGTACACC GATATGATAA 11880

ATATGTTGAT ATTTCAATTG ATAGTATCTT AAATCAGACA TACTCTGACT TTGAGTTAAT 11940

AATAATTGCA AATGGAGGGG ATTGTTTCGA GATAGCAAAA CAGCTGAAGC ATTATACAGA 12000

GCTGGATAAC AGAGTTAAAA TTTATACATT AGAAATAGGG CAGTTATCGT TTGCATTAAA 12060

TTACGCAGTA ACTAAGTGTA AATACTCTAT TATTGCCAGA ATGGATTCCG ACGATGTTTC 12120

ACTGCCGTTA CGTCTAGAAA AACAATATAT GTATATGTTG CAGAATGATT TAGAAATGGT 12180

GGGGACTGGG ATCAGACTTA TCAATGAAAA CGGTGAGTTT ATTAAAGAAT TAAAATATCC 12240

AAATCATAAT AAGATAAATA AGATACTTCC TTTTAAAAAT TGTTTTGCGC ATCCTACTTT 12300

GATGTTCAAG AAAGATGTTA TACTAAAGCA GCGAGGTTAT TGTGGTGGTT TTAATTCAGA 12360

AGATTATGAT CTATGGCTCA GAATCTTAAA TGAATGTCCG AATATACGCT GGGATAATCT 12420

AAGTGAGTGT TTGCTAAATT ATCGAATTCA TAACAAATCT ACGCAAAAAT CAGCACTCGC 12480

ATATTATGAA TGTGCTAGTT ATTCTCTGCG AGAATTCTTA AAAAAAAGAA CTATTACGAA 12540

It is initial that orf11 stops orf12

TTTTCTTTCT TGCCTCTATC ATTTTTGTAA AGCACTAATA AAA TAAAGGT TGACGCT ATG 12600

TATAGTTGTT TGTCTGGTGG ATTAGGTAAT CAAATGTTTC AGTATGCTGC GGCATATATC 12660

TTACAGAGAA AGCTTAAACA AAGATCATTA GTTTTAGACG ATAGCTATTT TTTAGATTGC 12720

TCAAATCGTG ATACACGTAG AAGATTTGAA TTGAATCAAT TTAACATATG TTATGATCGT 12780

CTGACTACAA GTAAGGAAAA AAAAGAGATA TCCATAATAC GACATGTAAA TAGATATCGT 12840

TTGCCCTTAT TTGTTACAAA TTCTATATTT GGAGTTCTAC TAAAAAAAAA CTATTTGCCT 12900

GAAGCAAAAT TTTATGAATT TTTGAACAAC TGTAAATTAC AGGTTAAAAA TGGTTATTGT 12960

CTATTTTCTT ATTTCCAGGA TGCTACATTG ATAGATAGTC ATCGTGATAT GATTCTCCCA 13020

TTATTCCAGA TTAATGAAGA TTTGCTCAAT TTATGTAATG ACTTGCATAT TTACAAAAAA 13080

GTGATATGTG AGAATGCTAA CACAACTTCA CTACATATCA GGCGTGGAGA CTACATCACC 13140

AACCCTCACG CCTCTAAATT TCATGGGGTG TTGCCCATGG ATTACTATGA AAAGGCTATT 13200

CGTTATATTG AGGATGTTCA AGGAGAACAG GTGATTATCG TATTTTCAGA TGATGTGAAA 13260

TGGGCTGAGA ATACATTTGC TAATCAACCT AATTATTACG TTGTTAATAA TTCTGAATGC 13320

GAGTACAGTG CGATTGATAT GTTTTTAATG TCAAAGTGTA AAAACAATAT AATAGCCAAT 13380

AGTACATATA GTTGGTGGGG GGCATGGTTA AATACTTTCG AAGATAAAAT AGTTGTTTCC 13440

CCTCGTAAGT GGTTTGCTGG AAATAATAAA TCTAAGTTGA CCATGGATAG TTGGATTAAT 13500

Orf12 stops

CTT TGATTAT GCTGTGTGTT TTTTTTAAAA TATGATATCC ATTCATAATT TGTAATTTTG 13560

ATATATTATT TTTAGACTAA AATGTCAATC TGCAACATTC CGTAGTAATC CCTGACAGGA 13620

GTAAACAATG TCAAAGCAAC AGATCGGCGT AGTCGGTATG GCAGTGATGG GGCGTAACCT 13680

TGCGCTCAAC ATCGAAAGCC GTGGTTATAC CGTCTCTATT TTCAACCGCT CCCGTGAGAA 13740

AACGGAAGAA GTAATTGCCG AAAACCCGGG CAAGAAACTG GTTCCTTACT ATACGGTGAA 13800

AGAGTTTGTT GAATCTCTGG AAACGCCTCG TCGCATCCTG TTAATGGTGA AAGCAGGTGC 13860

AGGCACGGAT GCTGCTATTG ATTCCCTCAA GCCATACCTC GATAAAGGTG ACATCATCAT 13920

TGATGGCGGT AACACCTTCT TCCAGGACAC CATTCGTCGT AACCGTGAGC TTTCTGCCGA 13980

AGGCTTTAAC TTCATTGGTA CCGGTGTTTC CGGTGGTGAA GAGGGCGCGC TGAAAGGTCC 14040

TTCCATTATG CCTGGTGGCC AGAAAGAAGC CTATGAACTG GTTGCTCCGA TCCTGACCAA 14100

AATCGCCGCA GTAGCTGAAG ACGGTGAGCC ATGCGTTACC TATATTGGTG CCGATG 14156

The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Claims (4)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O86 type, it is characterized in that: its wzx sequence is that one or more Nucleotide are inserted, lack or replaced to the Nucleotide of 8601 to 9803 bases among the SEQ ID NO:1 or Nucleotide that the wzy sequence is 10491 to 11831 bases among the SEQ IDNO:1 or above-mentioned sequence, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O86 type.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O86 type, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the nuclear battalion acid of the Nucleotide of 8987 to 9008 bases among the SEQ ID NO:1 and 9399 to 9419 bases, the Nucleotide of 9108 to 9130 bases among the SEQ ID NO:1 and the Nucleotide of 9532 to 9556 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 11055 to 11075 bases among the SEQ IDNO:1 and the Nucleotide of 11766 to 11786 bases; The Nucleotide of 10674 to 10692 bases among the SEQID NO:1 and the Nucleotide of 11319 to 11339 bases.

3, the application of oligonucleotide in detecting the O-antigen of expressing the antigenic bacterium of O-, identifying bacterium of claim 1, any described O-antigen-specific to intestinal bacteria O86 type of 2.

4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O86 type of 2, it 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, and the confession detection bodies reaches the bacterium in the environment outward.