CN1307306C - Nucleotide specific for Escherichia coli 011-type O-antigen - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O11. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O11 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 14180 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 oligonucleotide of glycosyl transferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O11. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O11 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O11 by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O11 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O11 type (Escherichia coli O11), particularly relate in the intestinal bacteria O11 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O11 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 Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inenteri cbacteria " .Microbiologicai Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " TheEscherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highlyconserved " .Journal of Bacteriology.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 ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of S.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiologicalinyestigation of an outbreak of Hemolytic-Uremic Syndrome caused bydry fermented sausage contaminated with Shiga-like toxin producingEscherichia 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.coliO111 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 Bast in D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet

Shigellae has 46 kinds of serotypes, 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; nichespecific selection and bacterial populatiohs " .FEMSMicrobiol.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 and Ewing ' sidentification of the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasive Escherichiacoli antigens 028ac; 0112ac; 0124; 0136,0143,0144; 0152 and andShigella 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 O11 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O11 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 O11 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O11 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, orf4, orf6, orf11 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of intestinal bacteria O11 type respectively comprises orf2, orf4, orf6, the orf11 gene; The gene that coming from coding transhipment enzyme 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 of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O11 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O11 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O11 type.

A further object of the present invention provides above-mentioned oligonucleotide and can be used as primer and be used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O11 type of these methods detections and identification of escherichia coli O11 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 O11 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 O11 type: it is the isolating Nucleotide shown in SEQ ID NO:1,14180 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 type, comprising called after wzx, orf2, wzy, orf4, gne, orf6, gmd, fcl gmm, manC, orf11,12 genomic constitutions of manB are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 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, orf4, orf6, orf11 gene; Wherein said gene: wzx is the Nucleotide of 1138 to 2373 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 3104 to 4339 bases among the SEQ IDNO:1; Orf2 is the Nucleotide of 2382 to 3107 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4341 to 5408 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6514 to 7521 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 11561 to 12307 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 type wherein also comprises coming from described wzx gene, wzy gene or glycosyltransferase gene orf2, orf4, orf6, orf11 gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 1573 to 1588 bases among the SEQ ID NO:1 and the Nucleotide of 1951 to 1967 bases; The Nucleotide of 1227 to 1243 bases among the SEQ ID NO:1 and the Nucleotide of 1888 to 1903 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 3154 to 3169 bases among the SEQ ID NO:1 and the Nucleotide of 3998 to 4013 bases; The Nucleotide of 3529 to 3544 bases among the SEQ ID NO:1 and the Nucleotide of 4034 to 4051 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 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 O11 type is providing the O-antigen of expressing intestinal bacteria O11 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 O11 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 the application of bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O11 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 O11 type bunch: with the genome of intestinal bacteria O11 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 O11 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O11 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O11 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O11, 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 O11.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O11 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O11 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul10mg/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 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O11 type bunch: with the genome of intestinal bacteria O11 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCAGGG ATC AAA GAA AT) 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 AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of ul 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 80ul, 70 ℃ of reactions 20 minutes make the 3 ' end of DNA 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 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of BiO-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of BiO-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds 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 O11 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 ResearchCouncil) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O11 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O11 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 O11 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 O11 type at last;

(6) specific gene screening: at wzx, the wzy gene gene design primer in the O-antigen gene of dysentery intestinal bacteria O11 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 O11 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 O11 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 O11 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 200 ml 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 1573 to 1588 bases among the SEQ ID NO:1 and the Nucleotide of 1951 to 1967 bases; The Nucleotide of 1227 to 1243 bases among the SEQ ID NO:1 and the Nucleotide of 1888 to 1903 bases; The Nucleotide of 3154 to 3169 bases among the SEQ ID NO:1 and the Nucleotide of 3998 to 4013 bases; The Nucleotide of 3529 to 3544 bases among the SEQ ID NO:1 and the Nucleotide of 4034 to 4051 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 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O11 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 O11 type, its complete sequence shown in SEQ ID NO:1,14180 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 O11 type by method of the present invention, as shown in table 3, it comprises called after wzx, orf2, wzy, orf4, gne, orf6, gmd, fcl, gmm, manC, orf11,12 genomic constitutions of manB are all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O11 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 (orf2, orf4, 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 O11 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O11 type is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf2, orf4, orf6, orf11 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are 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 O11 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O11 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O11 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 refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, 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 1138 to 2373 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 3104 to 4339 bases from SEQ ID NO:1); Orf2 gene (nucleotide position is the Nucleotide of 2382 to 3107 bases from SEQ ID NO:1); Orf4 gene (nucleotide position is the Nucleotide of 4341 to 5408 bases from SEQ ID NO:1); Orf6 gene (nucleotide position is the Nucleotide of 6514 to 7521 bases from SEQ ID NO:1); Orf11 gene (nucleotide position is the Nucleotide of 11561 to 12307 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O11 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 glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy 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 glycosyltransferase gene, 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 O11 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 O11 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 O11 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 O11 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 glycosyltransferase gene, 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 O11 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 O11 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 O11 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 ul 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 30ul 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 O11 type bunch:

With the genome of intestinal bacteria O11 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) 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 AGT TCGC) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 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, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 ul 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 80ul, 70 ℃ were reacted 20 minutes, made the 3 ' end of DNA 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 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, 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 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O11 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 Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O11 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O11 type is done 5 LongPCR 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 O11 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 O11 type at last, as shown in table 3.By retrieving and relatively, finding that Orf1 and orf3 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O11 kind.The O-antigen transferring enzyme of Orf1 encoded protein and e. coli k12 has 38% sequence identity, 62% similarity, 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 orf1 is wzx.The O-antigen polysaccharase of Orf3 encoded protein and intestinal bacteria (AAD50486) has 23% consistence, 46% similarity, it contains 10 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf3 is wzy.

The glcNAc4-epimerizase of coding has 61% consensus amino acid sequence among orf5 encoded protein and the Yersinia enterocolitica (type 0:8), 75% similarity, by search, find that the homology desired value of the consensus sequence of orf5 encoded protein and known glcNAc 4-epimerizase is 1.1e to Pfam protein-based order sequenced data storehouse ^-136GlcNAc 4-epimerizase is by the gne genes encoding, so we are gne with this unnamed gene.The GDP-mannose-4 of coding among Orf7 encoded protein and the Yersinia pseudotuberculosis (type O:1b), 6-dehydratase has 84% consensus amino acid sequence, 75% similarity, by search, find that the homology desired value of the consensus sequence of orf7 encoded protein and known NAD dependent epimerase/dehydratase family is Evalue=7.3e to Pfam protein-based order sequenced data storehouse ^-20GDP-mannose-4,6-dehydratase are by the gmd genes encoding, so we are gmd with this unnamed gene.The GDP-L-fucose synthetase of coding has 72% consensus amino acid sequence among Orf8 encoded protein and the Yersinia enterocolitica (type0:8), 86% similarity, by search, find that the homology desired value of the consensus sequence of ors8 encoded protein and known NAD dependent epimerase/dehydratase is Evalue=2.2e to Pfam protein-based order sequenced data storehouse ^-06GDP-L-fucose synthetase is by the fcl genes encoding, so we are fcl with this unnamed gene.The GDP-mannose mannosylhydrolase of coding has 50% consensus amino acid sequence among Orf9 encoded protein and the Klebsiella pneumoniae (BAD03940), 72% similarity, by search, find that the homology desired value of the consensus sequence of orf9 encoded protein and known NUDIXdomain is E value=1.2e to Pfam protein-based order sequenced data storehouse ^-20GDP-mannosemannosylhydrolase is by the gmm genes encoding, so we are gmm with this unnamed gene.The mannose-1-phosphateguanyltransferase of coding has 59% consensus amino acid sequence among Orf10 encoded protein and the Salmonella enterica (AAB49390), 78% similarity, by search, find that the homology desired value of the consensus sequence of orf10 encoded protein and known Nucleotidyl transferase is E value=6.5e to Pfam protein-based order sequenced data storehouse ^-124Mannose-1-phosphateguanyltransferase is by the manC genes encoding, so we are manC with this unnamed gene.The Orf12 encoded protein has 98% consensus amino acid sequence with the middle phosphomannomutase that encodes of Shigella sonnei (AAC80270) respectively, 99% similarity, by search, find that the homology desired value of the consensus sequence of orf12 encoded protein and known Phosphoglucomutase/phosphomannomutase is Evalue=1.5e to Pfam protein-based order sequenced data storehouse ^-64Phosphomannomutase is by the manB genes encoding, so we are manB with this unnamed gene

Albumen and other known glycosyltransferases of orf2, orf4, orf6, four genes encodings of orf11 have the sequence identity of 28-100% and the sequence similarity of 49-100%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these four genes encodings and known glycosyltransferase family 1 and 2 is respectively Evalue=6.4e ^-34, 2.6e ^-24, 2.7e ^-33, 3.3e ^-37, so we infer this three genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O11 may be made up of five monose.Because the definite function of these four genes can't be determined, so we are with the temporary called after orf2 of these four genes, orf4, orf6, orf11.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O11 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 O11 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 O11 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, 1 intestinal bacteria O11 is inoculated in the triangular flask of 20m1LB 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 1573 to 1588 bases among the SEQ ID NO:1 and the Nucleotide of 1951 to 1967 bases; The Nucleotide of 1227 to 1243 bases among the SEQ ID NO:1 and the Nucleotide of 1888 to 1903 bases; The Nucleotide of 3154 to 3169 bases among the SEQ ID NO:1 and the Nucleotide of 3998 to 4013 bases; The Nucleotide of 3529 to 3544 bases among the SEQ ID NO:1 and the Nucleotide of 4034 to 4051 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 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O11 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 O11 of the present invention can be applied to set up recombiant vaccine.

When molecular probe nucleotide sequence and target DNA sequence homology greater than 85% the time, can accurately aim sequence be hybridized out.The homology that requires both in the Southern of low preciseness hybridization is greater than 65% (" molecular cloning experiment guide " third edition, the 509th page, low preciseness hybridization).The homology search of specific nucleotide sequence among the present invention in Genebank shows does not have homology to exist greater than other genes of 65%.So in hybrid experiment, the specific nucleotide sequence among the present invention can only draw positive findings to the purpose bacterium as molecular probe.The Southern hybrid method is not strict with for the length of molecular probe, can use hybridization to whole sequence from 20bp or above oligonucleotide in the specific nucleotide sequence among the present invention.In a Southern experiment, utilize the relevant specific gene (more than the 1000bp) of Salmonellas to do molecular probe, success tell this bacterium (LiuD, Verma NK, RomanaLK, Reeves PR., 1991 Relationships among the rfb regions of Salmonel laserovars A, B, and D.J Bacteriol.173 (15): 4814-4819.), the experiment of a lot of this areas shows that all the gene order about 1000-2000bp can be used as molecular probe.Gene chip is the same with Southern hybridization ratio juris, also similar in the requirement of selecting molecular probe for use, so specific nucleotide sequence among the present invention and oligonucleotide fragment wherein can detect this purpose bacterium as molecular probe in hybridization, comprise the ordinary method of multiple hybridization such as Southern, gene chip.

Nucleotide sequence (shown in the SEQ ID NO:1) according to the O-antigen-specific to intestinal bacteria O11 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 oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O11 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O11 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 O11 type in the 13rd 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 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd 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 O11 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O11 type, screen gene by PCR: transhipment enzyme gene and pol gene (wzx and wzy gene) to the O-antigen high special of intestinal bacteria O11 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O11 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 O11 type.These all oligonucleotide all can be used for the intestinal bacteria O11 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 O11 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O11 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 galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O11 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O11 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)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O11 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O11 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>14180

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120

caactgctgg cggaagtaca atctatctgt ccgccgggcg tgaccattat gaacgtgcgt 180

cagggtgaac ctttaggttt gggccactcc attttgtgtg cacgccccgt cattggcgac 240

aacccatttg tcgtggtgct gccggacgtt gtgatcgacg acgccagcgc cgacccgttg 300

cgttacaacc ttgctgccat gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca tccagaccaa agaaccgctg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcaacc tggtgcatgg gggcgtattc agctgactga tgctattgct 600

gagctggcaa aaaaacaatc cgttgatgca atgctgatga ctggcgacag ttacgactgc 660

ggtaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gactacgcaa cttgaaagaa 720

ggggcgaagt tccgcaaagg tattgagaag ctgttaatcg aataatgaaa atctgaccga 780

atgtaacggg tgataagaaa attataacgg cggtgaagat tggtggggct aattgtactc 840

attgcctcat tctttcttta taccgttcta aatatgagta acaataactt ttcaataggc 900

atttattgtg ggaggctgag attttctcca tctctgaagc gatttggtaa aacgttcaga 960

gttaagcttt tcgaactaac cataacggtt aatgctagtt acaattagga catgcatgca 1020

gtacgctggt agctgttgag ccaggggcgg tagcgtgaat taaatgttta tcccatatta 1080

cttaacaaat tgtaaactat cgtgcaaaat aagacttgtt aagtacgtag gctcgttatg 1140

ttaaaaaaca tcatctattt attaagcgtt caaggtggca attatatatt tccattggtc 1200

acactgcctt atcttgtcag gatccttgaa cctacaggat acgggatata tgggtatagt 1260

tttgctgtgg tccaatattt tatattattt attgattacg gatttaacta ctcagcgccc 1320

aaaatcatat caatatcaag ggaaaatact gattccataa gtaaagtttt ttggaatgta 1380

acgtttatca aaataattgc agcatctctt ggtttgttaa ttatttattt aatttcggaa 1440

ttgaatatca ttgattacca aattaaattt atcattcttg catacatatc tgtcgtagga 1500

aatttaatat atccagtatg gttttttcaa gggctggaaa aaatgttagg aatagcgata 1560

agtatgtttt ccgcacgctt acttacacta tgtttaacat ttttattggt taaaagtgaa 1620

aaggatcttg gtatagcgat aacaatacaa tcatcaataa cagttattgc aggtataata 1680

tctacatatt ttttattttc tcttaaaaaa ataatatggg taccaccatc cttatccaaa 1740

atgaaagagt taatttatga tggatggcat tacttcattt cctctgcagc aataagcctt 1800

tatactacta gttcaacaat aatattaggt gcattatcag gaacaactgc agtgggatat 1860

tttatcgctg cagataaaat taggcttgca ctacaaggcg ttattggtcc tataacccaa 1920

gctgtttttc ctagagtaac ttatataatg aaggataatc atgacgatgg actattatta 1980

gccaggaaag tgtttcgatg gcaattttca ctgatgttag tactttctat atttctttac 2040

ttttttgcag acgatataat tttaatcatg tatggaaaaa cgtatcatga atctttagaa 2100

atactcaagg ttttagcttt tactccagca atagttacgg taagtaatta tattgcaatt 2160

ttagtaatga tccctattgg tatgaaaaaa gagtttacaa aaattattac taccagtgga 2220

ttaataggcc tactgataat tacttatttg actaatgaat actcaggagt tggtacagca 2280

ataggagtta tttcaactga gtttataata atggcgagat tttacttttt gattaagaaa 2340

acaaataaat taaataattt aaatttattg tagggctaac aatgaataaa ccaactttaa 2400

caatagttac cgttgtgtac aatggcgaaa tgtatatcga agatactata aaaagtgtct 2460

tatcacaaaa ctgcaatgat gacattgaat atattataat tgatggtgca tcaacggata 2520

atactctaaa tattgttaat tcttacaaaa atgaaatttc atatatcatt tctgaaaatg 2580

ataatggtat atatgatgca atgaataaag gtgtgttaaa agcatctgga aattatatag 2640

gatttataaa cgctgatgat ttttataata aagatgtatt atgtcaagta ataaaagtaa 2700

tgaaagaaaa taaatatgat gttatttatg gaaatatgtc tttggtagag gctgatagct 2760

tggatgttat tcaggtccgg aggccgaaag actggaaact acatatagat atgaatttaa 2820

gccatccggc cacatttatt aaaaaagata tccacagaga aaatttattt aatactagct 2880

ataaaattgc ttctgattat gatcttttgc taaaattgaa gaaacaaggt tatatctttc 2940

attatattga taaaaatatc tcttcgatga gaaatgccgg ccttagcgct aaaaatgtaa 3000

aattagcatt ggaggaagcg agacaaatcc gaaagaaaag aaacgggaaa ttatatgcgg 3060

cgttagcaga tatttaccta tttatgagaa aagttataaa aagatgatta atagaaacat 3120

aaaaattagt gcttcattta tttcaataat ttatttgcct cttgctttat tattagcaat 3180

gattgaaatg attaatgggg ccagatggag tgtttatgta tgttcattta tgctaggggt 3240

actagcattt ttgttaatac cttatgaaaa ttgggatttg actaggcatt atgaaacata 3300

tttatatgtt agtcaaacat cactaaacaa agtttttgaa aatagtgaca atagatatat 3360

tacaataaat actgttattt atttatttaa atctttagga ataaaaaaag aatttttacc 3420

ttttttttca acatttttat catatatttt ttattttaat atatatgtta attacattaa 3480

aaaatacaat ccggaattat ctcatagatg tatatattta tatattctaa taagcgtaat 3540

cccatttttt gctatagcta gttcattgcg gcaatatctt gcatttagta ttatgcttta 3600

tatgattgta cagtatttca ccagtgaaaa taataggtat aagtcacaaa aaattatatt 3660

ttctgcaatt atttcatgtt gtattcatcc cagcgcaata ctattgctag gtgttttttt 3720

tgtcagcaaa ttaataagac ttaacaaaac aatattgtat ttaataatat tagttctaat 3780

ccttaaccat gatggattaa tatcttcttt gctatttcaa ttgattaggc cattattgga 3840

aagcttaggt ttttactttc ccgaatatat ggactctgta accataggca taagcaatag 3900

cttgctgagc actaatgaat atatcttgaa taagatcata ttaccgtcag tattttttat 3960

ttttactttt atctatttaa ttttatttaa taaaaaaatg acgaggcaag agtcacaaat 4020

caaaaatttt tgcagtttac ttcttttgct gatatgcctt ttatcgttat cgcctgattt 4080

attttatcga ttttccattt tctgggtgtt attatatagt tatatatttt tctcctacga 4140

tattaataaa attagcaaac tatgtaagca tctgatcttc ttaatactca ttgccgcatg 4200

ttcaatcata aatttggcac aggcaaatat gggtaaagat attatttatg tgtcttggtg 4260

cgatatattg tataagccct cattattcct attcaataaa gaagttaaat cagcagaata 4320

tataagagtg agtcaataaa atgtccattg gagtctttaa gctgtataaa aataaaatag 4380

gtggcactga gaaagcagtt gaaaatctaa ttgatatatt aactaaaaac agcctacaag 4440

tgagatgttt ttcagcaata aacgcacaag agatacaagt tagagaagat aatcggaaaa 4500

actacttatt ccaagacaat actgcatatg aagaaagcaa attaaggttt tacattaaat 4560

atttgttttt tttgagtgcc aaaagtaaag accttgaatt aataatttcc acggatatag 4620

ctacatcatc attattgtta ttgattggtt tttttcggta taataagaat aaaattattt 4680

gctgggaaca ttatccattt gacaagaata gtattttttg gagaaaagta tttaaatttc 4740

ttttgtctct taataaaaaa gctgtagtag tttgtaattc tcacaaggaa aaagataagt 4800

ttagtttttg taaaagggtt gtaattatac caaattcaat caaaaagcaa aaatataagc 4860

gcatacatca aggaaaagaa actcttgatt tgatttatat tggtcggatt gttaaagata 4920

aaggagttca aaggttaatt aatgcagtaa taaatcaccc gtctacaaaa aagtatgttt 4980

tacacattgt aggaggtggt caagagctgg agtatctcaa atctaaatat aagaataaca 5040

accaaataca ctttcatggg ccgagtttaa acacagatgt ttttttggca aaaagtgatg 5100

tttttgtatc cggttcatac tttgaatgtt ttcctgttac aatacttgaa gctatgtcct 5160

acggattacc tgtaatttca atggatatat ctggagggac aagtagcgtg atttcttcat 5220

cgggtggcgg atttgtgtgt caatctcaaa ttgaatttca ggaaaaacta acttactttg 5280

aagataatgt taaacgaacc gaacacggaa atgcaggatt cctattcgtg aatagacatt 5340

attcagaaga aatagttagt gctcagtgga tgagtttgat caatcgagcc aatagtcaag 5400

aaaattaagg tattggagct attaaagtat gagcatactt gtgactggtg gggctggcta 5460

tattggctca catgctatat taactttatt gcaaaatgga tatgatgtta tatcgctaga 5520

taattattgc aattcctctc caaaatctct agaacgagta gaggaaattt gcttatctaa 5580

gataaaaaaa ttacgtggtg atattagaga tagacatata ttaaaaaata tcttttcaaa 5640

atataatgtt tctactgtaa tacattttgc gggtttaaaa tctgttaacg agtccattaa 5700

aaaaccatta gaatattacg acacaaatct tttggggact ctaatattac ttgatgaaat 5760

gcagaaagcg aatatttcta atctaatttt tagttcgtct gctactgtct atggcaatcc 5820

tcattatgtt ccaatatctg aaaaacaccc tgtcggtgag gtgatcaatc cgtacggtcg 5880

ttcaaagttt atggtggaac atatattgcg tgatttttgt aacgcgaata gcgacgcaaa 5940

tatcacaata ctaagatatt ttaatcctgt aggtgctcat gagactggat taattggaga 6000

agatccttta ggaactccta ataacctgtt tccctatgtt gcgcaagtcg caattggaaa 6060

attaccctat ctaaatgttt ttggtaataa atatatgaca aaagatggaa caggtgtaag 6120

agattatatc catgtagtcg atttagttga ggggcatctt gccgctttaa aaaatctcaa 6180

tagaaaccgg gggcttaaag tttttaattt aggcactggt caaggttata gtgtgttaga 6240

tatcatcgac gcttttaaac tggaaacagg acgcgatata cctttccgta tcgtttctga 6300

gcgaaatgga gatgtggctg aatgttggtc cgatccttcc ttagcaaacg agcagctaag 6360

ttgggttgct aaaaggactc ttaataatat ggtggaagat gcatggagat ggcaaacaat 6420

gaatccaaat ggatataaag agtaaattga aacaattata atcatccatt tcattgctta 6480

ctgtattgtt atctgtatat atgaaaacaa aaaatgaaaa tattatatga tgggattgtg 6540

aaaagtttac aatcatatgg tggtatcact gtatactttg aagaaatcat taagagatta 6600

aaaaatgatg aatacaaatt cattcaattt aatgataaaa atgaaaatca caatgatgat 6660

ctccatcggc catgccggtt tttagaacga tatagatgtt ttgaagcatt aaaaagtgat 6720

tcagattaca gtatatttca ttctacatat tatcgggttc cgaaagatgg ggaatatgcg 6780

gttgtgacca cggtccacga tttcacatat gaattgtata tgaaaggact tcggcgcaat 6840

gttcatagct tacaaaaaag atgggctgtg aaaaatagcg atgcaatcat ttgtgtctct 6900

aataatacag ccaaggattt aattaatttc tttaaggttg atgaaaaaaa gatacatgtt 6960

attcataatg gtgtatcgga agattataga cacttaaatc tagaaaaaga aaattttgtg 7020

ctttttgtcg gtgctcgttc aggatataaa aattttgagc ttgcagttaa agcagtttct 7080

aaactttttg atactaatct taaaatagtc ggcggaggtg gtttaaataa agcagaaatg 7140

aagttactat ctgaatatct tccagaaaga ttccagtact tagggaaggt atcaaatgaa 7200

aaactaaata tattatataa ttcggcttat tgcttaatct atccttcaga atatgaaggg 7260

tttggaattc ccttgcttga ggctatgcga actggctgtc ctgtgatagc cgtaaataga 7320

tcatcaatac ccgaggttgt gggggatagt gcgattttag tcgaagaagt atcaattgaa 7380

agtttattct cagctttatt aagtgtagaa gctaacgcta atgaattaaa aaataaagga 7440

ttagcgcaat ctaataaact ttcatggggg aagtgtttct ccgaaacata taatctatat 7500

aaatcactat ctaagttttg agaggttatt tatgtgtaag aaagctttaa ttacaggaat 7560

taccgggcaa gatggttcct acttggcaga atttctgctt aaaaaaggct atgaggttca 7620

tggaataaaa cgtcgatctt ccttgtttaa tactgaccgt attgatcata tatatcaaga 7680

cccacaccat cgtaatttaa aattacaact tcattatggt gatttaacag atacttctaa 7740

tttgactcgg atattatctg aagttcaacc tgatgaagtt tataatcttg gcgcaatgag 7800

tcacgtagct gtttccttcg aatccccaga atatacagct gatgttgatgc tataggtac 7860

tttacgatta ttggaagcta ttcgtttttt gggattagaa aaaaaaaccc gattttatca 7920

ggcgtcaacc tctgaattgt atggattagt tcaggaagtt ccacaaaaag agacaacgcc 7980

gttctatcct cgttcacctt atgccgtagc taaactgtat gcatattgga tcactgtaaa 8040

ctatcgtgaa tcttatggaa tgtatgcatg taacggtatt ttgttcaatc atgaatcccc 8100

acgtcgtggg gaaacgtttg taacacgtaa aatcacacgt gctatagcta atatttcgca 8160

ggggatagaa aaatgcttat atcttggcaa catggattct ttacgtgatt gggggcatgc 8220

gaaagattat gttcgtatgc aatggatgat gcttcaacaa gaccagccag aagactttgt 8280

tattgccacc ggcaaacaaa tatctgtacg tgaatttgtc cgtatgtctg ctaaagaagt 8340

tggacttgaa cttgagtttt ccggtactgg tgttgatgag attgcatttg ttgtaaataa 8400

aacatcagat tgcgccgttg gtgttaatat tggtgatgtt attgttcgcg ttgaccctcg 8460

ttattttcga ccagcagaag ttgaaactct tcttggtgat ccaacaaaag caaaaaatgt 8520

cttgggttgg gagccagaga ttacagttga agaaatgtgt gcagaaatgg tggctagtga 8580

tttagaaaaa gctaaacagc atgcacttct taaaagccat ggttttgacg tggcagtctc 8640

tttggagcgt taaagtatga ccaagaagcg aatttttgtg gctggacatc gtgggatggt 8700

tggttctgct atttgtcgtc aattattagc acgtaatgat gttgagttaa ttgtcaaaac 8760

tcacaaagaa ctcgatttga cagttcaaaa ggatgttgaa tgtttttttg agcaagagag 8820

aatagatcag gtttaccttg cagcagcaaa agtaggagga attcatgcta ataacacttt 8880

tccagctgaa tttatttatc agaaccttct tattgaaagc aatattattc actcatctta 8940

caaagcgggc ataaagaaac tattattctt agggtcaagt tgtatctatc caaagtttgc 9000

agaacagcca atgaaagaat ctgcactttt gacgggaact cttgagcctac taatgagcc 9060

ttatgctatt tccaaaatag caggtataaa gctatgtgaa tcttataatc gccaatatgg 9120

atgtgattat cgcagtgtaa tgcctactaa cttatatggt atgaatgata attttcatcc 9180

caataactca catgtaattc cagcgcttat gcgacgtttt catgaagcaa aggaacttgg 9240

tttaaatgaa gtggttgtct ggggaacggg aactccaaaa cgcgagtttt tatatgttga 9300

tgatatggca gctgcatcag tatatgtgat ggagcttgat gatgaaattt ataaaaaaaa 9360

tactcaacca atgttatctc atattaacgt aggtactggt gttgattgtt ccatacgtga 9420

aatggcagaa acaatggctt tagttgttgg ctatgatgga aaaattgttt ttgatataac 9480

aaaaccagat ggttcgcctc gtaaacttat ggatgttaca cgccttgaga atttgggttg 9540

gaaatatcgt tataatttaa aacaaggact agaattaaca tataaatggt ttataaataa 9600

tcttgactca tttaggagtt aatattgaac aagagattag aaacagagtt atttaaatca 9660

atagttgaac atactccact aatctcgata gaccttatta taagaaacga agatggtaag 9720

gcgctgcttg gtcagcgcct taatcgtcca gcacaaaatt attggtttgt gccgggggga 9780

cggattctta aagatgagtc tttcgagaac gcattcaaaa gagttacttt tgaagaattg 9840

ggtgttcaaa ttagcattaa tgaagcaaaa tttcttggga tttatgaaca tttctacagc 9900

gacaattttt ctgggacttg tttttcaact cattatgtcg tacacggata tgagattagt 9960

ttgatgccac atcaaattaa ctatccaaca ctacaacata gtacttacaa ttggttcgat 10020

atagccgaat tgttagctaa ttcctcagtt catcagtaca caaaaaatta ttttaagtga 10080

aggttattta tatgctactt cctgttgtaa tggccggtgg ttctggaact agattgtggc 10140

cactttctcg aacactatat cctaagcaat tcctatcgtt aactagtcgt ttgactatgc 10200

tacaagagac cttaagaagg cttgaaggag ttgaacatcg tcctgctctg gttatttgta 10260

atgaagttca tcgttttatc gttgcagagc agatgcgtaa tgtaaattta gcgaatagtg 10320

gcattttact agagccgata ggtcgtaata cagcccccgc agttgcgctt gctgctctta 10380

aagcaatttc atcaggtgag gatccaatac tgcttgtact agcagccgat catgaaatac 10440

aagaccaaaa gcgctttata tcgtcaatat tggctgcaaa agaatttgca gaggaaggta 10500

agctggtaac ttttggtatt gtgccaacca aacccgaaac tggatatggc tacataaaaa 10560

ccggagaaaa tttaaatgaa tatggtttta aagtttctgc ttttgttgaa aagcctgagc 10620

tagatgtggc caaaaaatat cttgaagatg gagattatct ttggaatagt ggaatattca 10680

tgtttagggc ttctgtgttt attgatgagt tgaataaatt tagaccagac atactaaaaa 10740

tatgccaaca agcattgaag tcctctacac aagatcttga ttttatccgt atagataatg 10800

actcatttag ttgttgccct gaagaatcca ttgattacgc tgttatggag aaaacaacag 10860

aggctgtggt agttccatta aatgcccact ggagcgatgt tggttcttgg tctgcattat 10920

gggaaattag cagaaaagat aaaagtggaa atgcaattag aggagatgta ttaattcatg 10980

attcttctga cagttatctt tactcacaac atagattaat tggtgtagta ggagttaaag 11040

atttagtagt tgttgaaact aaagatgcta ttctcgttgc gcataaagat aaagttcaac 11100

aggtaaaaaa tattgtagaa aaacttaaag ttaataaccg tacggaatat ctgcagcatc 11160

gtgaaatatt taggccatgg ggcagtcatg actctatagc ggaaggatct cgatttgagg 11220

ttaaacatgt cgttattcat cctggacata aaactgctaa acagattcat taccatcgta 11280

ctgagcattg gattgttgtt tctggaacag caaaagtcca ttatgaagat gaaatatttc 11340

ttgtttctga aaatgaatca acttatatac caataggtgt acctcatttt attgagaatc 11400

caggaaagat ccctttggag attattgagg tacgttcagg tgtctacttg gatgaagatg 11460

atgttgttag aatctaaaat gatgctggat actaatgcaa cattattatt cgtaatgctg 11520

tactgacgtt atttaattat gattattatt ggatgttaaa atgaaagtca gtattattac 11580

agtgacttat aatagtgaaa agactttaag ggatacttta gagtcaatag agttgcaaac 11640

ctataaagat atagagtata taattattga tggtggttct acagataata cgttaaaact 11700

aattaacgaa gtatcaacaa gagtaaccaa atgtctatca gaaagcgata acggtatcta 11760

tgatgcttta aataaaggta taaatttatc tacgggagat attattggat tcgttcattc 11820

tgatgacctt ttagcaagac ctgatattat tgaaactatt gtaagccgtt tccatgagac 11880

aaaagcggat gttgtatacg gtgatttggt ttttttcgaa aaaaaccaaa ttgatataat 11940

caaacgatac tggcgtagtg gtccttttaa acgttctaag ctatcattag gctgggcacc 12000

accgcatcca tctttctata tgaggaggga attatataaa gacgatggat attttgattt 12060

atcctatagg attgctgcag actatgatca gatggtcagg attttgaaac gtgatgatat 12120

taaagttatt tatgttcctc aagtatttgt aaagatgaga ttgggtgggg aaagtaccag 12180

aattgataac gcgatatcaa gtacaaagga aattgtagaa gttatgaaaa accataatgt 12240

aaattggaaa attgctatca ttatcagaaa aatctcaaaa ctaatgcaat tgtttgcgca 12300

taaataattt tattatcggt aaggttttta tatgaaaaaa ttaacctgct ttaaagccta 12360

tgatattcgc gggaaattag gcgaagaact gaatgaagat attgcgtggc gcattggtcg 12420

cgcctatggc gaatttctca aaccgaaaac cattgtgtta ggcggtgatg tccgcctcac 12480

cagcgaaacc ttaaaactgg cgctggcgaa aggtttacag gatgcgggcg tcgatgtgct 12540

ggatattggc atgtccggca ccgaagagat ctatttcgcc acgttccatc tcggcgtgga 12600

tggcggcatc gaagttaccg ccagccataa cccgatggat tacaacggca tgaaactcgt 12660

gcgcgaaggg gctcgcccga tcagcggtga taccggactg cgcgatgtcc agcgcctggc 12720

agaagccaac gactttcctc ccgtcgatga aaccaaacgc ggtcgctatc agcaaatcaa 12780

tctgcgtgac gcttacgttg atcacctgtt cggttatatc aacgtcaaaa acctcacgcc 12840

gctcaagctg gtgattaact ccgggaacgg cgcagcgggt ccggtggtgg acgccattga 12900

agcccgattt aaagccctcg gcgcaccagt ggaattcatc aaagtgcaca atacgccgga 12960

cggcaatttc cccaacggta ttcctaaccc gctgctgccg gaatgccgtg acgacacccg 13020

taatgcggt catcaaacacg gcgcggatat gggcattgcc tttgatggcg attttgaccg 13080

ctgtttcctg tttgacgaaa aagggcagtt tatcgagggc tactacattg tcggcctgct 13140

ggcagaagca ttcctcgaaa aaaatcccgg cgcgaagatc atccacgatc cgcgtctctc 13200

ctggaatacc gttgatgtgg tgactgctgc tggcggcact ccggtgatgt cgaaaaccgg 13260

acacgccttt attaaagaac gtatgcgcaa ggaagacgcc atctacggtg gcgaaatgag 13320

cgcccaccat tacttccgtg atttcgctta ctgcgacagc ggcatgatcc cgtggctgct 13380

ggtagccgaa ctggtgtgtc tgaaaggaaa aacgctgggc gaactggtgc gcgaccggat 13440

ggcggcgttt ccggcaagcg gtgagatcaa cagcaaactg gcgcaccccg ttgaggcgat 13500

taaccgcgtc gaacagcact ttacccgtgg ggcgctggcg gtggatcgca ccgatggcat 13560

cagcatgacc tttgccgatt ggcgctttaa cctgcgcacc tccaacaccg aaccggtggt 13620

gcggttgaat gtggaatctc acggtgatgt gccgctgatg gaagaaaaga caaaacttat 13680

ccttgagtta ctgaacaagt aattcagtaa tttcatataa ataagtttta aatgacggaa 13740

aaggtgagac attcagtgtg gtatagcaaa ggtaatgcta ttcaccatct ctatgagtga 13800

gttaacatct ataccacatt taagccgcac actcggcggt gaccaccccc tgacaggagt 13860

aaacaatgtc aaagcaacag atcggcgtag tcggtatggc agtgatggga cgcaaccttg 13920

cgctcaacat cgaaagccgt ggttataccg tctctatttt caaccgttcc cgtgaaaaga 13980

cggaagaagt gattgccgaa aatccaggca agaagctggt tccttactat acggtgaaag 14040

agtttgttga atctctggaa acgcctcgtc gcatcctgtt aatggtgaaa gcaggtgcag 14100

gcacggatgc tgctattgat tccctcaaac catatctcga taaaggtgac atcatcattg 14160

atggtggtaa caccttcttc 14180

Oligosaccharide unit treatment gene in the O antigen gene of table 1 intestinal bacteria O11 type bunch and primer and PCR data wherein

* only in intestinal bacteria O11 type, obtain a correct band

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	The transhipment enzyme	1138-2373	1573-1588	1951-1967	395bp	0*	48
			1227-1243	1888-1903	677bp	0*	52
								wzy	Polysaccharase	3104-4339	3154-3169	3998-4013	860bp	0*	52
			3529-3544	4034-4051	523bp	0*	52

Table 2 166 strain intestinal bacteria and 43 strain Shigellaes and their source

The bacterium source that contains in this group of group number

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

O19ab，O20，O21，O22，O23，O24

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

O36，O37，O38，O40，O41，O42，O43

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

O57，O58，O60，O61，O62，O53

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

O79，O80，O81，O82，O83，O68

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

O101，O102，O103，O104，O105，O106，O97

6, wild-type e. coli O107, O108, O109, O110, O111, O11 2ab, O112ac, O113, IMVS ^a

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

7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O136, O137, IMVS ^a

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

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

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

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

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

10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d

B16，B17，B18

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

DS，DR

12, wild-type e. coli O3, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVS ^a

O124，O167，O162，O121，O127，O149，O119

13, the 12nd group of bacterial strain adds intestinal bacteria reference culture O11 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 O11 type O antigen gene structure

E.coli O11 O-antigern cluster

#orfgalF wzx orf2 wzy orf4 gne orf6 gmd fcl gmm manC orf1l manB gnd

G+G％ 30.0 28.9 26.2 31.2 35.7 32.5 38.8 35.3 35.3 37.3 32.3 54.5

content

Table 4 intestinal bacteria O11 type O antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAACAGCG CGTGAAGCGT 120

CAACTGCTGG CGGAAGTACA ATCTATCTGT CCGCCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGTGAAC CTTTAGGTTT GGGCCACTCC ATTTTGTGTG CACGCCCCGT CATTGGCGAC 240

AACCCATTTG TCGTGGTGCT GCCGGACGTT GTGATCGACG ACGCCAGCGC CGACCCGTTG 300

CGTTACAACC TTGCTGCCAT GATTGCGCGC TTCAATGAAA CGGGCCGTAG CCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTCA TCCAGACCAA AGAACCGCTG 420

GATCGTGAAG GTAAAGTCAG CCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGCTGGACT CAGACATCAT GGCCGTTGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACTCAACC TGGTGCATGG GGGCGTATTC AGCTGACTGA TGCTATTGCT 600

GAGCTGGCAA AAAAACAATC CGTTGATGCA ATGCTGATGA CTGGCGACAG TTACGACTGC 660

GGTAAAAAAA TGGGCTATAT GCAGGCGTTT GTGAAGTATG GACTACGCAA CTTGAAAGAA 720

GGGGCGAAGT TCCGCAAAGG TATTGAGAAG CTGTTAATCG AATAATGAAA ATCTGACCGA 780

ATGTAACGGG TGATAAGAAA ATTATAACGG CGGTGAAGAT TGGTGGGGCT AATTGTACTC 840

ATTGCCTCAT TCTTTCTTTA TACCGTTCTA AATATGAGTA ACAATAACTT TTCAATAGGC 900

ATTTATTGTG GGAGGCTGAG ATTTTCTCCA TCTCTGAAGC GATTTGGTAA AACGTTCAGA 960

GTTAAGCTTT TCGAACTAAC CATAACGGTT AATGCTAGTT ACAATTAGGA CATGCATGCA 1020

GTACGCTGGT AGCTGTTGAG CCAGGGGCGG TAGCGTGAAT TAAATGTTTA TCCCATATTA 1080

Orf1's is initial

CTTAACAAAT TGTAAACTAT CGTGCAAAAT AAGACTTGTT AAGTACGTAG GCTCGTT ATG 1140

TTAAAAAACA TCATCTATTT ATTAAGCGTT CAAGGTGGCA ATTATATATT TCCATTGGTC 1200

ACACTGCCTT ATCTTGTCAG GATCCTTGAA CCTACAGGAT ACGGGATATA TGGGTATAGT 1260

TTTGCTGTGG TCCAATATTT TATATTATTT ATTGATTACG GATTTAACTA CTCAGCGCCC 1320

AAAATCATAT CAATATCAAG GGAAAATACT GATTCCATAA GTAAAGTTTT TTGGAATGTA 1380

ACGTTTATCA AAATAATTGC AGCATCTCTT GGTTTGTTAA TTATTTATTT AATTTCGGAA 1440

TTGAATATCA TTGATTACCA AATTAAATTT ATCATTCTTG CATACATATC TGTCGTAGGA 1500

AATTTAATAT ATCCAGTATG GTTTTTTCAA GGGCTGGAAA AAATGTTAGG AATAGCGATA 1560

AGTATGTTTT CCGCACGCTT ACTTACACTA TGTTTAACAT TTTTATTGGT TAAAAGTGAA 1620

AAGGATCTTG GTATAGCGAT AACAATACAA TCATCAATAA CAGTTATTGC AGGTATAATA 1680

TCTACATATT TTTTATTTTC TCTTAAAAAA ATAATATGGG TACCACCATC CTTATCCAAA 1740

ATGAAAGAGT TAATTTATGA TGGATGGCAT TACTTCATTT CCTCTGCAGC AATAAGCCTT 1800

TATACTACTA GTTCAACAAT AATATTAGGT GCATTATCAG GAACAACTGC AGTGGGATAT 1860

TTTATCGCTG CAGATAAAAT TAGGCTTGCA CTACAAGGCG TTATTGGTCC TATAACCCAA 1920

GCTGTTTTTC CTAGAGTAAC TTATATAATG AAGGATAATC ATGACGATGG ACTATTATTA 1980

GCCAGGAAAG TGTTTCGATG GCAATTTTCA CTGATGTTAG TACTTTCTAT ATTTCTTTAC 2040

TTTTTTGCAG ACGATATAAT TTTAATCATG TATGGAAAAA CGTATCATGA ATCTTTAGAA 2100

ATACTCAAGG TTTTAGCTTT TACTCCAGCA ATAGTTACGG TAAGTAATTA TATTGCAATT 2160

TTAGTAATGA TCCCTATTGG TATGAAAAAA GAGTTTACAA AAATTATTAC TACCAGTGGA 2220

TTAATAGGCC TACTGATAAT TACTTATTTG ACTAATGAAT ACTCAGGAGT TGGTACAGCA 2280

ATAGGAGTTA TTTCAACTGA GTTTATAATA ATGGCGAGAT TTTACTTTTT GATTAAGAAA 2340

The termination orf2's of Orf1 is initial

ACAAATAAAT TAAATAATTT AAATTTATTG TAGGGCTAAC A ATGAATAAA CCAACTTTAA 2400

CAATAGTTAC CGTTGTGTAC AATGGCGAAA TGTATATCGA AGATACTATA AAAAGTGTCT 2460

TATCACAAAA CTGCAATGAT GACATTGAAT ATATTATAAT TGATGGTGCA TCAACGGATA 2520

ATACTCTAAA TATTGTTAAT TCTTACAAAA ATGAAATTTC ATATATCATT TCTGAAAATG 2580

ATAATGGTAT ATATGATGCA ATGAATAAAG GTGTGTTAAA AGCATCTGGA AATTATATAG 2640

GATTTATAAA CGCTGATGAT TTTTATAATA AAGATGTATT ATGTCAAGTA ATAAAAGTAA 2700

TGAAAGAAAA TAAATATGAT GTTATTTATG GAAATATGTC TTTGGTAGAG GCTGATAGCT 2760

TGGATGTTAT TCAGGTCCGG AGGCCGAAAG ACTGGAAACT ACATATAGAT ATGAATTTAA 2820

GCCATCCGGC CACATTTATT AAAAAAGATA TCCACAGAGA AAATTTATTT AATACTAGCT 2880

ATAAAATTGC TTCTGATTAT GATCTTTTGC TAAAATTGAA GAAACAAGGT TATATCTTTC 2940

ATTATATTGA TAAAAATATC TCTTCGATGA GAAATGCCGG CCTTAGCGCT AAAAATGTAA 3000

AATTAGCATT GGAGGAAGCG AGACAAATCC GAAAGAAAAG AAACGGGAAA TTATATGCGG 3060

The termination of the initial Orf2 of orf3

CGTTAGCAGA TATTTACCTA TTTATGAGAA AAGTTATAAA AAG ATGATTA ATAGAAACAT 3120

AAAAATTAGT GCTTCATTTA TTTCAATAAT TTATTTGCCT CTTGCTTTAT TATTAGCAAT 3180

GATTGAAATG ATTAATGGGG CCAGATGGAG TGTTTATGTA TGTTCATTTA TGCTAGGGGT 3240

ACTAGCATTT TTGTTAATAC CTTATGAAAA TTGGGATTTG ACTAGGCATT ATGAAACATA 3300

TTTATATGTT AGTCAAACAT CACTAAACAA AGTTTTTGAA AATAGTGACA ATAGATATAT 3360

TACAATAAAT ACTGTTATTT ATTTATTTAA ATCTTTAGGA ATAAAAAAAG AATTTTTACC 3420

TTTTTTTTCA ACATTTTTAT CATATATTTT TTATTTTAAT ATATATGTTA ATTACATTAA 3480

AAAATACAAT CCGGAATTAT CTCATAGATG TATATATTTA TATATTCTAA TAAGCGTAAT 3540

CCCATTTTTT GCTATAGCTA GTTCATTGCG GCAATATCTT GCATTTAGTA TTATGCTTTA 3600

TATGATTGTA CAGTATTTCA CCAGTGAAAA TAATAGGTAT AAGTCACAAA AAATTATATT 3660

TTCTGCAATT ATTTCATGTT GTATTCATCC CAGCGCAATA CTATTGCTAG GTGTTTTTTT 3720

TGTCAGCAAA TTAATAAGAC TTAACAAAAC AATATTGTAT TTAATAATAT TAGTTCTAAT 3780

CCTTAACCAT GATGGATTAA TATCTTCTTT GCTATTTCAA TTGATTAGGC CATTATTGGA 3840

AAGCTTAGGT TTTTACTTTC CCGAATATAT GGACTCTGTA ACCATAGGCA TAAGCAATAG 3900

CTTGCTGAGC ACTAATGAAT ATATCTTGAA TAAGATCATA TTACCGTCAG TATTTTTTAT 3960

TTTTACTTTT ATCTATTTAA TTTTATTTAA TAAAAAAATG ACGAGGCAAG AGTCACAAAT 4020

CAAAAATTTT TGCAGTTTAC TTCTTTTGCT GATATGCCTT TTATCGTTAT CGCCTGATTT 4080

ATTTTATCGA TTTTCCATTT TCTGGGTGTT ATTATATAGT TATATATTTT TCTCCTACGA 4140

TATTAATAAA ATTAGCAAAC TATGTAAGCA TCTGATCTTC TTAATACTCA TTGCCGCATG 4200

TTCAATCATA AATTTGGCAC AGGCAAATAT GGGTAAAGAT ATTATTTATG TGTCTTGGTG 4260

CGATATATTG TATAAGCCCT CATTATTCCT ATTCAATAAA GAAGTTAAAT CAGCAGAATA 4320

The termination orf4's of orf3 is initial

TATAAGAGTG AGTCAA TAAA ATGTCCATTG GAGTCTTTAA GCTGTATAAA AATAAAATAG 4380

GTGGCACTGA GAAAGCAGTT GAAAATCTAA TTGATATATT AACTAAAAAC AGCCTACAAG 4440

TGAGATGTTT TTCAGCAATA AACGCACAAG AGATACAAGT TAGAGAAGAT AATCGGAAAA 4500

ACTACTTATT CCAAGACAAT ACTGCATATG AAGAAAGCAA ATTAAGGTTT TACATTAAAT 4560

ATTTGTTTTT TTTGAGTGCC AAAAGTAAAG ACCTTGAATT AATAATTTCC ACGGATATAG 4620

CTACATCATC ATTATTGTTA TTGATTGGTT TTTTTCGGTA TAATAAGAAT AAAATTATTT 4680

GCTGGGAACA TTATCCATTT GACAAGAATA GTATTTTTTG GAGAAAAGTA TTTAAATTTC 4740

TTTTGTCTCT TAATAAAAAA GCTGTAGTAG TTTGTAATTC TCACAAGGAA AAAGATAAGT 4800

TTAGTTTTTG TAAAAGGGTT GTAATTATAC CAAATTCAAT CAAAAAGCAA AAATATAAGC 4860

GCATACATCA AGGAAAAGAA ACTCTTGATT TGATTTATAT TGGTCGGATT GTTAAAGATA 4920

AAGGAGTTCA AAGGTTAATT AATGCAGTAA TAAATCACCC GTCTACAAAA AAGTATGTTT 4980

TACACATTGT AGGAGGTGGT CAAGAGCTGG AGTATCTCAA ATCTAAATAT AAGAATAACA 5040

ACCAAATACA CTTTCATGGG CCGAGTTTAA ACACAGATGT TTTTTTGGCA AAAAGTGATG 5100

TTTTTGTATC CGGTTCATAC TTTGAATGTT TTCCTGTTAC AATACTTGAA GCTATGTCCT 5160

ACGGATTACC TGTAATTTCA ATGGATATAT CTGGAGGGAC AAGTAGCGTG ATTTCTTCAT 5220

CGGGTGGCGG ATTTGTGTGT CAATCTCAAA TTGAATTTCA GGAAAAACTA ACTTACTTTG 5280

AAGATAATGT TAAACGAACC GAACACGGAA ATGCAGGATT CCTATTCGTG AATAGACATT 5340

ATTCAGAAGA AATAGTTAGT GCTCAGTGGA TGAGTTTGAT CAATCGAGCC AATAGTCAAG 5400

The termination orf5's of Orf4 is initial

AAAAT TAAGG TATTGGAGCT ATTAAAGT AT GAGCATACTT GTGACTGGTG GGGCTGGCTA 5460

TATTGGCTCA CATGCTATAT TAACTTTATT GCAAAATGGA TATGATGTTA TATCGCTAGA 5520

TAATTATTGC AATTCCTCTC CAAAATCTCT AGAACGAGTA GAGGAAATTT GCTTATCTAA 5580

GATAAAAAAA TTACGTGGTG ATATTAGAGA TAGACATATA TTAAAAAATA TCTTTTCAAA 5640

ATATAATGTT TCTACTGTAA TACATTTTGC GGGTTTAAAA TCTGTTAACG AGTCCATTAA 5700

AAAACCATTA GAATATTACG ACACAAATCT TTTGGGGACT CTAATATTAC TTGATGAAAT 5760

GCAGAAAGCG AATATTTCTA ATCTAATTTT TAGTTCGTCT GCTACTGTCT ATGGCAATCC 5820

TCATTATGTT CCAATATCTG AAAAACACCC TGTCGGTGAG GTGATCAATC CGTACGGTCG 5880

TTCAAAGTTT ATGGTGGAAC ATATATTGCG TGATTTTTGT AACGCGAATA GCGACGCAAA 5940

TATCACAATA CTAAGATATT TTAATCCTGT AGGTGCTCAT GAGACTGGAT TAATTGGAGA 6000

AGATCCTTTA GGAACTCCTA ATAACCTGTT TCCCTATGTT GCGCAAGTCG CAATTGGAAA 6060

ATTACCCTAT CTAAATGTTT TTGGTAATAA ATATATGACA AAAGATGGAA CAGGTGTAAG 6120

AGATTATATC CATGTAGTCG ATTTAGTTGA GGGGCATCTT GCCGCTTTAA AAAATCTCAA 6180

TAGAAACCGG GGGCTTAAAG TTTTTAATTT AGGCACTGGT CAAGGTTATA GTGTGTTAGA 6240

TATCATCGAC GCTTTTAAAC TGGAAACAGG ACGCGATATA CCTTTCCGTA TCGTTTCTGA 6300

GCGAAATGGA GATGTGGCTG AATGTTGGTC CGATCCTTCC TTAGCAAACG AGCAGCTAAG 6360

TTGGGTTGCT AAAAGGACTC TTAATAATAT GGTGGAAGAT GCATGGAGAT GGCAAACAAT 6420

The termination of Orf5

GAATCCAAAT GGATATAAAG AG TAAATTGA AACAATTATA ATCATCCATT TCATTGCTTA 6480

Orf6's is initial

CTGTATTGTT ATCTGTATAT ATGAAAACAA AAA ATGAAAA TATTATATGA TGGGATTGTG 6540

AAAAGTTTAC AATCATATGG TGGTATCACT GTATACTTTG AAGAAATCAT TAAGAGATTA 6600

AAAAATGATG AATACAAATT CATTCAATTT AATGATAAAA ATGAAAATCA CAATGATGAT 6660

CTCCATCGGC CATGCCGGTT TTTAGAACGA TATAGATGTT TTGAAGCATT AAAAAGTGAT 6720

TCAGATTACA GTATATTTCA TTCTACATAT TATCGGGTTC CGAAAGATGG GGAATATGCG 6780

GTTGTGACCA CGGTCCACGA TTTCACATAT GAATTGTATA TGAAAGGACT TCGGCGCAAT 6840

GTTCATAGCT TACAAAAAAG ATGGGCTGTG AAAAATAGCG ATGCAATCAT TTGTGTCTCT 6900

AATAATACAG CCAAGGATTT AATTAATTTC TTTAAGGTTG ATGAAAAAAA GATACATGTT 6960

ATTCATAATG GTGTATCGGA AGATTATAGA CACTTAAATC TAGAAAAAGA AAATTTTGTG 7020

CTTTTTGTCG GTGCTCGTTC AGGATATAAA AATTTTGAGC TTGCAGTTAA AGCAGTTTCT 7080

AAACTTTTTG ATACTAATCT TAAAATAGTC GGCGGAGGTG GTTTAAATAA AGCAGAAATG 7140

AAGTTACTAT CTGAATATCT TCCAGAAAGA TTCCAGTACT TAGGGAAGGT ATCAAATGAA 7200

AAACTAAATA TATTATATAA TTCGGCTTAT TGCTTAATCT ATCCTTCAGA ATATGAAGGG 7260

TTTGGAATTC CCTTGCTTGA GGCTATGCGA ACTGGCTGTC CTGTGATAGC CGTAAATAGA 7320

TCATCAATAC CCGAGGTTGT GGGGGATAGT GCGATTTTAG TCGAAGAAGT ATCAATTGAA 7380

AGTTTATTCT CAGCTTTATT AAGTGTAGAA GCTAACGCTA ATGAATTAAA AAATAAAGGA 7440

TTAGCGCAAT CTAATAAACT TTCATGGGGG AAGTGTTTCT CCGAAACATA TAATCTATAT 7500

The termination orf7's of Orf6 is initial

AAATCACTAT CTAAGTTT TG AGAGGTTATT T ATGTGTAAG AAAGCTTTAA TTACAGGAAT 7560

TACCGGGCAA GATGGTTCCT ACTTGGCAGA ATTTCTGCTT AAAAAAGGCT ATGAGGTTCA 7620

TGGAATAAAA CGTCGATCTT CCTTGTTTAA TACTGACCGT ATTGATCATA TATATCAAGA 7680

CCCACACCAT CGTAATTTAA AATTACAACT TCATTATGGT GATTTAACAG ATACTTCTAA 7740

TTTGACTCGG ATATTATCTG AAGTTCAACC TGATGAAGTT TATAATCTTG GCGCAATGAG 7800

TCACGTAGCT GTTTCCTTCG AATCCCCAGA ATATACAGCT GATGTTGATG CTATAGGTAC 7860

TTTACGATTA TTGGAAGCTA TTCGTTTTTT GGGATTAGAA AAAAAAACCC GATTTTATCA 7920

GGCGTCAACC TCTGAATTGT ATGGATTAGT TCAGGAAGTT CCACAAAAAG AGACAACGCC 7980

GTTCTATCCT CGTTCACCTT ATGCCGTAGC TAAACTGTAT GCATATTGGA TCACTGTAAA 8040

CTATCGTGAA TCTTATGGAA TGTATGCATG TAACGGTATT TTGTTCAATC ATGAATCCCC 8100

ACGTCGTGGG GAAACGTTTG TAACACGTAA AATCACACGT GCTATAGCTA ATATTTCGCA 8160

GGGGATAGAA AAATGCTTAT ATCTTGGCAA CATGGATTCT TTACGTGATT GGGGGCATGC 8220

GAAAGATTAT GTTCGTATGC AATGGATGAT GCTTCAACAA GACCAGCCAG AAGACTTTGT 8280

TATTGCCACC GGCAAACAAA TATCTGTACG TGAATTTGTC CGTATGTCTG CTAAAGAAGT 8340

TGGACTTGAA CTTGAGTTTT CCGGTACTGG TGTTGATGAG ATTGCATTTG TTGTAAATAA 8400

AACATCAGAT TGCGCCGTTG GTGTTAATAT TGGTGATGTT ATTGTTCGCG TTGACCCTCG 8460

TTATTTTCGA CCAGCAGAAG TTGAAACTCT TCTTGGTGAT CCAACAAAAG CAAAAAATGT 8520

CTTGGGTTGG GAGCCAGAGA TTACAGTTGA AGAAATGTGT GCAGAAATGG TGGCTAGTGA 8580

TTTAGAAAAA GCTAAACAGC ATGCACTTCT TAAAAGCCAT GGTTTTGACG TGGCAGTCTC 8640

The termination orf8's of Orf7 is initial

TTTGGAGCGT TAAAGT ATGA CCAAGAAGCG AATTTTTGTG GCTGGACATC GTGGGATGGT 8700

TGGTTCTGCT ATTTGTCGTC AATTATTAGC ACGTAATGAT GTTGAGTTAA TTGTCAAAAC 8760

TCACAAAGAA CTCGATTTGA CAGTTCAAAA GGATGTTGAA TGTTTTTTTG AGCAAGAGAG 8820

AATAGATCAG GTTTACCTTG CAGCAGCAAA AGTAGGAGGA ATTCATGCTA ATAACACTTT 8880

TCCAGCTGAA TTTATTTATC AGAACCTTCT TATTGAAAGC AATATTATTC ACTCATCTTA 8940

CAAAGCGGGC ATAAAGAAAC TATTATTCTT AGGGTCAAGT TGTATCTATC CAAAGTTTGC 9000

AGAACAGCCA ATGAAAGAAT CTGCACTTTT GACGGGAACT CTTGAGCCTA CTAATGAGCC 9060

TTATGCTATT TCCAAAATAG CAGGTATAAA GCTATGTGAA TCTTATAATC GCCAATATGG 9120

ATGTGATTAT CGCAGTGTAA TGCCTACTAA CTTATATGGT ATGAATGATA ATTTTCATCC 9180

CAATAACTCA CATGTAATTC CAGCGCTTAT GCGACGTTTT CATGAAGCAA AGGAACTTGG 9240

TTTAAATGAA GTGGTTGTCT GGGGAACGGG AACTCCAAAA CGCGAGTTTT TATATGTTGA 9300

TGATATGGCA GCTGCATCAG TATATGTGAT GGAGCTTGAT GATGAAATTT ATAAAAAAAA 9360

TACTCAACCA ATGTTATCTC ATATTAACGT AGGTACTGGT GTTGATTGTT CCATACGTGA 9420

AATGGCAGAA ACAATGGCTT TAGTTGTTGG CTATGATGGA AAAATTGTTT TTGATATAAC 9480

AAAACCAGAT GGTTCGCCTC GTAAACTTAT GGATGTTACA CGCCTTGAGA ATTTGGGTTG 9540

GAAATATCGT TATAATTTAA AACAAGGACT AGAATTAACA TATAAATGGT TTATAAATAA 9600

The termination orf9's of Orf8 is initial

TCTTGACTCA TTTAGGAGT T AATA TTGAAC AAGAGATTAG AAACAGAGTT ATTTAAATCA 9660

ATAGTTGAAC ATACTCCACT AATCTCGATA GACCTTATTA TAAGAAACGA AGATGGTAAG 9720

GCGCTGCTTG GTCAGCGCCT TAATCGTCCA GCACAAAATT ATTGGTTTGT GCCGGGGGGA 9780

CGGATTCTTA AAGATGAGTC TTTCGAGAAC GCATTCAAAA GAGTTACTTT TGAAGAATTG 9840

GGTGTTCAAA TTAGCATTAA TGAAGCAAAA TTTCTTGGGA TTTATGAACA TTTCTACAGC 9900

GACAATTTTT CTGGGACTTG TTTTTCAACT CATTATGTCG TACACGGATA TGAGATTAGT 9960

TTGATGCCAC ATCAAATTAA CTATCCAACA CTACAACATA GTACTTACAA TTGGTTCGAT 10020

The termination of Orf9

ATAGCCGAAT TGTTAGCTAA TTCCTCAGTT CATCAGTACA CAAAAAATTA TTTTAAG TGA 10080

Orf10's is initial

AGGTTATTTA T ATGCTACTT CCTGTTGTAA TGGCCGGTGG TTCTGGAACT AGATTGTGGC 10140

CACTTTCTCG AACACTATAT CCTAAGCAAT TCCTATCGTT AACTAGTCGT TTGACTATGC 10200

TACAAGAGAC CTTAAGAAGG CTTGAAGGAG TTGAACATCG TCCTGCTCTG GTTATTTGTA 10260

ATGAAGTTCA TCGTTTTATC GTTGCAGAGC AGATGCGTAA TGTAAATTTA GCGAATAGTG 10320

GCATTTTACT AGAGCCGATA GGTCGTAATA CAGCCCCCGC AGTTGCGCTT GCTGCTCTTA 10380

AAGCAATTTC ATCAGGTGAG GATCCAATAC TGCTTGTACT AGCAGCCGAT CATGAAATAC 10440

AAGACCAAAA GCGCTTTATA TCGTCAATAT TGGCTGCAAA AGAATTTGCA GAGGAAGGTA 10500

AGCTGGTAAC TTTTGGTATT GTGCCAACCA AACCCGAAAC TGGATATGGC TACATAAAAA 10560

CCGGAGAAAA TTTAAATGAA TATGGTTTTA AAGTTTCTGC TTTTGTTGAA AAGCCTGAGC 10620

TAGATGTGGC CAAAAAATAT CTTGAAGATG GAGATTATCT TTGGAATAGT GGAATATTCA 10680

TGTTTAGGGC TTCTGTGTTT ATTGATGAGT TGAATAAATT TAGACCAGAC ATACTAAAAA 10740

TATGCCAACA AGCATTGAAG TCCTCTACAC AAGATCTTGA TTTTATCCGT ATAGATAATG 10800

ACTCATTTAG TTGTTGCCCT GAAGAATCCA TTGATTACGC TGTTATGGAG AAAACAACAG 10860

AGGCTGTGGT AGTTCCATTA AATGCCCACT GGAGCGATGT TGGTTCTTGG TCTGCATTAT 10920

GGGAAATTAG CAGAAAAGAT AAAAGTGGAA ATGCAATTAG AGGAGATGTA TTAATTCATG 10980

ATTCTTCTGA CAGTTATCTT TACTCACAAC ATAGATTAAT TGGTGTAGTA GGAGTTAAAG 11040

ATTTAGTAGT TGTTGAAACT AAAGATGCTA TTCTCGTTGC GCATAAAGAT AAAGTTCAAC 11100

AGGTAAAAAA TATTGTAGAA AAACTTAAAG TTAATAACCG TACGGAATAT CTGCAGCATC 11160

GTGAAATATT TAGGCCATGG GGCAGTCATG ACTCTATAGC GGAAGGATCT CGATTTGAGG 11220

TTAAACATGT CGTTATTCAT CCTGGACATA AAACTGCTAA ACAGATTCAT TACCATCGTA 11280

CTGAGCATTG GATTGTTGTT TCTGGAACAG CAAA GTCCA TTATGAAGAT GAAATATTTC 11340

TTGTTTCTGA AAATGAATCA ACTTATATAC CAATAGGTGT ACCTCATTTT ATTGAGAATC 11400

CAGGAAAGAT CCCTTTGGAG ATTATTGAGG TACGTTCAGG TGTCTACTTG GATGAAGATG 11460

The termination of Orf10

ATGTTGTTAG AATC TAAAAT GATGCTGGAT ACTAATGCAA CATTATTATT CGTAATGCTG 11520

Orf11's is initial

TACTGACGTT ATTTAATTAT GATTATTATT GGATGTTAAA ATGAAAGTCA GTATTATTAC 11580

AGTGACTTAT AATAGTGAAA AGACTTTAAG GGATACTTTA GAGTCAATAG AGTTGCAAAC 11640

CTATAAAGAT ATAGAGTATA TAATTATTGA TGGTGGTTCT ACAGATAATA CGTTAAAACT 11700

AATTAACGAA GTATCAACAA GAGTAACCAA ATGTCTATCA GAAAGCGATA ACGGTATCTA 11760

TGATGCTTTA AATAAAGGTA TAAATTTATC TACGGGAGAT ATTATTGGAT TCGTTCATTC 11820

TGATGACCTT TTAGCAAGAC CTGATATTAT TGAAACTATT GTAAGCCGTT TCCATGAGAC 11880

AAAAGCGGAT GTTGTATACG GTGATTTGGT TTTTTTCGAA AAAAACCAAA TTGATATAAT 11940

CAAACGATAC TGGCGTAGTG GTCCTTTTAA ACGTTCTAAG CTATCATTAG GCTGGGCACC 12000

ACCGCATCCA TCTTTCTATA TGAGGAGGGA ATTATATAAA GACGATGGAT ATTTTGATTT 12060

ATCCTATAGG ATTGCTGCAG ACTATGATCA GATGGTCAGG ATTTTGAAAC GTGATGATAT 12120

TAAAGTTATT TATGTTCCTC AAGTATTTGT AAAGATGAGA TTGGGTGGGG AAAGTACCAG 12180

AATTGATAAC GCGATATCAA GTACAAAGGA AATTGTAGAA GTTATGAAAA ACCATAATGT 12240

AAATTGGAAA ATTGCTATCA TTATCAGAAA AATCTCAAAA CTAATGCAAT TGTTTGCGCA 12300

The termination orf12's of Orf11 is initial

TAAA TAATTT TATTATCGGT AAGGTTTTTA T ATGAAAAAA TTAACCTGCT TTAAAGCCTA 12360

TGATATTCGC GGGAAATTAG GCGAAGAACT GAATGAAGAT ATTGCGTGGC GCATTGGTCG 12420

CGCCTATGGC GAATTTCTCA AACCGAAAAC CATTGTGTTA GGCGGTGATG TCCGCCTCAC 12480

CAGCGAAACC TTAAAACTGG CGCTGGCGAA AGGTTTACAG GATGCGGGCG TCGATGTGCT 12540

GGATATTGGC ATGTCCGGCA CCGAAGAGAT CTATTTCGCC ACGTTCCATC TCGGCGTGGA 12600

TGGCGGCATC GAAGTTACCG CCAGCCATAA CCCGATGGAT TACAACGGCA TGAAACTCGT 12660

GCGCGAAGGG GCTCGCCCGA TCAGCGGTGA TACCGGACTG CGCGATGTCC AGCGCCTGGC 12720

AGAAGCCAAC GACTTTCCTC CCGTCGATGA AACCAAACGC GGTCGCTATC AGCAAATCAA 12780

TCTGCGTGAC GCTTACGTTG ATCACCTGTT CGGTTATATC AACGTCAAAA ACCTCACGCC 12840

GCTCAAGCTG GTGATTAACT CCGGGAACGG CGCAGCGGGT CCGGTGGTGG ACGCCATTGA 12900

AGCCCGATTT AAAGCCCTCG GCGCACCAGT GGAATTCATC AAAGTGCACA ATACGCCGGA 12960

CGGCAATTTC CCCAACGGTA TTCCTAACCC GCTGCTGCCG GAATGCCGTG ACGACACCCG 13020

TAATGCGGTC ATCAAACACG GCGCGGATAT GGGCATTGCC TTTGATGGCG ATTTTGACCG 13080

CTGTTTCCTG TTTGACGAAA AAGGGCAGTT TATCGAGGGC TACTACATTG TCGGCCTGCT 13140

GGCAGAAGCA TTCCTCGAAA AAAATCCCGG CGCGAAGATC ATCCACGATC CGCGTCTCTC 13200

CTGGAATACC GTTGATGTGG TGACTGCTGC TGGCGGCACT CCGGTGATGT CGAAAACCGG 13260

ACACGCCTTT ATTAAAGAAC GTATGCGCAA GGAAGACGCC ATCTACGGTG GCGAAATGAG 13320

CGCCCACCAT TACTTCCGTG ATTTCGCTTA CTGCGACAGC GGCATGATCC CGTGGCTGCT 13380

GGTAGCCGAA CTGGTGTGTC TGAAAGGAAA AACGCTGGGC GAACTGGTGC GCGACCGGAT 13440

GGCGGCGTTT CCGGCAAGCG GTGAGATCAA CAGCAAACTG GCGCACCCCG TTGAGGCGAT 13500

TAACCGCGTC GAACAGCACT TTACCCGTGG GGCGCTGGCG GTGGATCGCA CCGATGGCAT 13560

CAGCATGACC TTTGCCGATT GGCGCTTTAA CCTGCGCACC TCCAACACCG AACCGGTGGT 13620

GCGGTTGAAT GTGGAATCTC ACGGTGATGT GCCGCTGATG GAAGAAAAGA CAAAACTTAT 13680

The termination of Orf12

CCTTGAGTTA CTGAACAAG T AATTCAGTAA TTTCATATAA ATAAGTTTTA AATGACGGAA 13740

AAGGTGAGAC ATTCAGTGTG GTATAGCAAA GGTAATGCTA TTCACCATCT CTATGAGTGA 13800

GTTAACATCT ATACCACATT TAAGCCGCAC ACTCGGCGGT GACCACCCCC TGACAGGAGT 13860

AAACAATGTC AAAGCAACAG ATCGGCGTAG TCGGTATGGC AGTGATGGGA CGCAACCTTG 13920

CGCTCAACAT CGAAAGCCGT GGTTATACCG TCTCTATTTT CAACCGTTCC CGTGAAAAGA 13980

CGGAAGAAGT GATTGCCGAA AATCCAGGCA AGAAGCTGGT TCCTTACTAT ACGGTGAAAG 14040

AGTTTGTTGA ATCTCTGGAA ACGCCTCGTC GCATCCTGTT AATGGTGAAA GCAGGTGCAG 14100

GCACGGATGC TGCTATTGAT TCCCTCAAAC CATATCTCGA TAAAGGTGAC ATCATCATTG 14160

ATGGTGGTAA CACCTTCTTC 14180

The invention has the beneficial effects as follows: the special molecular marker (molecular probe) that the objective of the invention is to seek and use this bacterium, it is specific nucleotide sequence, therefore technical scheme of the present invention has uniqueness, the difference of itself and conventional study method is, search out and have specific nucleotide sequence, and guarantee the specificity of molecule marker by reliable experimental, can utilize and well known to a person skilled in the art mature technology (as PCR or gene chip etc.), use the technology of this marker bacterial detection, make all those skilled in the art can be, realize purpose of the present invention easily and obtain the result of use of expection according to technology contents provided by the invention.Already provided experimental data among the present invention, fully proved the specificity of this nucleotide sequence, and can be applied to the detection of this bacterium, use modern molecular biology method for determining bacteria of the present invention with respect to traditional serology immune response, have fast, accurately, advantage cheaply.The present invention order-checking is also inferred the function of each gene with information biology software, be in order to seek specificity Nucleotide, the gene that some specific functions are arranged in the bacterium is a high special, gene that utilizes above method to infer these specific functions and their position, utilize experiment to prove then, the function of these genes that will be inferred, remove to search out better faster specificity Nucleotide as a kind of road sign, like this, can reduce the blindness of research experiment, accelerate the progress of experiment, reduce experiment fees.

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 (3)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O11 type is characterized in that it is the Nucleotide that is selected from 1573 to 1588 bases among the SEQ ID NO:1; The Nucleotide of 1951 to 1967 bases among the SEQ IDNO:1; The Nucleotide of 1227 to 1243 bases among the SEQ ID NO:1; The Nucleotide of 1888 to 1903 bases among the SEQ ID NO:1; The Nucleotide of 3154 to 3169 bases among the SEQ IDNO:1; The Nucleotide of 3998 to 4013 bases among the SEQ ID NO:1; The Nucleotide of 3529 to 3544 bases among the SEQ ID NO:1; The Nucleotide of 4034 to 4051 bases among the SEQ IDNO:1.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O11 type is right, it is characterized in that it is to be selected from the Nucleotide of 1573 to 1588 bases among the SEQ ID NO:1 and the Nucleotide of 1951 to 1967 bases; The Nucleotide of 1227 to 1243 bases among the SEQ ID NO:1 and the Nucleotide of 1888 to 1903 bases; The Nucleotide of 3154 to 3169 bases among the SEQ ID NO:1 and the Nucleotide of 3998 to 4013 bases; The Nucleotide of 3529 to 3544 bases among the SEQ ID NO:1 and the Nucleotide of 4034 to 4051 bases.

3, the right application of the oligonucleotide of the oligonucleotide of claim 1 or claim 2 is characterized in that it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, manufacturing gene chip or microarray as probe, for detecting intestinal bacteria O11 type.