CN1249235C - Nucleotide specific against o-antigen of colibacillus 0150 - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O150. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O150 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 13552 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the oligonucleotide of glycosyltransferase genes and unit processing genes stemmed from the O-antigen gene clusters of Escherichia coli O150. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O150 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Escherichia coli O150 in livestock and environment.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O150 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O150 type (Escherichia coli O150), particularly relate in the intestinal bacteria O150 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide to detect the intestinal bacteria O150 type in livestock and the environment quickly and accurately and identify O-antigen in these pathogenic bacterium the O-antigen-specific.

Background technology

Intestinal bacteria O150 at first was detected [Furowicz A J in ill animal in 1972, Orskov F.Acta Pathol Microbiol Scand Microbiol Immunol, 1972,80 (3): 441-444.], be proved and cause the intestinal tract infections of livestocks such as ox, belong to enterotoxigenic E.Coli (Enterotoxigenic E.coli, ETEC), the explosive popular danger of its potential very big [Danbara H, Komase K, Arita H, et al.Infect Immun, 1988,56 (6): 1513-1517.], being badly in need of one on the agricultural can be quick, accurately detect the method for intestinal bacteria O150.

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O35gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, it is one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of will intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specificselection and bacterial populations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' s identification ofthe Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152and and Shigella O antigens " J.clinMicrobiol, 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 O150 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O150 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 O150 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O150 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 orf8, orf9, orf10 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 O150 type respectively comprises orf8, orf9, orf10 gene; The gene that coming from coding transhipment enzyme be the wzx gene or with wzx the gene of identity function, the gene that comes from the coding polysaccharase are arranged 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 O150 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O150 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O150 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect O-antigen and detection and identification of escherichia coli O150 type with identification of escherichia coli O150 type by these methods.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O150 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 to the Nucleotide of the O-antigen-specific of intestinal bacteria O150 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,13551 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type, it is by 11 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type, wherein said gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene wzy gene or the gene of identity function is arranged with wzy; Glycosyltransferase gene comprises orf8, orf9, orf10 gene; Wherein said gene: wzx is the Nucleotide of 5632 to 7134 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8307 to 9197 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9184 to 10098 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 10098 to 10994 bases among the SEQ IDNO:1; Wzy is the Nucleotide of 11012 to 12109 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type, wherein it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf8, orf9, orf10 gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 5706 to 5722 bases among the SEQ ID NO:1 and the Nucleotide of 6458 to 6475 bases; The Nucleotide of 5719 to 5736 bases among the SEQ ID NO:1 and the Nucleotide of 6681 to 6698 bases; The Nucleotide of 5978 to 5995 bases among the SEQ ID NO:1 and the Nucleotide of 6789 to 6806 bases; The oligonucleotide that comes from the orf8 gene is to being: the Nucleotide of 8681 to 8699 bases among the SEQ ID NO:1 and the Nucleotide of 9169 to 9185 bases; The Nucleotide of 8418 to 8435 bases among the SEQ ID NO:1 and the Nucleotide of 8995 to 9012 bases; The Nucleotide of 8333 to 8350 bases among the SEQ ID NO:1 and the Nucleotide of 9050 to 9067 bases; The oligonucleotide that comes from the orf9 gene is to being: the Nucleotide of 9397 to 9413 bases among the SEQ ID NO:1 and the Nucleotide of 9882 to 9899 bases; The Nucleotide of 9337 to 9354 bases among the SEQ ID NO:1 and the Nucleotide of 9899 to 9916 bases; The Nucleotide of 9489 to 9506 bases among the SEQ ID NO:1 and the Nucleotide of 10035 to 10051 bases; The oligonucleotide that comes from the orf10 gene is to being: the Nucleotide of 10302 to 10319 bases among the SEQ ID NO:1 and the Nucleotide of 10852 to 10869 bases; The Nucleotide of 10293 to 10310 bases among the SEQ ID NO:1 and the Nucleotide of 10954 to 10971 bases; The Nucleotide of 10377 to 10394 bases among the SEQ ID NO:1 and the Nucleotide of 10973 to 10989 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 11217 to 11234 bases among the SEQ IDNO:1 and the Nucleotide of 11985 to 12002 bases; The Nucleotide of 11225 to 11241 bases among the SEQ ID NO:1 and the Nucleotide of 12035 to 12052 bases; The Nucleotide of 11172 to 11189 bases among the SEQ ID NO:1 and the Nucleotide of 11960 to 11977 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type, and can provide the O-antigen of expressing intestinal bacteria O150 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type, wherein 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, detects the bacterium in livestock and the environment.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O150 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O150 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 that adds 3ul 20mg/ml afterwards, 15ul 10%SDS, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, 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 70% ethanol with glass yarn, 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 O150 type bunch: with the genome of intestinal bacteria O150 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT 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 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 30 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 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂The DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then add 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, use isopyknic ether extracting once again after, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, and be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares competence escherichia coli DH5a cell, get after 2-3ul connects product and 50ul competence escherichia coli DH5a mixes, forward in the electric shock cup of 0.2cm of Bi0-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 acillin 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 O150 type;

(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 90% fraction of coverage, residue 10% sequence is again by with the partial sequence backward sequencing, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O150 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O150 type is done 6 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 O150 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 11 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 O150 type at last;

(6) screening of specific gene: at wzx, orf8, orf9, orf10, the wzy gene design primer in the O-antigen gene of intestinal bacteria O 150 types bunch; Respectively designed three 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, the correct band of any size does not all increase in other groups, promptly do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PDR product band, but its size does not meet the expection size, so the O-antigen of wzx, orf8, orf9, orf10, wzy gene pairs intestinal bacteria O150 type all is high special.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O150 type, its complete sequence shown in SEQ ID NO:1,9227 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 O150 type by method of the present invention, as shown in table 3, it is altogether by 11 genomic constitutions, 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 O150 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 comprises orf8, orf9, orf10 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises the gne 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 are high specials to the O-antigen of intestinal bacteria O150 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O150 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, comprise orf8, orf9, the oligonucleotide of orf10 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 being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O150 type only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to the O-antigen of intestinal bacteria O150 type.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O150 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O150 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.

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 is meant the gene that derives from the encoding glycosyl transferring enzyme in the O-antigen gene bunch, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase, they can change on length, generally change in 10 to 20 Nucleotide scopes.More precisely these oligonucleotide are to come from wzx gene (nucleotide position is 5632 to 7134 bases from SEQ ID NO:1), orf8 gene (nucleotide position is 8307 to 9197 bases from SEQ ID NO:1), orf9 gene (nucleotide position is 9184 to 10098 bases from SEQ ID NO:1), orf10 gene (nucleotide position is 10098 to 10994 bases from SEQ ID NO:1), wzy gene (nucleotide position is 11012 to 12109 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide is high special to intestinal bacteria O150 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 O150 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 O150 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 O150 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 ill domestic animal.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 O150 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 O150 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 O150 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 O150 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 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, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O150 type bunch:

With the genome of intestinal bacteria O150 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (#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 CCTGGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long TemplatePCR 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 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul100mM 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 3 of DNA ' end add the dA tail; This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares competent cell bacillus coli DH 5.Get the single bacterium colony of a ring bacillus coli DH 5 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 about OD6000.5, 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 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 O150 type with the EcoRI enzyme.

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

From the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 90% fraction of coverage.Residue 10% sequence is again by with the partial sequence backward sequencing, thereby obtains all sequences of 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 O150 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O150 type is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O150 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 11 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 O150 type at last, as shown in table 3.

By retrieving and comparing, find that the orf6 encoded protein contains 14 potential transmembrane segments, algorithm [Eisenberg by Eisenberg etc., D, Schwarz, E.etal (1984) " Analysis of membraneand surface protein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that also Orf6 has 14 potential transmembrane domains; and with many Wzx protein similars; for example; and the Wzx of hot-bulb bacterium has 21% homogeny in 419 amino acid; 41% similarity is the wzx gene so can determine orf6, called after wzx.Orf8 and the 2 similar (pfam00535 of glycosyltransferase family, 3e-07), carry out blast relatively, with the glycosyltransferase of clostridium acetobutylicum 27% homogeny is arranged, 51% similarity, the homology that height is arranged between them is described, can infers that orf8 also is glycosyltransferase gene, called after orf8 temporarily.The rfbF gene of orf9 and shigella flexneri 2a has 47% homogeny in 285 amino acid, 63% similarity, the glycosyltransferase gene of rfbF genes encoding dTDP-rhamnosyl is so infer that orf9 is a glycosyltransferase gene, temporary called after orf9.The rfbG gene of orf10 and shigella flexneri 2a has 48% homogeny in 289 amino acid, 67% similarity, the glycosyltransferase gene of rfbG genes encoding dTDP-rhamnosyl is so infer that orf10 is a glycosyltransferase gene, temporary called after orf10.The orf11 encoded protein finds to contain 10 potential transmembrane segments by analyzing, the topological framework of its inner membrance has the characteristic feature of well-known O-antigen polysaccharase (Wzy), in addition, the Wzy of it and Shigella bogdii coding O-antigen polysaccharase has 25% homogeny in 317 amino acid, 47% similarity, the homology that height is arranged between them is described, so name orf11 is the wzy gene.

Embodiment 6: the screening of specific gene: at wzx, orf8, orf9, orf10, wzy gene design primer in the O-antigen gene of intestinal bacteria O150 type bunch, the position of these genes in nucleotide sequence sees Table 1.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O150 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O150 type in table, have been listed.In each gene, we have respectively designed three 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 IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer #101 (TTC ATC CTAAAC TCC TTA TT) and #102 (TAA TCG CAG GGG AAA GCA GG), extract genome then from 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genomic quality.

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 8-10 bacterium, and 27 groups altogether, all list in the table in their source.

The genomic dna that contains intestinal bacteria O150 type in the 24th 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: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, orf8, orf9, orf10, wzy gene, each gene all has three pairs of primers detected, every pair of primer has obtained except be PCR in the 24th 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, orf8, orf9, orf10, wzy gene pairs intestinal bacteria O150 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O150 type, screen gene by PCR: wzx, wzy and three glycosyltransferase genes to the O-antigen high special of intestinal bacteria O150 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O150 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 O150 type.These all oligonucleotide all can be used for detecting rapidly and accurately the intestinal bacteria O150 type in livestock and the environment, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O150 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O150 type, altogether by 11 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 O150 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O150 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.

Sequence list

＜110〉Nankai University

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O150 type

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O150 type

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<170>PatentIn version 3.1

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<211>13552

<212>DNA

<213>Escherichia coli

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attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaactgcg cgtgaagcgt 120

caactactgg cggaagttca gtccatctgc ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttgtgtg cgcgacccgc cattggtgac 240

aatccatttg tcgtggtgct gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgttg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccactg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtaggt cgctatgtgc tttctgccga tatttggccg 540

gaactggaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgctattgcc 600

gagctggcga aaaaacaatc cgttgatgtc atgctgatga ccggtgacag ttacgactgc 660

ggcaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gcctacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg tattgagaag ttgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt taacaagaaa attataacgg cagtgaagat ttgcagcaaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa accatcagaa tagcaacgag ttagcagtag 900

ggttttattc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc ataggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

tcaaactgag agccgcttat ttcacagcat gctctgaagt aatatggaat aaattaagtg 1140

aaaatacttg ttactggtgg cgcaggattt attggttctg ctgtagttcg tcacattata 1200

aataatacgc aggatagtgt tgttaatgtc gataaattaa cgtacgccgg aaacctggaa 1260

tcacttgctg atgtttctga ttccgaacgc tatgtttttg aacatgcgga tatttgcgat 1320

gcagctgtaa tggcacggat ttttgctcag catcagccgg atgcagtgat gcacctggct 1380

gctgaaagcc atgttgaccg ttcaattaca ggtcctgcgg catttattga aaccaatatt 1440

gttggtacat atgtcctttt ggaagccgct cgcaattact ggtctgctct tgatagcgac 1500

aagaaaaata gcttccgttt tcatcatatt tctactgacg aagtctatgg tgatttgcct 1560

catcctgacg aggtaaataa tacagaagaa ttacccttat ttactgagac aacagcttac 1620

gcgccaagca gcccttattc cgcatccaaa gcatccagcg atcatttagt ccgcgcgtgg 1680

aaacgtacct atggtttacc gaccattgtg actaattgct ctaacaatta tggtccttat 1740

catttcccgg aaaaattgat tccattggtt attctcaatg ctctggaagg taaagcatta 1800

cctatttatg gtaaagggga tcaaattcgc gactggctgt atgttgaaga tcatgcgcgt 1860

gcgttatata ccgtcgtaac cgaaggtaaa gcgggtgaaa cttataacat tggtgggcac 1920

aacgaaaaga aaaacatcga tgtagtgctc actatttgtg atttgttgga tgagattgta 1980

ccgaaagaga aatcttaccg cgagcaaatt acttatgttg ccgatcgtcc gggacacgat 2040

cgccgttatg ccattgatgc tgagaagatt ggtcgcgaat tgggatggaa accacaggaa 2100

acgtttgaga gcgggattcg gaagacagtg gaatggtacc tgtccaatac aaaatgggtt 2160

gataatgtga aaagtggtgc ctatcaatcg tggattgaac agaactatga gggccgccag 2220

taatgaatat cctccttttt ggcaaaacag ggcaggtagg ttgggaacta cagcgtgctc 2280

tggtaccttt gggtaatttg attgctcttg atgttcactc cactgattac tgtggtgatt 2340

ttagtaatcc tgaaggtgta gctgaaaccg taagaagcat tcggccggat attattgtca 2400

acgcagccgc tcacaccgca gtagacaaag cagaatcaga accgaagttt gcacaattac 2460

tgaacgcgac gagtgtcgaa gcgatcgcga aagcagccaa tgaagtcggc gcctgggtta 2520

ttcactactc tactgactac gtatttccgg ggaccggtga aataccatgg caggaggagg 2580

atgcaaccgc accgctaaat gtttacggtg aaaccaagtt agcgggagaa aaagcattac 2640

aagagcattg tgtgaagcac cttattttcc ggaccagctg ggtctatgca ggtaaaggaa 2700

ataacttcgc caaaacaatg ttacgtctgg caaaagagcg tgaagaattg gccgttatta 2760

acgatcagtt tggtgcgcca acaggtgctg agctgctggc tgattgtaca gcacatgcca 2820

ttcgtgtcgc attgaataaa ccggaagtcg caggcttgta ccatctggta gccagtggta 2880

ccacaacctg gtacgattat gctgcgctgg tttttgaaga ggcgcgcaaa gcagatattc 2940

cccttgcact caacaaactc aatgcggtac caacaacagc ctatcctaca tcagctcgtc 3000

gtccacataa ctctcgcctt aatacagaaa aatttcagca gaattttgcg cttgttttgc 3060

ctgactggca ggttggcgtg aaacgaatgc ttaacgaatt atttacgact acagcaattt 3120

aatagttttt gcatcttgtt cgtgatgatg gagcaagatg aattaaaagg aatgatgaaa 3180

tgaaaacgcg taaaggtatt attttagcgg gtggctctgg tactcgtctt tatcctgtga 3240

ctatggctgt cagtaaacag ctattaccta tttatgataa gccgatgatc tattacccgc 3300

tctctacact gatgttggcg ggtattcgcg atattttgat tatcagcacg ccacaggata 3360

ctcctcgttt tcaacaactg ctgggtgatg ggagccagtg ggggctaaat cttcactaca 3420

aagtgcaacc gagtccggat ggtcttgcgc aggcgtttat tatcggtgaa gagtttattg 3480

gtggtgatga ttgtgctttg gtacttggtg ataatatctt ctacggtcac gacctgccta 3540

agttaatgga tgccgctgtt aacaaagaaa gtggtgcaac ggtatttgcc tatcacgtta 3600

atgatcctga acgctacggt gtcgttgagt ttgataaaaa cggtacggca ataagcctgg 3660

aagaaaaacc gttacaacca aaaagtaatt atgcggtaac tgggctttat ttctatgata 3720

acgacgttgt cgaaatggcg aaaaacctta agccttctgc ccgtggtgaa ctagaaatta 3780

ccgatattaa ccgtatttat atggaacagg ggcgtttatc tgttgccatg atgggacgtg 3840

gttatgcatg gttggacacg gggacacatc aaagtcttat tgaggcaagc aatttcatcg 3900

cgacaataga agaacgacag gggctgaaag tttcctgccc ggaagaaatt gcttaccgta 3960

aggggtttat cgatgctgag caggtgaaag tattagctga accgttgaag aaaaatgctt 4020

atggccagta cctgctaaaa atgattaaag gttattaata aaatgaacgt aattaaaaca 4080

gagattccag acgtattaat tttcgaaccg aaagtttttg gtgatgagcg tggtttcttt 4140

atggaaagct ttaatcagaa agttttcgaa gaagctgtag gacgtaaggt tgaatttgtt 4200

caggataacc attcgaagtc ttgtaaaggt gttttacgcg ggctgcatta tcagttagaa 4260

ccttatgcgc aagggaaatt ggtacgttgc gttgttggtg aggtttttga tgttgcggtt 4320

gatattcgta aatggtcacc tacctttggc aaatgggttg gggtgaattt atctgctgag 4380

aataagcgcc agttgtggat ccctgaagga tttgcacatg gttttttggt gctaagtgag 4440

acggcagaat ttgtttataa gacgacgaat tattatgcac ctgggtatga aggtagcatc 4500

atttggaatg ataaaatact atctattgat tggcctgagt taacactgga gtatattctt 4560

tccgataaag atctaaaggc cccaattcta agtgaatcta tagagaatat ttaaaatgtt 4620

tattagtaga gtaaaacgta aaattcaatc tttgataagc cttgagttga attttttaca 4680

cgattattgg aggtttaaaa aatattacac gaagggatct caagcttcga aagataagca 4740

gaaacttgaa tcttggatat tgcaagataa acatcgaata gaaaaagctt tctctttacc 4800

taagccaaaa cttggatttg gtaaagatgt aatacctcgt ctcattgaca atcttgttaa 4860

ttatagcagt aaatttggaa atgatcaggt atattatatt ggcttaggtg cattacaatc 4920

gtataagaga ttccacgacg aacaaaaatt tttattgcct gaattttatt caaagaatat 4980

ttgtaaaata cggaatgatg attttctcga tccccgatgc aatgtcgctg gctattttaa 5040

aaaagatgat tcattagtta caggtaagat aacagtagaa tcattgatga acgaacggcg 5100

tagctgtcgg cattttgatg ttgatgaaag tcttaatatc ggagataaac ttttaaagga 5160

tgttacaaaa ctatctatta cagccccttc tgtttgtaat agacaacatt ggcgcattca 5220

ctatttttca ggtgaattaa agaataaaat tcttagttat caaaatggaa attcaggatt 5280

tacagaaaac ataccttatg tagcagttat aacaagtgat cttagagcat tttattctac 5340

tgacgaaaga aaccagccct atacagacgg tgggatcttt gccatgaatt ttatgtatgc 5400

tctacaacac tatggtcttg cttcttgtcc attgaattgg tgtaattcat ctcacattga 5460

gaatgagttt agagaaacta agttgattcc tgattacgaa gtagtagtac ttgtggttgc 5520

ttttggctat ccaaataaag atgccttata tgctaaatca ccacgattgt ctctaaataa 5580

cctttacaca attaattagg cgaaggccac ctttaaaggt ggctgacaat aatgaagaaa 5640

caaatcatta aaaatatttc agccaatttt actaatttga ttgttagtat ttttattgga 5700

ctattattga cgcctttact cgtcaaaagt ttaggcattg aagtatatgg agttctaccg 5760

atagctttat ttttaacatt ttatatggga gtcattgctc aagcattaac tgcatctgta 5820

aatagatttt taattgagga ttttattaaa ggagcatata ctgaggcaag tgtcgtattt 5880

agtacatcgt ttattcttat tctaggatat ctatctgtta ttagtattgg atttatttat 5940

ccaatattgc atataagtga tttttttaac ataccaccgg gatatgaaca tgatgcagtt 6000

cttctattct cgtgtgtaat attttcgttt gttattgcta tgatcagttc atcattgtca 6060

gtttctatgt atgcaaaaaa taggattgac ttgatgcaat atggaaacat tttaaggaat 6120

attactaaag tcttaatcat tataatgttt gtttatttat ctgaaataaa tttaacaaaa 6180

ataggaatat caattgtttt atctgagctg ttttatctaa tctatgtcat tataacttgg 6240

aaaatattga caccacaact ttctctgcag ataggatttt tcagaaagga taaggtaaag 6300

agattaacat ctttcagtgg ctggatattg tttgatcaaa ttgggtcaat tctttttctt 6360

aaaacagatc tgattttagt taattctcta agtggtagta aagctaatgg tgaatattca 6420

atagctacac aatttagtga tcttttgcgt tcacttgctg gtttggtagc tggtagttta 6480

ggtcctatga tggtaatttt atatgaagaa aagaaattag aagaacttac ttcattgacc 6540

attacatttg ttaaattcct aagcttgacg atagctattc ctattattat tctttgtgta 6600

tactctaaag aaattcttga gctttggtta ggttcggggt ttgtatttct ttcaccatta 6660

atatgtatcg taacgcttcc actgattatc aatttgggca cactgccaat attatcgatc 6720

aatattgcag tcaaaaaaat tcaagtgcct gcactcataa atttctttct tgcaatcgtt 6780

ggaattatag gctctgtagt attgttcaaa accactacat taggttatta tgctgtagct 6840

ataagttatg tgattacatt aactattaag aactccattt tcataccatg gtatgcaaca 6900

attatattaa ataaaccaag gtatcttttt tttaaaactc atctaattac tatcatcttc 6960

tcttctctat tttttatttt tatatatata cttaaaagta taatgcagat tactaattta 7020

aatttgcttt ttgaattgat attggttggt tgctttggac ttctatctac atttcctttc 7080

tatagtaaag aggaaaggcg atcatttatg ttaatgctca agaggaaagt gtaataatgg 7140

attcgactca gaaaataggc gttctgaatt tccagtattc ggatcataat tatggtgctg 7200

ttttacaagc cgcagcaata gaacaatttc tgaaaagcaa tggatataat gctgaacata 7260

tagactatat atctagtcca gagataaagg gtaatagggt aattatacaa ctaaaatgta 7320

tattgaaaaa attgggacta tctgattctg taagaaaaat gcttggaaaa caagtccact 7380

tgacacctgt ggtcacaaac gaaaaagtat ttgaggaatt tagaaaaaat tggttggtga 7440

gaacgaagtg tttcagaaac tttgaagaat taaaaaaaac tgattttaat tttaaagcgg 7500

ttatagttgg gagtgatcag gtttggcgac catctcaatt tactcggttt tccgattata 7560

aggtatattt cttaagcttt ttaccttcgc aggttaagaa aatatcttat gcagcaagtt 7620

ttggtattga taagtgggaa gttagtgatg ctaacgtcac gaacgaaata aaaaaatata 7680

ttcaagattt caatgctgtt tcagtcagag aagatagtgg cgttaatatt tgtgcaacca 7740

tttttaatgt cgacgctgaa catgttcttt atcctacttt actggttggt agtgactttt 7800

ttaacagtat aatagctaag gaaatacgtg ctgatgctta ttacgatgct atagtttatt 7860

acaaacttga tattgatcaa cattttatag attcagttac tatactaggt agaaagaaat 7920

gcaagccagt aaaaaatatt tattacaacc aagtagatgg ccgaaatgag tattattctg 7980

ttcctgaatg gttaagcaat attaagaatg ctaattttgt tatcacagat tcttttcatt 8040

gtgtatgttt ttgtcttctt tttcaaaaag agtttttatg ctgtgtgaat gaaagtagag 8100

gattatctag attgcaaagt ttacttggaa tgcttgagct tactgatcgt atttgctcgt 8160

caaaagatga ttttgataat aagttaaatt cgttaaagcc aatagactat tctaaagtgg 8220

gcgatatatt gaataagcat agagatattt ctaaggcgtt tttattaagt aatttatctt 8280

gagtttgagt gtgggttaaa tatttaatga atgcaaataa tagaaatgca tttccgatca 8340

ttgttatgac tcgcaacgaa ggcgccgact tacaacgttg cgttgattct atattacata 8400

ctgtatctat agatgtacat atatatatca tagacaataa ttctgatgat ttatcacata 8460

ttcagatcct tgctaacctt gaagacctga atcgtaaaaa tttaacagtt ataagaaata 8520

caaggaattt atggatttta ggtgtaaata acacactatt gaaaatcaag aaaaatcatg 8580

atactaaata ttttattctc actgatggtg atatagactt tcatggtttt aaagaagaaa 8640

gttgttggtt aacatattta atcaataaaa tggaatccaa cgtgataatc ggtaagctcg 8700

gaatttctct ggattggtca tttctggcct caaaaacgga gatgaaagat atttaccggc 8760

aagaacaaaa tctctataat gaaagaagaa aaatcggtga tctttatata tctgccgttg 8820

acacgacagt agccatttat cgttgggatt ggtctataga gcgtaaagct ttattttatc 8880

ctgaccatat gaggtattta cgtccagaac tgtactcttg ccgtactaaa cgtggtttaa 8940

cagtagagca tttaggatgg tatagatacc ttgataactc cctgtctacg caaagtataa 9000

actcgaaagt tttttgtttt acaattgttg gtggtactgt taaacctgag gttttaaaac 9060

tggctagtaa accctatcag tatttccata ttttgttttc aaaacctttc gctatgcttt 9120

ggtatttcag acgatatttt aaacttttta tatattttat tactaaaggg agacgtggat 9180

ttgatggaca gtcataataa taagttatca aaacgtattc cgtcacctgc actacgttgt 9240

tatgcagtaa tagttacatt taaccctgat attaataagg ttagaagttt aatagatatt 9300

ctacaaaaga attttatcac cgcagttgta gtagataata cgattggagc aataaattat 9360

ccattctcat gtcatgttat taatttaggt gataatttag gtattgccac agcacaaaat 9420

aaaggcatag aatattgttt gactaaacaa gctgaagcta tctggttttt tgatcaggac 9480

agcgtaatca cttcttcatt tgtcgaaaga tttctatcta ctgtccatga aaatcctcat 9540

gataagatct ttgctcctgt gttctgggat gagaagaaag ggtttgaata tgcaattaca 9600

aatatagatt caaagggcaa cagacaaaag ttgttatcat catcttttcc atcggatttt 9660

tatagttcga ttgttatttc atcagggtgc ttaataaaaa gtgatttgct gatgaaaatt 9720

ggtccaatgc tagatttttt atttatagat tacgttgata cggaatggtg tttacgtgct 9780

ttctattatg gctacaaagt tcacataatc aaaaatgcaa ggatgaaaca ttctataggt 9840

gataacacta tttctttaat cggttttaat gtaccaattc actctcctct tcgacgttat 9900

tatcgaataa gaaatgcatt tttattgatt aggtttaagc atatcccaaa aaaactagca 9960

tttagagaag tgatattttg ttgctgccat caattaataa ttatcctgac ccaagaaaaa 10020

aaactggcat atgttaagta ttttttacgg gctgtatatg atggccttag aaataaaact 10080

gggaagtttg taaactaatg agttttgtca tgaagccaaa ggtattagtg ctagttgcag 10140

tttataatgg tgaaaagtgg ttaatagagc aacttacatc tataataaat caaaaaaatg 10200

ttgatattga tgtgattatt aatatcgata aatcaactga ttcttcatac aaattgtgtg 10260

agaattttat tgcagataat gtcaagatac taccgtacgg agctgtattt ggcggtgcag 10320

gtgccaattt ttatcatcta atccatgaat ctgaattttc tggttacgat tacattgcat 10380

tttccgatca agacgatatt tggtttgaaa ctaaaatata tgatgccatc aataggatgc 10440

atgattatga tgcatattca agtaatgtaa tagcattctg ggaaaatggc aaacaatgtc 10500

taattgagaa atctcaacca cagacagaat ttgattattt ttttgaggca gcaggacccg 10560

ggtgcactta tatagtgaga caggaactag cactcgcatt taaatatttc gtttgcaaga 10620

attatgtggc tgtaaataaa gtttgtttac atgattggct gttatatgcg tttgccagag 10680

aaagaaaata taaatggttt attgatcctc aacctggtat gttgtataga cagcattcga 10740

ataatcaagt tggggcaaat gcttcattat taggtgctat caagcgtata aaaaaaatta 10800

aagattcttg gtatagaaat gaggtaaaaa aaatagtcaa tttaacttcg attgataata 10860

atttaataag tgaatgcctt atgagagata gatacttatc atctttaaaa ttggctttta 10920

tagttagaaa attaagacgt cgtcctcgag atcaatttgc tatgtttata gccttagttc 10980

tgggctggtt ttaacatcga tcgggagcac tatgctcatt ttaacaaaca atattatatt 11040

gctgattttt ataatctata cttttttggt atttataagt gcacttggag taaaattaaa 11100

tagcagaggg cttgataatt tttcgtgcct cattacagtc ctgacatttg ttatactcat 11160

tttcgctcgt acaggattag gtgttgacga aagtacatat ttagaatctt atcattatta 11220

tgttcaagga ggtgaagcag attttgaata cggatttaat atcctttttt ggggagtcag 11280

actgttcggg gtaacagaat cttcattcaa taccatattc ccgttattga tattaacagc 11340

attatacttc gctgttactg tatcagttaa aaatcctttt agatcttttg tattaatatt 11400

tacaatattt tcttctttct ttctggattt ttcatttaat gcatataggc aggggctttc 11460

attctggttt gttctacttg ccatagaatc acatctgtct gggaataggt ggaaatttat 11520

agtttattgc ttaattggac ttggtttcca ctggtcagca gcagttgttt tattcatgct 11580

tttcctaaca agatttttat ctacaaggtt agcgatattc tgtaatattt ttctattttt 11640

cttaacgcta gtggcagcaa tgatgccatt acatgtactt tcaatgcttg taacgatgat 11700

acaatacttg ccattaaact catcttactt gcaaaaagtt attttttatc ttactacaat 11760

taagtcttcg ttttacgatt taaatttctt tggacgtgct ccattaataa tttatgcgct 11820

tattcttatt tctctcttgg ctatatatag aaaactatta ccaattgtgc aatttaaatt 11880

gttaattctt ttattagtct acagtgtgtt gttacttgag atgtcataca gttttagaaa 11940

ttactactgg gtgctaccat ttactccatt tattatggcg aaaattttga gttgttactc 12000

taataagaaa aaggtaatgg taatgtatac tatgttttct acagggttgt ggtctctatc 12060

aatagctagt ttctattcat ttcctatact atctatgata tttcagtgaa atcatataat 12120

aatatggttc attataaata cttttttggt taataatatt ttatataaac tgtgcattaa 12180

cgcacggtga ccacccatga caggagtaaa caatgtcaaa gcaacagatc ggcgtcgtcg 12240

gtatggcagt gatggggcgc aacctagcgc tcaacatcga aagccgtggt tataccgtct 12300

ctattttcaa ccgctcccgt gagaagacgg aagaagtgat tgccgaaaat ccaggcaaga 12360

aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg cctcgtcgca 12420

tcctgttaat ggtgaaagca ggcgcaggca cggatgctgc tactgattct cccaaaccat 12480

acctcgataa aggcgacatc areattgatg gtggtaacac cttcttccag gacaccattc 12540

gtcgtaaccg tgagctttct gctgaaggct ttaacttcat tggtaccggt gtctctggag 12600

gcgaagaagg tgcgctgaaa ggtccttcca ttatgcctgg tgggcagaaa gaagcctatg 12660

aactggttgc gccgatcctg accaaaatcg ccgcagcggc tgaagacggt gagccatgcg l2720

ttacctatat cggtgccgat ggcgcaggcc actatgtgaa gatggttcac aatggtattg 12780

aatacggaga tatgcaactg attgctgaag cccactctct gcttaaaggt ggcccgaacc 12840

tcaccaacga agaactggct cagacctcta ccgagtggaa caacggtgaa ctgagcagtt 12900

acctgatcga catcaccaaa gatatcttca ccaaaaaaga tgaagacggc aactacctgg 12960

ttgatgtgat tctggatgaa gcggctaaca aagglaccgg taaatggacc agccagagcg 13020

cgctggatct cggcgaaccg ctgtcgctaa ttacegagtc tgtgtttgct cgttatatat 13080

cttctctgaa agatcagcgc gttgccgcgt ctaaagttct ctctggtccg caagcgcagc 13140

cagcaggcga caaagctgag tttatcgaga aagttcgtcg tgcgctgtat ctgggcaaaa 13200

tcgtttccta cgctcagggc ttctctcagc tgcgcgcggc gtctgaagag tacaactggg 13260

atctgaacfa cggcgaaatc gcgaagattt tccgtgctgg ctgcatcatc cgtgcgcagt 13320

tcctacagaa aalcaccgat gcttatgccg aaaatccgca gatcgctaac ctgctgctgg 13380

ctccgtactt taaacaaatt gccgatgatt accagcaggc gctgcgtgat gtcgttgctt 13440

atgcggtaca gaacggtatc ccggttccga ccttcgccgc tgcggttgca tattatgaca 13500

gctaccgttc cgctgttctg cctgcgaacc tgatccaggc acagcgtgac ta 13552

Ward off based transferase gene and oligosaccharide unit treatment gene and wherein primer and PCR data in the O antigen gene of table 1 intestinal bacteria O15O type bunch

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	5632-7134	5706-5721	6458-6475	770bp	0 *	50
			5719-5736	6681-6698	980bp	0 *	52
											5978-5995	6789-6806	829bp	0 *	54
orf8	Glycosyltransferase	8307-9197	8681-8699	9169-9185	505bp	0 *	48
											8418-8435	8995-9012	595bp	0 *	48
			8333-8350	9050-9067	735bp	0 *	48
								orf9	The a little transferring enzymes of sugar	9184-10098	9397-9413	9882-9899	503bp	0 *	50
			9337-9354	9899-9916	580bp	0 *	54
											9489-9506	10035-10051	563bp	0 *	54
orf10	Before glycosyl shifts	10098-10994	10302-40319	10852-10869	568bp	0 *	50
											10193-10310	10954-10971	679bp	0 *	44
			10377-10394	10973-10989	613bp	0 *	48
								Wzy	Polysaccharase	11012-12109	112l7-11234	11985-12002	786bp	0 *	52
			11225-11241	12035-12052	828bp	0 *	53
											11172-11189	11960-11977	806bp	0 *	54

^*In intestinal bacteria Ol50 type, obtain a correct band

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, O3, O4, O10, O16, O18, O39 IMVS ^a

2 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS

3 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 IMVS

4 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 IMVS

5 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 IMVS

6 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 IMVS

7 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 IMVS

8 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 IMVS

9 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 IMVS

10 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS

11 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS

12 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS

13 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS

14 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS

15 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 See b

16 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, See c

17 wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS

18 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS

19 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, IMVS

20 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 IMVS

21 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 IMVS

22 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 See d

23 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 See d

24 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 See d

25 shigella dysenteriae serum D9, D10, D11, D12, D13 See d

26 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) See d

27 last Nei Shi Shigellae D5, DR See d

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

b. O123 from IMVS；the rest from Statens Serum Institut，Copenhagen，Denmark

c. 172 and 173 from Statens Serum Institut，Copenhagen，Denmark，the rest from IMVS

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

Table 3 intestinal bacteria O150 type O antigen gene structure iron

Table 4 intestinal bacteria O150 type O antigen gene cluster gene position

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaactgcg cgtgaagcgt 120

caactactgg cggaagttca gtccatctgc ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttgtgtg cgcgacccgc cattggtgac 240

aatccatttg tcgtggtgct gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgttg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccactg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtaggt cgctatgtgc tttctgccga tatttggccg 540

gaactggaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgctattgcc 600

gagctggcga aaaaacaatc cgttgatgtc atgctgatga ccggtgacag ttacgactgc 660

ggcaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gcctacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg tattgagaag ttgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt taacaagaaa attataacgg cagtgaagat ttgcagcaaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa accatcagaa tagcaacgag ttagcagtag 900

ggttttattc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc ataggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

Or orf1 is initial

tcaaactgag agccgcttat ttcacagcat gctctgaagt aatatggaat aaattaa gtg 1140

aaaatacttg ttactggtgg cgcaggattt attggttctg ctgtagttcg tcacattata 1200

aataatacgc aggatagtgt tgttaatgtc gataaattaa cgtacgccgg aaacctggaa 1260

tcacttgctg atgtttctga ttccgaacgc tatgtttttg aacatgcgga tatttgcgat 1320

gcagctgtaa tggcacggat ttttgctcag catcagccgg atgcagtgat gcacctggct 1380

gctgaaagcc atgttgaccg ttcaattaca ggtcctgcgg catttattga aaccaatatt 1440

gttggtacat atgtcctttt ggaagccgct cgcaattact ggtctgctct tgatagcgac 1500

aagaaaaata gcttccgttt tcatcatatt tctactgacg aagtctatgg tgatttgcct 1560

catcctgacg aggtaaataa tacagaagaa ttacccttat ttactgagac aacagcttac 1620

gcgccaagca gcccttattc cgcatccaaa gcatccagcg atcatttagt ccgcgcgtgg 1680

aaacgtacct atggtttacc gaccattgtg actaattgct ctaacaatta tggtccttat 1740

catttcccgg aaaaattgat tccattggtt attctcaatg ctctggaagg taaagcatta 1800

cctatttatg gtaaagggga tcaaattcgc gactggctgt atgttgaaga tcatgcgcgt 1860

gcgttatata ccgtcgtaac cgaaggtaaa gcgggtgaaa cttataacat tggtgggcac 1920

aacgaaaaga aaaacatcga tgtagtgctc actatttgtg atttgttgga tgagattgta 1980

ccgaaagaga aatcttaccg cgagcaaatt acttatgttg ccgatcgtcc gggacacgat 2040

cgccgttatg ccattgatgc tgagaagatt ggtcgcgaat tgggatggaa accacaggaa 2100

acgtttgaga gcgggattcg gaagacagtg gaatggtacc tgtccaatac aaaatgggtt 2160

Orf1 stops, and orf2 is initial

ga taatgtga aaagtggtgc ctatcaatcg tggattgaac agaactatga gggccgccag 2220

taatgaatat cctccttttt ggcaaaacag ggcaggtagg ttgggaacta cagcgtgctc 2280

tggtaccttt gggtaatttg attgctcttg atgttcactc cactgattac tgtggtgatt 2340

ttagtaatcc tgaaggtgta gctgaaaccg taagaagcat tcggccggat attattgtca 2400

acgcagccgc tcacaccgca gtagacaaag cagaatcaga accgaagttt gcacaattac 2460

tgaacgcgac gagtgtcgaa gcgatcgcga aagcagccaa tgaagtcggc gcctgggtta 2520

ttcactactc tactgactac gtatttccgg ggaccggtga aataccatgg caggaggagg 2580

atgcaaccgc accgctaaat gtttacggtg aaaccaagtt agcgggagaa aaagcattac 2640

aagagcattg tgtgaagcac cttattttcc ggaccagctg ggtctatgca ggtaaaggaa 2700

ataacttcgc caaaacaatg ttacgtctgg caaaagagcg tgaagaattg gccgttatta 2760

acgatcagtt tggtgcgcca acaggtgctg agctgctggc tgattgtaca gcacatgcca 2820

ttcgtgtcgc attgaataaa ccggaagtcg caggcttgta ccatctggta gccagtggta 2880

ccacaacctg gtacgattat gctgcgctgg tttttgaaga ggcgcgcaaa gcagatattc 2940

cccttgcact caacaaactc aatgcggtac caacaacagc ctatcctaca tcagctcgtc 3000

gtccacataa ctctcgcctt aatacagaaa aatttcagca gaattttgcg cttgttttgc 3060

ctgactggca ggttggcgtg aaacgaatgc ttaacgaatt atttacgact acagcaattt 3120

It is initial that orf2 stops orf3

aatagttttt gcatcttgtt cgtgatgatg gagcaagatg aattaaaagg aatgatgaaa 3180

tgaaaacgcg taaaggtatt attttagcgg tgggctctgg tactcgtctt tatcctgtga 3240

ctatggctgt cagtaaacag ctattaccta tttatgataa gccgatgatc tattacccgc 3300

tctctacact gatgttggcg ggtattcgcg atattttgat tatcagcacg ccacaggata 3360

ctcctcgttt tcaacaactg ctgggtgatg ggagccagtg ggggctaaat cttcactaca 3420

aagtgcaacc gagtccggat ggtcttgcgc aggcgtttat tatcggtgaa gagtttattg 3480

gtggtgatga ttgtgctttg gtacttggtg ataatatctt ctacggtcac gacctgccta 3540

agttaatgga tgccgctgtt aacaaagaaa gtggtgcaac ggtatttgcc tatcacgtta 3600

atgatcctga acgctacggt gtcgttgagt ttgataaaaa cggtacggca ataagcctgg 3660

aagaaaaacc gttacaacca aaaagtaatt atgcggtaac tgggctttat ttctatgata 3720

acgacgttgt cgaaatggcg aaaaacctta agccttctgc ccgtggtgaa ctagaaatta 3780

ccgatattaa ccgtatttat atggaacagg ggcgtttatc tgttgccatg atgggacgtg 3840

gttatgcatg gttggacacg gggacacatc aaagtcttat tgaggcaagc aatttcatcg 3900

cgacaataga agaacgacag gggctgaaag tttcctgccc ggaagaaatt gcttaccgta 3960

aggggtttat cgatgctgag caggtgaaag tattagctga accgttgaag aaaaatgctt 4020

It is initial that orf3 stops orf4

atggccagta cctgctaaaa atgattaaag gttattaata aaatgaacgt aattaaaaca 4080

gagattccag acgtattaat tttcgaaccg aaagtttttg gtgatgagcg tggtttcttt 4140

atggaaagct ttaatcagaa agttttcgaa gaagctgtag gacgtaaggt tgaatttgtt 4200

caggataacc attcgaagtc ttgtaaaggt gttttacgcg ggctgcatta tcagttagaa 4260

ccttatgcgc aagggaaatt ggtacgttgc gttgttggtg aggtttttga tgttgcggtt 4320

gatattcgta aatggtcacc tacctttggc aaatgggttg gggtgaattt atctgctgag 4380

aataagcgcc agttgtggat ccctgaagga tttgcacatg gttttttggt gctaagtgag 4440

acggcagaat ttgtttataa gacgacgaat tattatgcac ctgggtatga aggtagcatc 4500

atttggaatg ataaaatact atctattgat tggcctgagt taacactgga gtatattctt 4560

Orf4 stops, and orf5 is initial

tccgataaag atctaaaggc cccaattcta agtgaatcta tagagaatat t taaa atgtt 4620

tattagtaga gtaaaacgta aaattcaatc tttgataagc cttgagttga attttttaca 4680

cgattattgg aggtttaaaa aatattacac gaagggatct caagcttcga aagataagca 4740

gaaacttgaa tcttggatat tgcaagataa acatcgaata gaaaaagctt tctctttacc 4800

taagccaaaa cttggatttg gtaaagatgt aatacctcgt ctcattgaca atcttgttaa 4860

ttatagcagt aaatttggaa atgatcaggt atattatatt ggcttaggtg cattacaatc 4920

gtataagaga ttccacgacg aacaaaaatt tttattgcct gaattttatt caaagaatat 4980

ttgtaaaata cggaatgatg attttctcga tccccgatgc aatgtcgctg gctattttaa 5040

aaaagatgat tcattagtta caggtaagat aacagtagaa tcattgatga acgaacggcg 5100

tagctgtcgg cattttgatg ttgatgaaag tcttaatatc ggagataaac ttttaaagga 5160

tgttacaaaa ctatctatta cagccccttc tgtttgtaat agacaacatt ggcgcattca 5220

ctatttttca ggtgaattaa agaataaaat tcttagttat caaaatggaa attcaggatt 5280

tacagaaaac ataccttatg tagcagttat aacaagtgat cttagagcat tttattctac 5340

tgacgaaaga aaccagccct atacagacgg tgggatcttt gccatgaatt ttatgtatgc 5400

tctacaacac tatggtcttg cttcttgtcc attgaattgg tgtaattcat ctcacattga 5460

gaatgagttt agagaaacta agttgattcc tgattacgaa gtagtagtac ttgtggttgc 5520

ttttggctat ccaaataaag atgccttata tgctaaatca ccacgattgt ctctaaataa 5580

It is initial that orf5 stops orf6

cctttacaca attaat tagg cgaaggccac ctttaaaggt ggctgacaat a atgaagaaa 5640

caaatcatta aaaatatttc agccaatttt actaatttga ttgttagtat ttttattgga 5700

ctattattga cgcctttact cgtcaaaagt ttaggcattg aagtatatgg agttctaccg 5760

atagctttat ttttaacatt ttatatggga gtcattgctc aagcattaac tgcatctgta 5820

aatagatttt taattgagga ttttattaaa ggagcatata ctgaggcaag tgtcgtattt 5880

agtacatcgt ttattcttat tctaggatat ctatctgtta ttagtattgg atttatttat 5940

ccaatattgc atataagtga tttttttaac ataccaccgg gatatgaaca tgatgcagtt 6000

cttctattct cgtgtgtaat attttcgttt gttattgcta tgatcagttc atcattgtca 6060

gtttctatgt atgcaaaaaa taggattgac ttgatgcaat atggaaacat tttaaggaat 6120

attactaaag tcttaatcat tataatgttt gtttatttat ctgaaataaa tttaacaaaa 6180

ataggaatat caattgtttt atctgagctg ttttatctaa tctatgtcat tataacttgg 6240

aaaatattga caccacaact ttctctgcag ataggatttt tcagaaagga taaggtaaag 6300

agattaacat ctttcagtgg ctggatattg tttgatcaaa ttgggtcaat tctttttctt 6360

aaaacagatc tgattttagt taattctcta agtggtagta aagctaatgg tgaatattca 6420

atagctacac aatttagtga tcttttgcgt tcacttgctg gtttggtagc tggtagttta 6480

ggtcctatga tggtaatttt atatgaagaa aagaaattag aagaacttac ttcattgacc 6540

attacatttg ttaaattcct aagcttgacg atagctattc ctattattat tctttgtgta 6600

tactctaaag aaattcttga gctttggtta ggttcggggt ttgtatttct ttcaccatta 6660

atatgtatcg taacgcttcc actgattatc aatttgggca cactgccaat attatcgatc 6720

aatattgcag tcaaaaaaat tcaagtgcct gcactcataa atttctttct tgcaatcgtt 6780

ggaattatag gctctgtagt attgttcaaa accactacat taggttatta tgctgtagct 6840

ataagttatg tgattacatt aactattaag aactccattt tcataccatg gtatgcaaca 6900

attatattaa ataaaccaag gtatcttttt tttaaaactc atctaattac tatcatcttc 6960

tcttctctat tttttatttt tatatatata cttaaaagta taatgcagat tactaattta 7020

aatttgcttt ttgaattgat attggttggt tgctttggac ttctatctac atttcctttc 7080

Orf6 stops, and orf7 is initial

tatagtaaag aggaaaggcg atcatttatg ttaatgctca agaggaaagt gtaataatgg 7140

attcgactca gaaaataggc gttctgaatt tccagtattc ggatcataat tatggtgctg 7200

ttttacaagc cgcagcaata gaacaatttc tgaaaagcaa tggatataat gctgaacata 7260

tagactatat atctagtcca gagataaagg gtaatagggt aattatacaa ctaaaatgta 7320

tattgaaaaa attgggacta tctgattctg taagaaaaat gcttggaaaa caagtccact 7380

tgacacctgt ggtcacaaac gaaaaagtat ttgaggaatt tagaaaaaat tggttggtga 7440

gaacgaagtg tttcagaaac tttgaagaat taaaaaaaac tgattttaat tttaaagcgg 7500

ttatagttgg gagtgatcag gtttggcgac catctcaatt tactcggttt tccgattata 7560

aggtatattt cttaagcttt ttaccttcgc aggttaagaa aatatcttat gcagcaagtt 7620

ttggtattga taagtgggaa gttagtgatg ctaacgtcac gaacgaaata aaaaaatata 7680

ttcaagattt caatgctgtt tcagtcagag aagatagtgg cgttaatatt tgtgcaacca 7740

tttttaatgt cgacgctgaa catgttcttt atcctacttt actggttggt agtgactttt 7800

ttaacagtat aatagctaag gaaatacgtg ctgatgctta ttacgatgct atagtttatt 7860

acaaacttga tattgatcaa cattttatag attcagttac tatactaggt agaaagaaat 7920

gcaagccagt aaaaaatatt tattacaacc aagtagatgg ccgaaatgag tattattctg 7980

ttcctgaatg gttaagcaat attaagaatg ctaattttgt tatcacagat tcttttcatt 8040

gtgtatgttt ttgtcttctt tttcaaaaag agtttttatg ctgtgtgaat gaaagtagag 8100

gattatctag attgcaaagt ttacttggaa tgcttgagct tactgatcgt atttgctcgt 8160

caaaagatga ttttgataat aagttaaatt cgttaaagcc aatagactat tctaaagtgg 8220

Orf7 stops

gcgatatatt gaataagcat agagatattt ctaaggcgtt tttattaagt aatttatctt 8280

Orf8 is initial

gagtttgagt gtgggttaaa tatttaatga atgcaaataa tagaaatgca tttccgatca 8340

ttgttatgac tcgcaacgaa ggcgccgact tacaacgttg cgttgattct atattacata 8400

ctgtatctat agatgtacat atatatatca tagacaataa ttctgatgat ttatcacata 8460

ttcagatcct tgctaacctt gaagacctga atcgtaaaaa tttaacagtt ataagaaata 8520

caaggaattt atggatttta ggtgtaaata acacactatt gaaaatcaag aaaaatcatg 8580

atactaaata ttttattctc actgatggtg atatagactt tcatggtttt aaagaagaaa 8640

gttgttggtt aacatattta atcaataaaa tggaatccaa cgtgataatc ggtaagctcg 8700

gaatttctct ggattggtca tttctggcct caaaaacgga gatgaaagat atttaccggc 8760

aagaacaaaa tctctataat gaaagaagaa aaatcggtga tctttatata tctgccgttg 8820

acacgacagt agccatttat cgttgggatt ggtctataga gcgtaaagct ttattttatc 8880

ctgaccatat gaggtattta cgtccagaac tgtactcttg ccgtactaaa cgtggtttaa 8940

cagtagagca tttaggatgg tatagatacc ttgataactc cctgtctacg caaagtataa 9000

actcgaaagt tttttgtttt acaattgttg gtggtactgt taaacctgag gttttaaaac 9060

tggctagtaa accctatcag tatttccata ttttgttttc aaaacctttc gctatgcttt 9120

ggtatttcag acgatatttt aaacttttta tatattttat tactaaaggg agacgtggat 9180

The initial orf8 of orf9 stops

ttgatggaca gtcataataa taagttatca aaacgtattc cgtcacctgc actacgttgt 9240

tatgcagtaa tagttacatt taaccctgat attaataagg ttagaagttt aatagatatt 9300

ctacaaaaga attttatcac cgcagttgta gtagataata cgattggagc aataaattat 9360

ccattctcat gtcatgttat taatttaggt gataatttag gtattgccac agcacaaaat 9420

aaaggcatag aatattgttt gactaaacaa gctgaagcta tctggttttt tgatcaggac 9480

agcgtaatca cttcttcatt tgtcgaaaga tttctatcta ctgtccatga aaatcctcat 9540

gataagatct ttgctcctgt gttctgggat gagaagaaag ggtttgaata tgcaattaca 9600

aatatagatt caaagggcaa cagacaaaag ttgttatcat catcttttcc atcggatttt 9660

tatagttcga ttgttatttc atcagggtgc ttaataaaaa gtgatttgct gatgaaaatt 9720

ggtccaatgc tagatttttt atttatagat tacgttgata cggaatggtg tttacgtgct 9780

ttctattatg gctacaaagt tcacataatc aaaaatgcaa ggatgaaaca ttctataggt 9840

gataacacta tttctttaat cggttttaat gtaccaattc actctcctct tcgacgttat 9900

tatcgaataa gaaatgcatt tttattgatt aggtttaagc atatcccaaa aaaactagca 9960

tttagagaag tgatattttg ttgctgccat caattaataa ttatcctgac ccaagaaaaa 10020

aaactggcat atgttaagta ttttttacgg gctgtatatg atggccttag aaataaaact 10080

Orf9 stops, and orf10 is initial

gggaagtttg taaactaatg agttttgtca tgaagccaaa ggtattagtg ctagttgcag 10140

tttataatgg tgaaaagtgg ttaatagagc aacttacatc tataataaat caaaaaaatg 10200

ttgatattga tgtgattatt aatatcgata aatcaactga ttcttcatac aaattgtgtg 10260

agaattttat tgcagataat gtcaagatac taccgtacgg agctgtattt ggcggtgcag 10320

gtgccaattt ttatcatcta atccatgaat ctgaattttc tggttacgat tacattgcat 10380

tttccgatca agacgatatt tggtttgaaa ctaaaatata tgatgccatc aataggatgc 10440

atgattatga tgcatattca agtaatgtaa tagcattctg ggaaaatggc aaacaatgtc 10500

taattgagaa atctcaacca cagacagaat ttgattattt ttttgaggca gcaggacccg 10560

ggtgcactta tatagtgaga caggaactag cactcgcatt taaatatttc gtttgcaaga 10620

attatgtggc tgtaaataaa gtttgtttac atgattggct gttatatgcg tttgccagag 10680

aaagaaaata taaatggttt attgatcctc aacctggtat gttgtataga cagcattcga 10740

ataatcaagt tggggcaaat gcttcattat taggtgctat caagcgtata aaaaaaatta 10800

aagattcttg gtatagaaat gaggtaaaaa aaatagtcaa tttaacttcg attgataata 10860

atttaataag tgaatgcctt atgagagata gatacttatc atctttaaaa ttggctttta 10920

tagttagaaa attaagacgt cgtcctcgag atcaatttgc tatgtttata gccttagttc 10980

It is initial that orf10 stops orf11

tgggctggtt ttaacatcga tcgggagcac tatgctcatt ttaacaaaca atattatatt 11040

gctgattttt ataatctata cttttttggt atttataagt gcacttggag taaaattaaa 11100

tagcagaggg cttgataatt tttcgtgcct cattacagtc ctgacatttg ttatactcat 11160

tttcgctcgt acaggattag gtgttgacga aagtacatat ttagaatctt atcattatta 11220

tgttcaagga ggtgaagcag attttgaata cggatttaat atcctttttt ggggagtcag 11280

actgttcggg gtaacagaat cttcattcaa taccatattc ccgttattga tattaacagc 11340

attatacttc gctgttactg tatcagttaa aaatcctttt agatcttttg tattaatatt 11400

tacaatattt tcttctttct ttctggattt ttcatttaat gcatataggc aggggctttc 11460

attctggttt gttctacttg ccatagaatc acatctgtct gggaataggt ggaaatttat 11520

agtttattgc ttaattggac ttggtttcca ctggtcagca gcagttgttt tattcatgct 11580

tttcctaaca agatttttat ctacaaggtt agcgatattc tgtaatattt ttctattttt 11640

cttaacgcta gtggcagcaa tgatgccatt acatgtactt tcaatgcttg taacgatgat 11700

acaatacttg ccattaaact catcttactt gcaaaaagtt attttttatc ttactacaat 11760

taagtcttcg ttttacgatt taaatttctt tggacgtgct ccattaataa tttatgcgct 11820

tattcttatt tctctcttgg ctatatatag aaaactatta ccaattgtgc aatttaaatt 11880

gttaattctt ttattagtct acagtgtgtt gttacttgag atgtcataca gttttagaaa 11940

ttactactgg gtgctaccat ttactccatt tattatggcg aaaattttga gttgttactc 12000

taataagaaa aaggtaatgg taatgtatac tatgttttct acagggttgt ggtctctatc 12060

Orf11 stops

aatagctagt ttctattcat ttcctatact atctatgata tttcagtgaa atcatataat 12120

aatatggttc attataaata cttttttggt taataatatt ttatataaac tgtgcattaa 12180

cgcacggtga ccacccatga caggagtaaa caatgtcaaa gcaacagatc ggcgtcgtcg 12240

gtatggcagt gatggggcgc aacctagcgc tcaacatcga aagccgtggt tataccgtct 12300

ctattttcaa ccgctcccgt gagaagacgg aagaagtgat tgccgaaaat ccaggcaaga 12360

aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg cctcgtcgca 12420

tcctgttaat ggtgaaagca ggtgcaggca cggatgctgc tattgattct ctcaaaccat 12480

acctcgataa aggcgacatc atcattgatg gtggtaacac cttcttccag gacaccattc 12540

gtcgtaaccg tgagctttct gctgaaggct ttaacttcat tggtaccggt gtctctggag 12600

gcgaagaagg tgcgctgaaa ggtccttcca ttatgcctgg tgggcagaaa gaagcctatg 12660

aactggttgc gccgatcctg accaaaatcg ccgcagtggc tgaagacggt gagccatgcg 12720

ttacctatat cggtgccgat ggcgcaggcc attatgtgaa gatggttcac aatggtattg 12780

aatacggaga tatgcaactg attgctgaag cctattctct gcttaaaggt ggcctgaacc 12840

tcaccaacga agaactggct cagaccttta ccgagtggaa taacggtgaa ctgagcagtt 12900

acctgatcga catcaccaaa gatatcttca ccaaaaaaga tgaagacggt aactacctgg 12960

ttgatgtgat tctggatgaa gcggctaaca aaggtaccgg taaatggacc agccagagcg 13020

cgctggatct cggcgaaccg ctgtcgctaa ttaccgagtc tgtgtttgct cgttatatat 13080

cttctctgaa agatcagcgc gttgccgcgt ctaaagttct ctctggtccg caagcgcagc 13140

cagcaggcga caaagctgag tttatcgaga aagttcgtcg tgcgctgtat ctgggcaaaa 13200

tcgtttctta tgctcagggc ttctctcagc tgcgtgcggc gtctgaagag tacaactggg 13260

atctgaacta cggcgaaatc gcgaagattt tccgtgctgg ctgcatcatc cgtgcgcagt 13320

tcctacagaa aatcaccgat gcttatgccg aaaatccgca gatcgctaac ctgctgctgg 13380

ctccgtactt taaacaaatt gccgatgatt accagcaggc gctgcgtgat gtcgttgctt 13440

atgcggtaca gaacggtatc ccggttccga ccttcgccgc tgcggttgca tattatgaca 13500

gctaccgttc cgctgttctg cctgcgaacc tgatccaggc acagcgtgac ta 13552

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 (2)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O150 type is characterized in that described oligonucleotide is to being: the Nucleotide of 5706 to 5722 bases among the SEQ ID NO:1 and the Nucleotide of 6458 to 6475 bases; The Nucleotide of 5719 to 5736 bases among the SEQ ID NO:1 and the Nucleotide of 6681 to 6698 bases; The Nucleotide of 5978 to 5995 bases among the SEQ ID NO:1 and the Nucleotide of 6789 to 6806 bases; The Nucleotide of 8681 to 8699 bases among the SEQ ID NO:1 and the Nucleotide of 9169 to 9185 bases; The Nucleotide of 8418 to 8435 bases among the SEQ ID NO:1 and the Nucleotide of 8995 to 9012 bases; The Nucleotide of 8333 to 8350 bases among the SEQ ID NO:1 and the Nucleotide of 9050 to 9067 bases; The Nucleotide of 9397 to 9413 bases among the SEQ ID NO:1 and the Nucleotide of 9882 to 9899 bases; The Nucleotide of 9337 to 9354 bases among the SEQ ID NO:1 and the Nucleotide of 9899 to 9916 bases; The Nucleotide of 9489 to 9506 bases among the SEQ ID NO:1 and the Nucleotide of 10035 to 10051 bases; The Nucleotide of 10302 to 10319 bases among the SEQ ID NO:1 and the Nucleotide of 10852 to 10869 bases; The Nucleotide of 10293 to 10310 bases among the SEQ ID NO:1 and the Nucleotide of 10954 to 10971 bases; The Nucleotide of 10377 to 10394 bases among the SEQ ID NO:1 and the Nucleotide of 10973 to 10989 bases; The Nucleotide of 11217 to 11234 bases among the SEQ ID NO:1 and the Nucleotide of 11985 to 12002 bases; The Nucleotide of 11225 to 11241 bases among the SEQ ID NO:1 and the Nucleotide of 12035 to 12052 bases; The Nucleotide of 11172 to 11189 bases among the SEQ ID NO:1 and the Nucleotide of 11960 to 11977 bases.

2, the application of the described Nucleotide of claim 1 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray confession detection intestinal bacteria O150 type as probe.