CN1252268C - Nucleotide specific against 0-antigen of colibacillus 0107 - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O107. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O107 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 13188 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 O107. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O107 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Escherichia coli O107 in human bodies and environment.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O107 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O107 type (Escherichia coli O107), particularly relate in the intestinal bacteria O107 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O107 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Intestinal bacteria O107 at first found to cause infant and turista in Finland in 1997, belong to enterotoxigenic E.Coli, very big [the Keskimaki M of the explosive popular danger of its potential, Saari M, Heiskanen T.Shiga toxin-producing Escherichia coli in finland from 1990 through1997:prevalence and characteristics of isolates[J] .J Clin Microbiol, 1998,36 (12): 3641-3646], being badly in need of one can be quick, accurately detect the method for intestinal bacteria O107.

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, 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 Ecoli 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 Ecoli 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 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; 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 of the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152 and and Shigella O antigens " J.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 O107 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O107 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 O107 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O107 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 orf7, orf8, orf9, orf10 gene; Sugar synthesis path gene comprises the gne 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 O107 type respectively comprises orf7, 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 O107 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O107 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O107 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 detect O-antigen and detection and identification of escherichia coli O107 type with identification of escherichia coli O107 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 O107 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 O107 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,13188 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 O107 type, it is by 11 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O107 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 orf7, orf8, orf9, orf10 gene; Wherein said gene: wzx is the Nucleotide of 4604 to 5821 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 5814 to 7136 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 7129 to 7926 bases among the SEQID NO:1; Orf8 is the Nucleotide of 7913 to 8857 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 8854 to 9915 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9912 to 10679 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O107 type, wherein it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf7, orf8, orf9, orf10 gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O107 type, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 4649 to 4666 bases among the SEQ ID NO:1 and the Nucleotide of 5664 to 5681 bases; The Nucleotide of 4700 to 4717 bases among the SEQ ID NO:1 and the Nucleotide of 5626 to 5643 bases; The Nucleotide of 4753 to 4770 bases among the SEQ ID NO:1 and the Nucleotide of 5266 to 5283 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 5864 to 5882 bases among the SEQ ID NO:1 and the Nucleotide of 6852 to 6869 bases; The Nucleotide of 5895 to 5916 bases among the SEQ ID NO:1 and the Nucleotide of 6813 to 6831 bases; The Nucleotide of 6072 to 6090 bases among the SEQ ID NO:1 and the Nucleotide of 6762 to 6779 bases; The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 7141 to 7158 bases among the SEQ ID NO:1 and the Nucleotide of 7737 to 7754 bases; The Nucleotide of 7167 to 7185 bases among the SEQ ID NO:1 and the Nucleotide of 7710 to 7727 bases; The Nucleotide of 7190 to 7207 bases among the SEQ ID NO:1 and the Nucleotide of 7686 to 7703 bases; The oligonucleotide that comes from the orf8 gene is to being: the Nucleotide of 7936 to 7954 bases among the SEQ ID NO:1 and the Nucleotide of 8774 to 8791 bases; The Nucleotide of 8029 to 8046 bases among the SEQ ID NO:1 and the Nucleotide of 8652 to 8669 bases; The Nucleotide of 8101 to 8118 bases among the SEQ ID NO:1 and the Nucleotide of 8623 to 8640 bases; The oligonucleotide that comes from the orf9 gene is to being: the Nucleotide of 8862 to 8879 bases among the SEQ ID NO:1 and the Nucleotide of 9774 to 9791 bases; The Nucleotide of 8916 to 8933 bases among the SEQ ID NO:1 and the Nucleotide of 9681 to 9697 bases; The Nucleotide of 9025 to 9043 bases among the SEQ ID NO:1 and the Nucleotide of 9647 to 9664 bases; The oligonucleotide that comes from the orf10 gene is to being: the Nucleotide of 9942 to 9959 bases among the SEQ ID NO:1 and the Nucleotide of 10586 to 10603 bases; The Nucleotide of 10019 to 10036 bases among the SEQ ID NO:1 and the Nucleotide of 10502 to 10520 bases; The Nucleotide of 10095 to 10112 bases among the SEQ ID NO:1 and the Nucleotide of 10454 to 10471 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O107 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 O107 type, and can provide the O-antigen of expressing intestinal bacteria O107 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O107 type, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O107 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O107 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 glass yarn, DNA is resuspended among the 30ul TE with 70% ethanol; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O107 type bunch: with the genome of intestinal bacteria O107 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 lkb-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.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 Bi0-Rad company prepares competence bacillus coli DH 5 a cell, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 a 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 O107 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 ResearchCouncil) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O107 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O107 type is done 6 Long PCR reactions, mix these products then to produce library, 2) to each base, guarantee high-quality fraction of coverage more than 3.After obtaining the nucleotide sequence of intestinal bacteria O107 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 O107 type at last;

(6) screening of specific gene: at wzx, wzy, orf7, orf8, orf9, the orf10 gene design primer in the O-antigen gene of intestinal bacteria O107 type 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, wzy, orf7, orf8, orf9, orf10 gene pairs intestinal bacteria O107 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 O107 type, its complete sequence shown in SEQ ID NO:1,13188 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 O107 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 O107 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 orf7, 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, 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 O107 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O107 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 orf7, orf8, orf9, 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 O107 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 O107 type.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O107 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O107 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 definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 4604 to 5821 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 5814 to 7136 bases from SEQ ID NO:1); Orf7 gene (nucleotide position is the Nucleotide of 7129 to 7926 bases from SEQ ID NO:1); Orf8 gene (nucleotide position is the Nucleotide of 7913 to 8857 bases from SEQ ID NO:1); Orf9 gene (nucleotide position is the Nucleotide of 8854 to 9915 bases from SEQ ID NO:1); Orf10 gene (nucleotide position is the Nucleotide of 9912 to 10679 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O107 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 O107 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 O107 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 O107 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 O107 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 O107 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 O107 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 O107 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 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, 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 O107 type bunch:

With the genome of intestinal bacteria O107 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 1.25ul 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.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 O107 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 O107 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O107 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 O107 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 O107 type at last, as shown in table 3.

By retrieving and comparing, find that the orf5 encoded protein contains 12 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 Orf5 has 12 potential transmembrane domains; and with many Wzx protein similars; for example; and the Wzx of E.coli has 23% homogeny in 290 amino acid; 45% similarity, in addition and S.thermophilus, the Wzx similarity of B.fragilis is also very high; we carry out BLOCKMAKER with these four Wzx sequences and analyze, find 4 conservative motifs (respectively by 6,18,37,45 amino acid are formed), with 3 round-robin PSI-BLAST programs of the concensus sequence that obtains operation, except the sequence of submitting to, also and the sequence of the Wzx in a lot of other sources very high similarity (E value≤5 * e is arranged ^-14), be the wzx gene so can determine orf5, called after wzx.Orf6 contains 11 potential transmembrane segments, and 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 E.coli coding O-antigen polysaccharase has 24% homogeny in 415 amino acid, 45% similarity, and illustrating has certain homology between them, so name orf6 is the wzy gene.Orf7 by blast relatively has 41% homogeny with the glycosyltransferase of L.interrogans, and 63% similarity illustrates the homology that height is arranged between them, can infer that orf7 also is glycosyltransferase gene, called after orf7 temporarily.Orf8 by blast relatively has 35% homogeny with the glycosyltransferase of L.plantarum, and 53% similarity illustrates the homology that height is arranged between them, can infer that orf8 also is glycosyltransferase gene, called after orf8 temporarily.The glycosyltransferase gene of orf9 and T.tengcongensis has 27% homogeny in 317 amino acid, 51% similarity is so infer that orf9 is a glycosyltransferase gene, temporary called after orf9.The glycosyltransferase gene of orf10 and S.enterica has 48% homogeny in 249 amino acid, 65% similarity is so infer that orf10 is a glycosyltransferase gene, temporary called after orf10.

Embodiment 6: the screening of specific gene:

At wzx, wzy, orf7, orf8, orf9, orf10 gene design primer in the O-antigen gene of intestinal bacteria O107 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 O107 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 O107 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 O107 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, wzy, orf7, orf8, orf9, orf10 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, wzy, orf7, orf8, orf9, orf10 gene pairs intestinal bacteria O107 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O107 type, screen gene by PCR: wzx, wzy and three glycosyltransferase genes to the O-antigen high special of intestinal bacteria O107 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O107 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 O107 type.These all oligonucleotide all can be used for the intestinal bacteria O107 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 O107 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O107 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 O107 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O107 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 O107 type

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O107 type

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<170>PatentIn version 3.1

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<211>13188

<212>DNA

<213>Escherichia coli

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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 cacgccccgc cattggcgac 240

aacccatttg tcgtggtgct gccggacgtt gttatcgatg atgccagcgc cgacccgctg 300

cgctacaacc ttgctgctat gattgcgcgc ttcaatgaaa cgggccgtag tcaagtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca tccagaccaa agaaccgctg 420

gaccgtgaag gcaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtaggt cgttatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcaacc tggtgcatgg gggcgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgca atgctgatga ctggtgacag ctacgactgc 660

ggtaaaaaaa tgggttatat gcaggcgttt gtgaagtatg gcttacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaggat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgagt taagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

tcaaactaag agccgcttat ttcacagcat gctctgaagt aatatggaat aaaagagtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc tgtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgat tctgaacgct atatttttga acatgcggat atttgcgatg 1320

ctgctgcaat ggcacggatt tttgctcagc atcagccgga tgcagtgatg cacctggcag 1380

ctgaaagcca tgttgaccgt tcaattacag gtcctgcggc atttattgaa accaatattg 1440

ttggtactta tgtccttttg gaagccgctc gcaattactg gtctgctctt gatagcgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggt gatttgcctc 1560

atccagatga agtaaataat aacgaagaat tacccttatt tactgagacg acagcttacg 1620

caccaagcag cccttattcc gcatcaaaag catccagcga tcatttagtc cgcgcgtgga 1680

aacgtaccta tggtttaccg accattgtga ctaactgttc gaataactac ggtccttatc 1740

actttccgga aaaattgatt ccactagtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg actggctgta tgttgaagat catgcgcgtg 1860

cgttatatac cgtcgtaacc gaaggtaaag cgggtgaaac ttataacatt ggtggacaca 1920

acgaaaagaa aaacatcgat gtagtgctca ctatttgtga tttgttggat gagattgtac 1980

cgaaagagaa atcttatcgt gagcaaatta cttatgttgc cgaccgcccg ggacatgatc 2040

gccgttatgc gattgatgct gagaagatta gccgcgaatt gggttggaaa ccacaggaaa 2100

cgtttgagag cgggattcgt aaaacagtgg aatggtacct gtccaataca aaatgggttg 2160

ataatgtgaa aagtggtgcc tatcaatcgt ggattgaaca gaactatgag ggccgccagt 2220

aatgaatatc ctccttttcg gcaaaacagg gcaggtaggt tgggaactac agcgtgctct 2280

ggcacctctg ggtaatttga ttgctcttga tgttcactcc actgattact gtggtgattt 2340

tagtaatcct gaaggtgtag ctgaaaccgt aagaagcatt cggcctgata ttattgtcaa 2400

cgcagcagct cacaccgcag tagacaaagc agaatcagaa ccggagtttg cacaattact 2460

taacgcgacg agtgtcgaag cgatcgcgaa agcagccaat gaagtcggcg catgggttat 2520

tcactactct actgactacg tatttccggg gaccggtgaa ataccatggc aggaggcgga 2580

tgcaaccgca ccgctaaatg tttacggtga aaccaagtta gctggagaaa aagcattaca 2640

agagcattgt gcgaagcacc ttatttttcg taccagctgg gtatatgcag gtaaaggaaa 2700

caattttgcc aagacaatgt tacgtctggc aaaagagcgc gaagaattag ctgtcattaa 2760

cgatcagttt ggtgcaccaa caggtgctga actgctggct gattgtacgg cacatgcaat 2820

tcgtatggca ctgaataaac cagaagtcgc aggcttgtac catctggtag ccagtggtac 2880

cacaacctgg cacgattatg ctgcgctggt ttttgaagag gcacgaaaag caggtattcc 2940

ccttgcactc aacaagctca aggcagtacc aacaacagcc tatcctacac cagctcgtcg 3000

tccacataac tctcgcctta atacagaaaa atttcagcag aattttgcgc ttgtcttgcc 3060

tgactggcag gttggtgtga aacgaatgct caacgaatta tttacgacta cagcaattta 3120

atagtttttg catcttgttc gtgatggtgg agcaagatga attaaaagga atgatgaaat 3180

gaaaacgcgt aaaggtatta ttttagcggg tggttctggt acacgtcttt atcctgtgac 3240

tatggctgtc agtaaacagc tattacctat ttatgataag ccgatgatct attacccgct 3300

ctctacactg atgttggcgg gtattcgcga tattctgatt attagtacgc cacaggatac 3360

tcctcgtttt caacaactgc tgggtgacgg tagccagtgg gggctaaatc ttcagtacaa 3420

agtgcaaccg actccagatg ggcttgcgca ggcgtttatt atcggtgaag agtttatcgg 3480

tggtgatgat tgtgctttgg tacttggtga taatatcttc tacggtcacg acctgcctaa 3540

gttaatggat gccgctgtta acaaagaaag tggtgcaacg gtatttgcct atcacgttaa 3600

tgatcctgaa cgctacggtg tcgttgagtt tgataaaaac ggtacggcga tcagcctgga 3660

agaaaaaccg ctacaaccaa aaagtaatta tgcggtaacc gggctttatt tctatgataa 3720

cgacgttgtc gaaatggcga aaaatcttaa gccttctgcc cgcggtgaac tggaaattac 3780

cgatattaac cgtatttata tggaacaggg gcgtttatcc gttgccatga tgggacgtgg 3840

ttatgcatgg ctggacacgg ggacacatca aagtcttatt gaagcaagca acttcattgc 3900

aacaattgaa gagcgtcaag gcttgaaagt ttcctgcccg gaagaaattg ctttcaggaa 3960

agggtttatt gatgctgagc aggtgaaagt gttagccgaa ccgctgaaga aaaatgctta 4020

tggtcagtat ctgctcaaaa tgattaaagg tgattaataa aatgaacgta attaaaacag 4080

aaattccaga tgtactgatt tttgaaccaa aagtgtttgg ggatgaacgt ggttttttta 4140

tggaaagctt taatcaacga caatttgaag aaattgttgg acgcaaaatt tcgtttgttc 4200

aggataatca ttcaaagtca aaaaaaggtg ttttacgtgg ccttcattat cagttgcctc 4260

catttgcaca aggtaaatta gttagatgta tcaatggtga agtatatgat gttgcagttg 4320

atctacgacc tgaatcaatt aattatggta aatgggtagg catcattctt tctgctgaaa 4380

ataagaagca gctatggata ccagaaggtt ttgcacatgg ttttttagtt ataagcgaat 4440

ttgctgaatt tgcgtacaaa accacaaatt attattcaaa agaaagtgaa agaggcatac 4500

gttggaatga tccaacgata aatatccaat ggccaaaaat gggacaatat ttactttctc 4560

cgaaagatgc gttagcacca tttatagaat taagaattga ttaatggaaa acaagaaaaa 4620

tcatatttat ggaattgtaa ataatattat tgtgatgtta tctcccatta taataattcc 4680

gtttgcaatt aaaaatgtcg gcattgatga atatggctat tttgtacaag ttaatataat 4740

ttattcgtgt ttgatatcca tattcactgc ttctctatcg ggatatttta taaagagcta 4800

tcttgagaaa aaattatcct ttcatgatat ttttataatt caattgttta ttaatttgtt 4860

ctccactgcg atcataggta tatatatact gctaagattt gatgtcgttg ctccgtatat 4920

atttgttgtt gctatgctaa ctaatacttt aaatttcgaa tggttttttc atgcaattgg 4980

cgcacagaaa caattattaa taaggaattt aattataaaa acaatgtttg taattatagc 5040

aatatttatc ttagaagtat ataaaaatat tgctgtttat tttgttattt ttgggttgaa 5100

tattattatt gcaaattttt tattgggtat atatagtttt acgaaaataa aaaataagga 5160

atatatactt ttttccaatg ggaggggggg gaatcggttg ttttgtttta ttaaggatgc 5220

aaaattcttc tttttgtcac cgacaattgg tgcagtatat caatatggcg atcaaatatt 5280

agtttcatta ttattcccaa aaactgcttt ggtttttata aatttagcta aacaaataat 5340

tggtgcgtca gtgatggtgt cgggtactct atgtagagtt gaacaaaaga acattttttc 5400

atctgagaaa attgaaagat tgacaaaaat aaaaaaaaca cttatggcat atgtttttta 5460

tttgatgact acatctatat taataatatt aattggtcct ttaattcttg gcatgttaat 5520

taaagaaact gtatcacttg gattaaacta ttacattcta attgctggta tatatgtctt 5580

tacttcattg agtatattta tcgattatat aataggattg actttcagga ctgagcatta 5640

cacttctatt tcgaatattt gttgtgcaac catagttact ttattgaacg caatgttttt 5700

agaaaaatat ggtgcaactt tttcgctttt ttcattattc attggagagc ttttggtttt 5760

tattttatta tctacaatgc attatataaa tataaagcga aggacatatt ataatggtta 5820

atattctgtt atgtattaat ataataatac tcttgatttg catttgtatc tatttgatcg 5880

ccttaaaaag taaaaaagct ccacgtttat tcgcactata tttaggagct tttattcttt 5940

ttattgagtc ccacataatt ctagcaataa caacttcttt taattttggt acgtcagaat 6000

ggttttttaa tggagagttt gattataata caaaaacaga agttataaca agcatcaatc 6060

tatttataat aggtatgata ttaggttcgg tatttattgc atcaacaata acctataaaa 6120

gttcttccta tgatgtgact tttgagaaca aatcgatagc tagattttcg tggctactgt 6180

tagtatctat cttaccattt gtagttgttt atttaattaa tttaatagct tttatttcat 6240

caaacggttt ttattcttta tatattaacg gtaataaaat atcgggtgga tacattcttg 6300

atttgttttt tctaacactt tactcattat tgatatcttt aaaaaataag aaaaagatac 6360

tgttcattat cttgtgtgta gcctgtgttt atttattcat aggaacaaga ttggaattta 6420

tgtttaaagt atttcctgtg ttaatctatt atatacttat ttcaaaaaat atacacaaat 6480

atttcaggtt gaaaaatatc cttgctatat caatactctt ttggggttta atatttagta 6540

tgcaatactc ggtttcagca agagataata ttgaaatggg gtcgaatatc atcacaacgt 6600

tcttaaaaca acaaggcgta tcagtcaatg ttattggtat tgctataaag gacaaaaata 6660

attcattatt aagtgaatct gtgattttgt cgccactata tgatagtgct atttctttag 6720

ctaattcttt agtaggtgtc caaagcaatg gaaattcggt agagttcgca gagaattcat 6780

tctcattgtc gcacaaattg agttatttag aagatccttc agcatacctt gctggctatg 6840

gtgttggggg agccgctatt gctgagctgt acatagttgg tggttacttg gcttgtttaa 6900

ttggaggtat gcttacatat atttttatct ctatactaga aaaaatcgcc aaaaaatcct 6960

tttttaattt tatttttgtt atgttgataa ctgggaaaat attatactca cctcgagggg 7020

agtttctttc atttatgtct gctgacagaa tgttaatatt atttttaatt tttacgtttt 7080

cttataaatt tctattggca acttcaaata aaaaaatgag ttttaaaaat gaatgaactt 7140

gtatctataa ttataccgtt atacaaccaa ggagagtata tcgaggaaac tctgaattcg 7200

gtcaagcaac agtcttattc aaattatgaa ttgatcattg tagatgatgg cagtaacgat 7260

caattcacaa ttgaaaagat aaaagaactc aggttcaaag gatatacgat tatctcaaag 7320

caaaacggcg gattagcatc agcaagaaat gaaggtatta gaaatgctat aggcaaatat 7380

attattgttc ttgattctga tgatataatt catcgcgatt atattaaaaa tcttcttgaa 7440

gttataacta atacaaaagc acgaatcgta tatagtaagg ctttattgtt tgaatacaga 7500

acgggaatgt ggatgctacc atcatttaca atgcgaagaa tgctgaaaag taatattatt 7560

ttttgttctg ctatgtatta tcgtgaagat tggtttaatg tgggaggcta tgatgaatct 7620

ttaagaacag gccttgagga ctgggatttt tggctatcat taataggttt atacaataat 7680

cataacgata tcgttgcgag ggtaaataag cctctgtttt tttatcgaat tagaaaggaa 7740

ttcaatgcta agataaatta cactgaaaaa gaaaaaattg ttaattatat acataaaaaa 7800

cacattcaat tatttgaact gtataaagtt aagcccctta tttatagtaa acagaattta 7860

atatccaaaa tttttaataa aattgcctcc aaaatatgtg gaataggata caatgttaaa 7920

cgataaaaag gaaatggttg ctattgttct tgcctcatat aatggacagt tatatattaa 7980

agaacaagtt ttaagtattt taaaccagag ttatcaatat attaaattgt attgtagcga 8040

tgatggttct actgatggaa caatcgacta tttaaaggag tatcaaaaaa ataatattgt 8100

tattgcaaag aatgatggaa aaaaaggccc tgcgtataat tttataaacg gattaaaatt 8160

ggttggtgat gaaaagtaca tcatttttag cgatcaggat gatgtttgga tgcctgaaaa 8220

agtttcaaca ttgaagtcgt atgcagataa attattgaat aatgacaaac cgggggcaat 8280

ttattgcgac gctttaattg ttgatcattc tcttactcaa cttggtaaaa gaatgtatgg 8340

tgagcaccat atatgtccta ccaaaatctc tgaactttta tatttaaatg gtggagtgca 8400

gggagcatct atgcttatta atcgaaaaat gatggatgaa atgttatcat ataataaata 8460

tatttatatg catgatcagt tagcgacata tttagctgtt gtttataaga atatctatta 8520

tataaatata ccattaatgt tgtacaggca acattctaaa aatgtcattg gagttaatac 8580

taataaaata gagaaaatga aactgttatt tagttctaat atgattgata gtcgatcata 8640

taaatttata actgaattta gtcaacacta tgcttgtaac aatcatatca atagtagaaa 8700

tagtctagaa tattatatcg agctgaaaga aaatatatta aaatctataa taaaggaatt 8760

tttgaatggt aatgctacac ttcataacag tcgaagtaaa ttactaataa agcttttctt 8820

taaattcatt attaataaag tcgttcaaaa aaaatgaaaa tagcgtatgt tgtgtcttct 8880

aagaaaaaat gtggcccaaa tattgtcata ttgaacattg ttaaagaatt ggctaataaa 8940

catgaaatgg aaatattttt ccttgatgag tctgatgatg atgtatttga atgtgtgaac 9000

gtaaagtcta cacaaataaa aaaagcttct gatttgaagg agcatctaaa aaggttcgat 9060

ataattcatt ctagtggaat acgtcccgat gcattggttg ttttatgcaa agttatttat 9120

cgtgtgaaat gtaaaataat tacaacaata cataactatg tatttcaaga cctttattac 9180

tcatatggtt tggttaaatc cttaatttgg ggcttacttt ggtgttctat ctggctattt 9240

ttcgataaat tagtaattct ttcaaaaaat gccgataatt attactggtt tctcccgtct 9300

gcaaaaaaaa atatcattta taatgggata gatgataatg attgccttca aaataaaaaa 9360

tgtaattatc gtaaggaatt taatattcct gatgatggga tattagcagg ctcttgtgct 9420

aatttaacca agtgtaaagg gatcgatctt gttattcaga ccttaactaa agaacataaa 9480

atttactata tagttgccgg tgatggtatt gaaaaacata atcttattaa tcttgtaaaa 9540

gcgcgtaagc tgcatgaaag agtgtatttt atcgatttcc ttgatgaacc agaaagtttt 9600

atgtcccagt tggatgtttt tttgatgcca tccagaagtg aaggatttgg acttactgtt 9660

ttggaatcaa ctaagttagg tatacctgta ataacgtcta atattccaat atttatggag 9720

ttatttgatc aaatgtgtct cacatttgat attaaaaatc catctacatt aatcgatgta 9780

ataacctacg caaaaaaaaa tagattgcat ctttcgcaaa aattccatgc tatatttcaa 9840

gataggttta cttcttcaaa gatggctaca aaatatgaaa acgtctataa taatttattt 9900

agagaggttt tatgagcttt tccgttttaa tgtctttata taatggtgaa agttccgagt 9960

atcttaatga ttgcttaagt agcattaatt cgcaaatatt aaaaccgaac caaattgtaa 10020

tcgtatatga tggcccaatt agaaaagaat tggacgatat agttacaaga tgggaaaagc 10080

aattgccaat acattgtgta aagctcactt cctgtacagg attaggtaat gcattgaatt 10140

atggtttgaa attttgtgat tacgatattg tctgtcgtat ggacactgat gatatatgta 10200

gaaatgacag atttttaaga caagttaaaa tacttgaaag tgataataat ctggctttga 10260

ttgggtctta tatctcagag tttgactcta ctccctcaaa cagtcatgct gttaggaccg 10320

ttcccattga gcataatgat atttgtttat atgcaaaaaa aagaaatccg tttaaccata 10380

tgactgtagc atttagaaaa agtgtagtaa cttctctcgg tgggtataag gatgaatatt 10440

tatatgaaga ctacgctctg tggattaaga tgcttaaaaa tggatttttg actaaaaata 10500

ttccagaggc gctagtttat gcgcgggttg gtaataatat ggagcttaaa cgcggtgggt 10560

ggaaatattt tacatcagaa gttaaggctc aatatggctt ctataaaatt ggttttctta 10620

acaaacagga aatgattatt aatattgttt tgagagcacc cggttcgttt aatgcctaac 10680

tttatgagaa agattattta tagaaatttc cttagaaact gatataggta tataaatgag 10740

tgtattagtg acaggtgggg caggttatat tggtagtcat acagttttga ctctcttaca 10800

aaaaggaata gatgtcattg tgattgatga tttttctaat tctagcttag agtctttgga 10860

aagagttaaa aaaattacaa atcgagatat taaagtatat aagggggatg tagcagatct 10920

tcgtttattg aatcttattt tttctaatca tgatattcat acggttattc attttgctgg 10980

ttctaaatct gttggggagt ctatatcaaa accaattcat tactataata acaatgttgt 11040

agctagcctt gttcttatta atgaaatgat gaaaagaggt atttataatt taatttttag 11100

ttcatctgca acagtatacg gtaactcgaa taccatgcca gttactgaaa atgcacctat 11160

tgggatgact acaaatcctt atggcacttc taaacttatg attgaaaaaa tattagatga 11220

tgttacaagg gcaaattcga gctttagagt gacggttttg aggtatttta accctgtggg 11280

tgctcatttc tctggagaga ttggtgagga tccaaatggt attccaaata atcttatgcc 11340

atatgtttgt caggtggcaa ttggtaaata taaacaagta tactatagta taagtatact 11400

atacatgtat atatgcactg gtgtccgtga ttttatccat gtaatggatc tcgctgaagg 11460

tcatgttgct gcgttggaac atagaaacga aggtcccaat cataaagttt acaacctcgg 11520

aacaggaaag gggtattctg ttttggatct tttgaccact tttgaacgag taacctttcg 11580

atccgtacct tacgttttga gtgaaagacg ccctggagat atcgccgaat gttggtctga 11640

tccttctaag gcttataggg aacttggatg gaaagcgaag cgtgggttgg aagaaatggt 11700

tcgagatgcc tggaattggc aacaaaagaa tccgaacggt tatataaaag catgaatgct 11760

ctatggtatt tttgtcaacg gtaaaaatta tcgattttat gtatcctgag ttaatatagc 11820

atctacattg acctgagtta tgtttcgcag tatcaccctg acaggagtaa acaatgtcaa 11880

agcaacagat cggcgtcgtc ggtatggcag tgatggggcg caaccttgcg ctcaacatcg 11940

aaagccgtgg ttataccgtc tctattttca accgttcccg tgaaaagacg gaagaagtga 12000

ttgccgaaaa tccaggcaag aaactggttc cttactatac ggtgaaagag tttgttgaat 12060

ctctggaaac gcctcgtcgc atcctgttaa tggtgaaagc aggtgctggc acggatgctg 12120

ctattgattc cctcaagccg tacctcgata aaggtgacat catcattgat ggtggtaata 12180

cattcttcca ggacaccatt cgccgtaacc gtgagctttc tgccgaaggc tttaacttca 12240

tcggtaccgg tgtttccggt ggtgaagagg gcgcgctgaa aggtccttcc attatgcctg 12300

gtggccagaa agaagcctat gaactggttg ctccgatcct gaccaaaatc gccgcagtag 12360

ctgaagacgg tgagccatgc gttacctata ttggtgccga tggcgcaggt cactatgtga 12420

agatggttca caacggtatt gaatacggtg atatgcagct gattgctgaa gcctattctc 12480

tgcttaaagg tggcctgaat ctctctaacg aagaactggc acagaccttt accgagtgga 12540

ataacggtga actgagcagc tacctgatcg acatcaccaa agacatcttc actaaaaaag 12600

atgaaggcgg taactacctg gttgatgtga tcctggatga agcggctaac aaaggtaccg 12660

gtaaatggac cagccagagc gcgctggatc tcggcgaacc gctgtcgctg attaccgagt 12720

ctgtgtttgc acgttatatc tcttctctga aagatcagcg cgttgccgca tctaaagttc 12780

tctctggtcc gcaagctcag tcagcaggcg acaaggctga gttcatcgaa aaagttcgcc 12840

gtgcgctgta tctgggcaaa atcgtttctt acgctcaggg cttctctcag ctacgcgccg 12900

cgtctgaaga gtacaactgg gatctgaact acggcgaaat cgcgaagatt ttccgtgctg 12960

gctgcatcat ccgtgcgcag ttcctgcaga aaatcaccga tgcttatgcc gaagatccgc 13020

agatcgctaa cctgctgctg gctccgtact tcaagcaaat tgccgatgac taccagcagg 13080

cgctgcgcga tgtcgttgct tatgcagtac aaaacggtat cccggttccg accttcgccg 13140

ctgcggttgc ctattacgat agctaccgtg ccgctgttct gcctgcga 13188

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O107 type bunch and oligosaccharide unit treatment gene and wherein

Primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								Wzx Wzy orf7 orf8 orf9 orf10	Transhipment enzymatic polymerization enzyme glycosyl transferase glycosyl transferase glycosyl transferase glycosyl transferase	4604-5821 5814-7136 7129-7926 7913-8857 8854-9915 9912-10679	4649-4666 4700-4717 4753-4770 5864-5882 5895-5916 6072-6090 7141-7158 7167-7185 7190-7207 7936-7954 8029-8046 8101-8118 8862-8879 8916-8933 9025-9043 9942-9959 10019-10036 10095-10112	5664-5681 5626-5643 5266-5283 6852-6869 6813-6831 6762-6779 7737-7754 7710-7727 7686-7703 8774-8791 8652-8669 8623-8640 9774-9791 9681-9697 9647-9664 10586-10603 10502-10520 10454-10471	1033 943 530 1006 934 708 614 561 514 856 641 540 930 782 640 662 502 377	0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 *	48 52 48 52 52 53 52 53 51 52 53 50 51 54 53 52 48 52

^*In intestinal bacteria O107 type, obtain a correct band

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

Group number	The bacterial strain that contains in this group	The source
			1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23	Wild-type e. coli O1, O2, O3, O4, O10, O16, O18, O39 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, wild-type e. coli O160, O161, O163, O8, O9, O124, O111 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18	IMVS a IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS See b See c IMVS IMVS IMVS IMVS IMVS See d 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 bacillus ceylonensis A D5, DR See d

a.1nstitude 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 O107 type O antigen gene structure iron

1Kb

orf# 1 2 3 4 5 6 7 8 9 10 11

Table 4 intestinal bacteria O107 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 cacgccccgc cattggcgac 240

aacccatttg tcgtggtgct gccggacgtt gttatcgatg atgccagcgc cgacccgctg 300

cgctacaacc ttgctgctat gattgcgcgc ttcaatgaaa cgggccgtag tcaagtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca tccagaccaa agaaccgctg 420

gaccgtgaag gcaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtaggt cgttatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcaacc tggtgcatgg gggcgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgca atgctgatga ctggtgacag ctacgactgc 660

ggtaaaaaaa tgggttatat gcaggcgttt gtgaagtatg gcttacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaggat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgagt taagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

Orf1 is initial

tcaaactaag agccgcttat ttcacagcat gctctgaagt aatatggaat aaaagagtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc tgtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgat tctgaacgct atatttttga acatgcggat atttgcgatg 1320

ctgctgcaat ggcacggatt tttgctcagc atcagccgga tgcagtgatg cacctggcag 1380

ctgaaagcca tgttgaccgt tcaattacag gtcctgcggc atttattgaa accaatattg 1440

ttggtactta tgtccttttg gaagccgctc gcaattactg gtctgctctt gatagcgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggt gatttgcctc 1560

atccagatga agtaaataat aacgaagaat tacccttatt tactgagacg acagcttacg 1620

caccaagcag cccttattcc gcatcaaaag catccagcga tcatttagtc cgcgcgtgga 1680

aacgtaccta tggtttaccg accattgtga ctaactgttc gaataactac ggtccttatc 1740

actttccgga aaaattgatt ccactagtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg actggctgta tgttgaagat catgcgcgtg 1860

cgttatatac cgtcgtaacc gaaggtaaag cgggtgaaac ttataacatt ggtggacaca 1920

acgaaaagaa aaacatcgat gtagtgctca ctatttgtga tttgttggat gagattgtac 1980

cgaaagagaa atcttatcgt gagcaaatta cttatgttgc cgaccgcccg ggacatgatc 2040

gccgttatgc gattgatgct gagaagatta gccgcgaatt gggttggaaa ccacaggaaa 2100

cgtttgagag cgggattcgt aaaacagtgg aatggtacct gtccaataca aaatgggttg 2160

Orf1 stops

ataatgtgaa aagtggtgcc tatcaatcgt ggattgaaca gaactatgag ggccgccagt 2220

Orf2 is initial

aatgaatatc ctccttttcg gcaaaacagg gcaggtaggt tgggaactac agcgtgctct 2280

ggcacctctg ggtaatttga ttgctcttga tgttcactcc actgattact gtggtgattt 2340

tagtaatcct gaaggtgtag ctgaaaccgt aagaagcatt cggcctgata ttattgtcaa 2400

cgcagcagct cacaccgcag tagacaaagc agaatcagaa ccggagtttg cacaattact 2460

taacgcgacg agtgtcgaag cgatcgcgaa agcagccaat gaagtcggcg catgggttat 2520

tcactactct actgactacg tatttccggg gaccggtgaa ataccatggc aggaggcgga 2580

tgcaaccgca ccgctaaatg tttacggtga aaccaagtta gctggagaaa aagcattaca 2640

agagcattgt gcgaagcacc ttatttttcg taccagctgg gtatatgcag gtaaaggaaa 2700

caattttgcc aagacaatgt tacgtctggc aaaagagcgc gaagaattag ctgtcattaa 2760

cgatcagttt ggtgcaccaa caggtgctga actgctggct gattgtacgg cacatgcaat 2820

tcgtatggca ctgaataaac cagaagtcgc aggcttgtac catctggtag ccagtggtac 2880

cacaacctgg cacgattatg ctgcgctggt ttttgaagag gcacgaaaag caggtattcc 2940

ccttgcactc aacaagctca aggcagtacc aacaacagcc tatcctacac cagctcgtcg 3000

tccacataac tctcgcctta atacagaaaa atttcagcag aattttgcgc ttgtcttgcc 3060

Orf2 stops

tgactggcag gttggtgtga aacgaatgct caacgaatta tttacgacta cagcaatt ta 3120

Orf3 is initial

atagtttttg catcttgttc gtgatggtgg agcaagatga attaaaagga atgatgaa at 3180

gaaaacgcgt aaaggtatta ttttagcggg tggttctggt acacgtcttt atcctgtgac 3240

tatggctgtc agtaaacagc tattacctat ttatgataag ccgatgatct attacccgct 3300

ctctacactg atgttggcgg gtattcgcga tattctgatt attagtacgc cacaggatac 3360

tcctcgtttt caacaactgc tgggtgacgg tagccagtgg gggctaaatc ttcagtacaa 3420

agtgcaaccg actccagatg ggcttgcgca ggcgtttatt atcggtgaag agtttatcgg 3480

tggtgatgat tgtgctttgg tacttggtga taatatcttc tacggtcacg acctgcctaa 3540

gttaatggat gccgctgtta acaaagaaag tggtgcaacg gtatttgcct atcacgttaa 3600

tgatcctgaa cgctacggtg tcgttgagtt tgataaaaac ggtacggcga tcagcctgga 3660

agaaaaaccg ctacaaccaa aaagtaatta tgcggtaacc gggctttatt tctatgataa 3720

cgacgttgtc gaaatggcga aaaatcttaa gccttctgcc cgcggtgaac tggaaattac 3780

cgatattaac cgtatttata tggaacaggg gcgtttatcc gttgccatga tgggacgtgg 3840

ttatgcatgg ctggacacgg ggacacatca aagtcttatt gaagcaagca acttcattgc 3900

aacaattgaa gagcgtcaag gcttgaaagt ttcctgcccg gaagaaattg ctttcaggaa 3960

agggtttatt gatgctgagc aggtgaaagt gttagccgaa ccgctgaaga aaaatgctta 4020

Orf3 stops, and orf4 is initial

tggtcagtat ctgctcaaaa tgattaaagg tgat taataa a atgaacgta attaaaacag4080

aaattccaga tgtactgatt tttgaaccaa aagtgtttgg ggatgaacgt ggttttttta 4140

tggaaagctt taatcaacga caatttgaag aaattgttgg acgcaaaatt tcgtttgttc 4200

aggataatca ttcaaagtca aaaaaaggtg ttttacgtgg ccttcattat cagttgcctc 4260

catttgcaca aggtaaatta gttagatgta tcaatggtga agtatatgat gttgcagttg 4320

atctacgacc tgaatcaatt aattatggta aatgggtagg catcattctt tctgctgaaa 4380

ataagaagca gctatggata ccagaaggtt ttgcacatgg ttttttagtt ataagcgaat 4440

ttgctgaatt tgcgtacaaa accacaaatt attattcaaa agaaagtgaa agaggcatac 4500

gttggaatga tccaacgata aatatccaat ggccaaaaat gggacaatat ttactttctc 4560

Orf4 stops, and orf5 is initial

cgaaagatgc gttagcacca tttatagaat taagaattga t taatggaaa acaagaaaaa 4620

tcatatttat ggaattgtaa ataatattat tgtgatgtta tctcccatta taataattcc 4680

gtttgcaatt aaaaatgtcg gcattgatga atatggctat tttgtacaag ttaatataat 4740

ttattcgtgt ttgatatcca tattcactgc ttctctatcg ggatatttta taaagagcta 4800

tcttgagaaa aaattatcct ttcatgatat ttttataatt caattgttta ttaatttgtt 4860

ctccactgcg atcataggta tatatatact gctaagattt gatgtcgttg ctccgtatat 4920

atttgttgtt gctatgctaa ctaatacttt aaatttcgaa tggttttttc atgcaattgg 4980

cgcacagaaa caattattaa taaggaattt aattataaaa acaatgtttg taattatagc 5040

aatatttatc ttagaagtat ataaaaatat tgctgtttat tttgttattt ttgggttgaa 5100

tattattatt gcaaattttt tattgggtat atatagtttt acgaaaataa aaaataagga 5160

atatatactt ttttccaatg ggaggggggg gaatcggttg ttttgtttta ttaaggatgc 5220

aaaattcttc tttttgtcac cgacaattgg tgcagtatat caatatggcg atcaaatatt 5280

agtttcatta ttattcccaa aaactgcttt ggtttttata aatttagcta aacaaataat 5340

tggtgcgtca gtgatggtgt cgggtactct atgtagagtt gaacaaaaga acattttttc 5400

atctgagaaa attgaaagat tgacaaaaat aaaaaaaaca cttatggcat atgtttttta 5460

tttgatgact acatctatat taataatatt aattggtcct ttaattcttg gcatgttaat 5520

taaagaaact gtatcacttg gattaaacta ttacattcta attgctggta tatatgtctt 5580

tacttcattg agtatattta tcgattatat aataggattg actttcagga ctgagcatta 5640

cacttctatt tcgaatattt gttgtgcaac catagttact ttattgaacg caatgttttt 5700

agaaaaatat ggtgcaactt tttcgctttt ttcattattc attggagagc ttttggtttt 5760

Orf6 is initial, and orf5 stops

tattttatta tctacaatgc attatataaa tataaagcga aggacatatt ata atggt ta5820

atattctgtt atgtattaat ataataatac tcttgatttg catttgtatc tatttgatcg 5880

ccttaaaaag taaaaaagct ccacgtttat tcgcactata tttaggagct tttattcttt 5940

ttattgagtc ccacataatt ctagcaataa caacttcttt taattttggt acgtcagaat 6000

ggttttttaa tggagagttt gattataata caaaaacaga agttataaca agcatcaatc 6060

tatttataat aggtatgata ttaggttcgg tatttattgc atcaacaata acctataaaa 6120

gttcttccta tgatgtgact tttgagaaca aatcgatagc tagattttcg tggctactgt 6180

tagtatctat cttaccattt gtagttgttt atttaattaa tttaatagct tttatttcat 6240

caaacggttt ttattcttta tatattaacg gtaataaaat atcgggtgga tacattcttg 6300

atttgttttt tctaacactt tactcattat tgatatcttt aaaaaataag aaaaagatac 6360

tgttcattat cttgtgtgta gcctgtgttt atttattcat aggaacaaga ttggaattta 6420

tgtttaaagt atttcctgtg ttaatctatt atatacttat ttcaaaaaat atacacaaat 6480

atttcaggtt gaaaaatatc cttgctatat caatactctt ttggggttta atatttagta 6540

tgcaatactc ggtttcagca agagataata ttgaaatggg gtcgaatatc atcacaacgt 6600

tcttaaaaca acaaggcgta tcagtcaatg ttattggtat tgctataaag gacaaaaata 6660

attcattatt aagtgaatct gtgattttgt cgccactata tgatagtgct atttctttag 6720

ctaattcttt agtaggtgtc caaagcaatg gaaattcggt agagttcgca gagaattcat 6780

tctcattgtc gcacaaattg agttatttag aagatccttc agcatacctt gctggctatg 6840

gtgttggggg agccgctatt gctgagctgt acatagttgg tggttacttg gcttgtttaa 6900

ttggaggtat gcttacatat atttttatct ctatactaga aaaaatcgcc aaaaaatcct 6960

tttttaattt tatttttgtt atgttgataa ctgggaaaat attatactca cctcgagggg 7020

agtttctttc atttatgtct gctgacagaa tgttaatatt atttttaatt tttacgtttt 7080

Orf7 is initial, and orf6 stops

cttataaatt tctattggca acttcaaata aaaaaatgag ttttaaaa at gaa tgaactt7140

gtatctataa ttataccgtt atacaaccaa ggagagtata tcgaggaaac tctgaattcg 7200

gtcaagcaac agtcttattc aaattatgaa ttgatcattg tagatgatgg cagtaacgat 7260

caattcacaa ttgaaaagat aaaagaactc aggttcaaag gatatacgat tatctcaaag 7320

caaaacggcg gattagcatc agcaagaaat gaaggtatta gaaatgctat aggcaaatat 7380

attattgttc ttgattctga tgatataatt catcgcgatt atattaaaaa tcttcttgaa 7440

gttataacta atacaaaagc acgaatcgta tatagtaagg ctttattgtt tgaatacaga 7500

acgggaatgt ggatgctacc atcatttaca atgcgaagaa tgctgaaaag taatattatt 7560

ttttgttctg ctatgtatta tcgtgaagat tggtttaatg tgggaggcta tgatgaatct 7620

ttaagaacag gccttgagga ctgggatttt tggctatcat taataggttt atacaataat 7680

cataacgata tcgttgcgag ggtaaataag cctctgtttt tttatcgaat tagaaaggaa 7740

ttcaatgcta agataaatta cactgaaaaa gaaaaaattg ttaattatat acataaaaaa 7800

cacattcaat tatttgaact gtataaagtt aagcccctta tttatagtaa acagaattta 7860

Orf8 is initial

atatccaaaa tttttaataa aattgcctcc aaaatatgtg gaataggata ca atgttaaa 7920

Orf7 stops

cga taaaaag gaaatggttg ctattgttct tgcctcatat aatggacagt tatatattaa 7980

agaacaagtt ttaagtattt taaaccagag ttatcaatat attaaattgt attgtagcga 8040

tgatggttct actgatggaa caatcgacta tttaaaggag tatcaaaaaa ataatattgt 8100

tattgcaaag aatgatggaa aaaaaggccc tgcgtataat tttataaacg gattaaaatt 8160

ggttggtgat gaaaagtaca tcatttttag cgatcaggat gatgtttgga tgcctgaaaa 8220

agtttcaaca ttgaagtcgt atgcagataa attattgaat aatgacaaac cgggggcaat 8280

ttattgcgac gctttaattg ttgatcattc tcttactcaa cttggtaaaa gaatgtatgg 8340

tgagcaccat atatgtccta ccaaaatctc tgaactttta tatttaaatg gtggagtgca 8400

gggagcatct atgcttatta atcgaaaaat gatggatgaa atgttatcat ataataaata 8460

tatttatatg catgatcagt tagcgacata tttagctgtt gtttataaga atatctatta 8520

tataaatata ccattaatgt tgtacaggca acattctaaa aatgtcattg gagttaatac 8580

taataaaata gagaaaatga aactgttatt tagttctaat atgattgata gtcgatcata 8640

taaatttata actgaattta gtcaacacta tgcttgtaac aatcatatca atagtagaaa 8700

tagtctagaa tattatatcg agctgaaaga aaatatatta aaatctataa taaaggaatt 8760

tttgaatggt aatgctacac ttcataacag tcgaagtaaa ttactaataa agcttttctt 8820

Orf9 is initial, and orf8 stops

taaattcatt attaataaag tcgttcaaaa aaa atgaaaa tagcgtatgt tgtgtcttct 8880

aagaaaaaat gtggcccaaa tattgtcata ttgaacattg ttaaagaatt ggctaataaa 8940

catgaaatgg aaatattttt ccttgatgag tctgatgatg atgtatttga atgtgtgaac 9000

gtaaagtcta cacaaataaa aaaagcttct gatttgaagg agcatctaaa aaggttcgat 9060

ataattcatt ctagtggaat acgtcccgat gcattggttg ttttatgcaa agttatttat 9120

cgtgtgaaat gtaaaataat tacaacaata cataactatg tatttcaaga cctttattac 9180

tcatatggtt tggttaaatc cttaatttgg ggcttacttt ggtgttctat ctggctattt 9240

ttcgataaat tagtaattct ttcaaaaaat gccgataatt attactggtt tctcccgtct 9300

gcaaaaaaaa atatcattta taatgggata gatgataatg attgccttca aaataaaaaa 9360

tgtaattatc gtaaggaatt taatattcct gatgatggga tattagcagg ctcttgtgct 9420

aatttaacca agtgtaaagg gatcgatctt gttattcaga ccttaactaa agaacataaa 9480

atttactata tagttgccgg tgatggtatt gaaaaacata atcttattaa tcttgtaaaa 9540

gcgcgtaagc tgcatgaaag agtgtatttt atcgatttcc ttgatgaacc agaaagtttt 9600

atgtcccagt tggatgtttt tttgatgcca tccagaagtg aaggatttgg acttactgtt 9660

ttggaatcaa ctaagttagg tatacctgta ataacgtcta atattccaat atttatggag 9720

ttatttgatc aaatgtgtct cacatttgat attaaaaatc catctacatt aatcgatgta 9780

ataacctacg caaaaaaaaa tagattgcat ctttcgcaaa aattccatgc tatatttcaa 9840

gataggttta cttcttcaaa gatggctaca aaatatgaaa acgtctataa taatttattt 9900

Orf10 is initial, and orf9 stops

agagaggttt t atgagcttt tccgttttaa tgtctttata taatggtgaa agttccgagt 9960

atcttaatga ttgcttaagt agcattaatt cgcaaatatt aaaaccgaac caaattgtaa 10020

tcgtatatga tggcccaatt agaaaagaat tggacgatat agttacaaga tgggaaaagc 10080

aattgccaat acattgtgta aagctcactt cctgtacagg attaggtaat gcattgaatt 10140

atggtttgaa attttgtgat tacgatattg tctgtcgtat ggacactgat gatatatgta 10200

gaaatgacag atttttaaga caagttaaaa tacttgaaag tgataataat ctggctttga 10260

ttgggtctta tatctcagag tttgactcta ctccctcaaa cagtcatgct gttaggaccg 10320

ttcccattga gcataatgat atttgtttat atgcaaaaaa aagaaatccg tttaaccata 10380

tgactgtagc atttagaaaa agtgtagtaa cttctctcgg tgggtataag gatgaatatt 10440

tatatgaaga ctacgctctg tggattaaga tgcttaaaaa tggatttttg actaaaaata 10500

ttccagaggc gctagtttat gcgcgggttg gtaataatat ggagcttaaa cgcggtgggt 10560

ggaaatattt tacatcagaa gttaaggctc aatatggctt ctataaaatt ggttttctta 10620

Orf10 stops

acaaacagga aatgattatt aatattgttt tgagagcacc cggttcgttt aatgcc taac 10680

Orr11 is initial

tttatgagaa agattattta tagaaatttc cttagaaact gatataggta tataaatgag 10740

tgtattagtg acaggtgggg caggttatat tggtagtcat acagttttga ctctcttaca 10800

aaaaggaata gatgtcattg tgattgatga tttttctaat tctagcttag agtctttgga 10860

aagagttaaa aaaattacaa atcgagatat taaagtatat aagggggatg tagcagatct 10920

tcgtttattg aatcttattt tttctaatca tgatattcat acggttattc attttgctgg 10980

ttctaaatct gttggggagt ctatatcaaa accaattcat tactataata acaatgttgt 11040

agctagcctt gttcttatta atgaaatgat gaaaagaggt atttataatt taatttttag 11100

ttcatctgca acagtatacg gtaactcgaa taccatgcca gttactgaaa atgcacctat 11160

tgggatgact acaaatcctt atggcacttc taaacttatg attgaaaaaa tattagatga 11220

tgttacaagg gcaaattcga gctttagagt gacggttttg aggtatttta accctgtggg 11280

tgctcatttc tctggagaga ttggtgagga tccaaatggt attccaaata atcttatgcc 11340

atatgtttgt caggtggcaa ttggtaaata taaacaagta tactatagta taagtatact 11400

atacatgtat atatgcactg gtgtccgtga ttttatccat gtaatggatc tcgctgaagg 11460

tcatgttgct gcgttggaac atagaaacga aggtcccaat cataaagttt acaacctcgg 11520

aacaggaaag gggtattctg ttttggatct tttgaccact tttgaacgag taacctttcg 11580

atccgtacct tacgttttga gtgaaagacg ccctggagat atcgccgaat gttggtctga 11640

tccttctaag gcttataggg aacttggatg gaaagcgaag cgtgggttgg aagaaatggt 11700

Orf11 stops

tcgagatgcc tggaattggc aacaaaagaa tccgaacggt tatataaaag ca tgaatgct 11760

ctatggtatt tttgtcaacg gtaaaaatta tcgattttat gtatcctgag ttaatatagc 11820

atctacattg acctgagtta tgtttcgcag tatcaccctg acaggagtaa acaatgtcaa 11880

agcaacagat cggcgtcgtc ggtatggcag tgatggggcg caaccttgcg ctcaacatcg 11940

aaagccgtgg ttataccgtc tctattttca accgttcccg tgaaaagacg gaagaagtga 12000

ttgccgaaaa tccaggcaag aaactggttc cttactatac ggtgaaagag tttgttgaat 12060

ctctggaaac gcctcgtcgc atcctgttaa tggtgaaagc aggtgctggc acggatgctg 12120

ctattgattc cctcaagccg tacctcgata aaggtgacat catcattgat ggtggtaata 12180

cattcttcca ggacaccatt cgccgtaacc gtgagctttc tgccgaaggc tttaacttca 12240

tcggtaccgg tgtttccggt ggtgaagagg gcgcgctgaa aggtccttcc attatgcctg 12300

gtggccagaa agaagcctat gaactggttg ctccgatcct gaccaaaatc gccgcagtag 12360

ctgaagacgg tgagccatgc gttacctata ttggtgccga tggcgcaggt cactatgtga 12420

agatggttca caacggtatt gaatacggtg atatgcagct gattgctgaa gcctattctc 12480

tgcttaaagg tggcctgaat ctctctaacg aagaactggc acagaccttt accgagtgga 12540

ataacggtga actgagcagc tacctgatcg acatcaccaa agacatcttc actaaaaaag 12600

atgaaggcgg taactacctg gttgatgtga tcctggatga agcggctaac aaaggtaccg 12660

gtaaatggac cagccagagc gcgctggatc tcggcgaacc gctgtcgctg attaccgagt 12720

ctgtgtttgc acgttatatc tcttctctga aagatcagcg cgttgccgca tctaaagttc 12780

tctctggtcc gcaagctcag tcagcaggcg acaaggctga gttcatcgaa aaagttcgcc 12840

gtgcgctgta tctgggcaaa atcgtttctt acgctcaggg cttctctcag ctacgcgccg 12900

cgtctgaaga gtacaactgg gatctgaact acggcgaaat cgcgaagatt ttccgtgctg 12960

gctgcatcat ccgtgcgcag ttcctgcaga aaatcaccga tgcttatgcc gaagatccgc 13020

agatcgctaa cctgctgctg gctccgtact tcaagcaaat tgccgatgac taccagcagg 13080

cgctgcgcga tgtcgttgct tatgcagtac aaaacggtat cccggttccg accttcgccg 13140

ctgcggttgc ctattacgat agctaccgtg ccgctgttct gcctgcga 13188

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 O107 type is characterized in that described oligonucleotide is to being: the Nucleotide of 4649 to 4666 bases among the SEQ ID NO:1 and the Nucleotide of 5664 to 5681 bases; The Nucleotide of 4700 to 4717 bases among the SEQ ID NO:1 and the Nucleotide of 5626 to 5643 bases; The Nucleotide of 4753 to 4770 bases among the SEQ ID NO:1 and the Nucleotide of 5266 to 5283 bases; The Nucleotide of 5864 to 5882 bases among the SEQ ID NO:1 and the Nucleotide of 6852 to 6869 bases; The Nucleotide of 5895 to 5916 bases among the SEQ ID NO:1 and the Nucleotide of 6813 to 6831 bases; The Nucleotide of 6072 to 6090 bases among the SEQ ID NO:1 and the Nucleotide of 6762 to 6779 bases; The Nucleotide of 7141 to 7158 bases among the SEQ ID NO:1 and the Nucleotide of 7737 to 7754 bases; The Nucleotide of 7167 to 7185 bases among the SEQ ID NO:1 and the Nucleotide of 7710 to 7727 bases; The Nucleotide of 7190 to 7207 bases among the SEQ ID NO:1 and the Nucleotide of 7686 to 7703 bases; The Nucleotide of 7936 to 7954 bases among the SEQ ID NO:1 and the Nucleotide of 8774 to 8791 bases; The Nucleotide of 8029 to 8046 bases among the SEQ ID NO:1 and the Nucleotide of 8652 to 8669 bases; The Nucleotide of 8101 to 8118 bases among the SEQ ID NO:1 and the Nucleotide of 8623 to 8640 bases; The Nucleotide of 8862 to 8879 bases among the SEQ ID NO:1 and the Nucleotide of 9774 to 9791 bases; The Nucleotide of 8916 to 8933 bases among the SEQ ID NO:1 and the Nucleotide of 9681 to 9697 bases; The Nucleotide of 9025 to 9043 bases among the SEQ ID NO:1 and the Nucleotide of 9647 to 9664 bases; The Nucleotide of 9942 to 9959 bases among the SEQ ID NO:1 and the Nucleotide of 10586 to 10603 bases; The Nucleotide of 10019 to 10036 bases among the SEQ ID NO:1 and the Nucleotide of 10502 to 10520 bases; The Nucleotide of 10095 to 10112 bases among the SEQ ID NO:1 and the Nucleotide of 10454 to 10471 bases.

2, the application of the 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 O107 type as probe.