CN1234861C - Nucleotide peculiar to 0-antigen of 095 type bacillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O95. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O95 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 13932 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotides of glycosyl transferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O95. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O95 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O95 in the human body and the environment by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O95 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O95 type (Escherichia coli O95), particularly relate in the intestinal bacteria O95 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O95 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

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

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.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 O95 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O95 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 O95 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O95 type: i.e. the gene of abc transport system comprises wzm, wzt gene; Glycosyltransferase gene comprises the orf5 gene.

Another purpose of the present invention has provided oligonucleotide, and they come from the gene of coding abc transport system in the O-antigen gene bunch of intestinal bacteria O95 type respectively, comprise wzm, wzt gene; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O95 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O95 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O95 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 pass through O-antigen and the detection and the identification of escherichia coli O95 type of these methods detections and identification of escherichia coli O95 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O95 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 O95 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,13932 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 O95 type is comprising called after rmlB, rmlA, orf3, orf4, orf5, orf6, orf7, wzm, wzt, orf11, orf12,13 genomic constitutions of orf13 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type, the gene that has high degree of specificity in the wherein said gene is: the wzm gene of abc transport system; Glycosyltransferase gene comprises the orf5 gene; Wherein said gene wzm is the Nucleotide of 8944 to 9762 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 4268 to 5914 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type, wherein it is to come from described wzm gene, glycosyltransferase gene orf5 gene; And their mixing or their reorganization.The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type is characterized in that the oligonucleotide that wherein comes from the orf5 gene is to being: the Nucleotide of 4350 to 4365 bases among the SEQ ID NO:1 and the Nucleotide of 4888 to 4903 bases; The Nucleotide of 5246 to 5263 bases among the SEQ ID NO:1 and the Nucleotide of 5690 to 5707 bases.The oligonucleotide that comes from the wzm gene is to being: the Nucleotide of 9347 to 9364 bases among the SEQ ID NO:1 and the Nucleotide of 9743 to 9758 bases; The Nucleotide of 9160 to 9175 bases among the SEQ ID NO:1 and the Nucleotide of 9650 to 9665 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type is providing the O-antigen of expressing intestinal bacteria O95 type by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O95 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O95 type bunch: with the genome of intestinal bacteria O95 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

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

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

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O95 type;

(6) screening of specific gene: at wzm, the orf5 gene design primer in the O-antigen gene of intestinal bacteria O95 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzm, orf5 gene pairs intestinal bacteria O95 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O95, after the bacterial count respectively with 5 * 10 ³, 5 * 10 ², 5 * 10 ¹5 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O95.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O95 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O95 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 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, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O95 type bunch: with the genome of intestinal bacteria O95 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF sequences Design upstream primer (#1523-ATT GTG GCTGCA 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 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 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 the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of 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 penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O95 type;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O95 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O95 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O95 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 13 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 O95 type at last;

(6) screening of specific gene: at wzm, the orf5 gene design primer in the O-antigen gene of intestinal bacteria O95 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing the 13rd group of intestinal bacteria O95, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzm, orf5 gene pairs intestinal bacteria O95 type all is high special.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O95 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4350 to 4365 bases among the SEQ ID NO:1 and the Nucleotide of 4888 to 4903 bases; The Nucleotide of 5246 to 5263 bases among the SEQ ID NO:1 and the Nucleotide of 5690 to 5707 bases.The Nucleotide of 9347 to 9364 bases among the SEQ ID NO:1 and the Nucleotide of 9743 to 9758 bases; The Nucleotide of 9160 to 9175 bases among the SEQ IDNO:1 and the Nucleotide of 9650 to 9665 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O95 in the pork filling when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O95 type, its complete sequence shown in SEQ ID NO:1,13932 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 O95 type by method of the present invention, as shown in table 3, it comprises called after rmlB, rmlA, orf3, orf4, orf5, orf6, orf7, wzm, wzt, orf11, orf12,13 genomic constitutions of orf13 are all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O95 type, i.e. the gene of abc transport system (comprising wzm, wzt 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 transhipment enzyme gene that the present invention relates to and pol gene are high specials to the O-antigen of intestinal bacteria O95 type.

The 3rd aspect of the present invention provides the gene of the abc transport system in the O-antigen gene bunch that comes from intestinal bacteria O95 type, comprises wzm, wzt gene; They are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzm, orf5 gene pairs intestinal bacteria O95 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O95 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O95 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As used herein, " oligonucleotide " mainly refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzm gene (nucleotide position is the Nucleotide of 8944 to 9762 bases from SEQ ID NO:1); Orf5 gene (nucleotide position is the Nucleotide of 4268 to 5914 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide is high special to intestinal bacteria O95 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 the gene of abc transport system, comprise wzm, wzt gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is the genes that come from glycosyltransferase gene, abc transport system, comprises the combination of wzm, wzt gene.

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) the (ii) gene of abc transport system of the gene of encoding glycosyl transferring enzyme comprises wzm, wzt gene.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O95 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, and the gene of abc transport system comprises wzm, wzt gene.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O95 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 O95 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 the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding abc transport system to oligonucleotide, comprise wzm, wzt gene, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) the (ii) gene of abc transport system of the gene of encoding glycosyl transferring enzyme comprises wzm, wzt gene.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O95 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 the gene that derives from glycosyltransferase gene, abc transport system that one of them oligonucleotide molecules can hybridize to one, comprises on the sequence of wzm, wzt gene.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 O95 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 O95 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 O95 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 is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O95 type bunch:

With the genome of intestinal bacteria O95 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGTTCG 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, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCRPreps 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 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mMDTT 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 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated 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 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O95 type with the EcoRI enzyme.

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

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

Embodiment 5: the splicing of nucleotide sequence and analysis:

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O95 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O95 type is done 5 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O95 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 13 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 O95 type at last, as shown in table 3.

By retrieving and relatively, finding that the rmlB gene of orf1 and Escherichia coli has 97% homogeny in 297 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf1 is the rmlB gene.The rmlA gene of orf2 and Vibrio cholerae has 82% homogeny in 289 amino acid whose sequences, show the homology that height is arranged between them.So, can determine that orf2 is the rmlA gene.

Orf3, orf6, orf8, orf11, orf12 and orf13 encoded protein are not more found the function that it is determined by retrieval, so our tentative these several genes are orf3, orf6, orf8, orf11, orf12 and orf13.

NTP transferring enzyme among Orf4 encoded protein and the Pseudomonas syringae pv.tomato str.DC3000 (AE016858) has 58% sequence identity and 71% sequence similarity, but its exact function is unclear, so our tentative this gene is orf4.

Orf5 encoded protein and other known glycosyltransferases have 25% sequence identity and 43% sequence similarity.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of this genes encoding and known glycosyltransferase family is very high, but its exact function is unclear, so our tentative this gene is orf5.

The orf7 encoded protein respectively in the Escherichia coli O52 antigen gene bunch the albumen of Orf3 genes encoding 31% sequence identity and 45% sequence similarity are arranged, but its exact function is unclear, so our tentative this gene is orf7.

Orf9 and Legionella pneumophila, the translocase of abc transport (AJ007311) in 283 amino acid, have 32% sequence identity and 58% sequence similarity, this membranin is by the wzm genes encoding, so determine that orf9 is the translocase gene of abc transport, called after wzm.Orf10 and Legionella pneumophila, (AJ007311) translocator has 49% sequence identity and 73% sequence similarity in 474 amino acid, this albumen be in the abc transport system in conjunction with the subunit of ATP, be the transhipment polysaccharide the albumen with atpase activity.Orf 10 also with Klebsiellapneumoniae, (L31775) translocator in the abc transport system has 42% sequence identity and 67% sequence similarity in 246 amino acid, so determine orf10 be in the abc transport system in conjunction with the translocator of ATP with atpase activity, called after wzt.

Embodiment 6: the screening of specific gene:

Wzm, orf5 gene design primer in the O-antigen gene of intestinal bacteria O95 type bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O95 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzm, orf5 gene pairs intestinal bacteria O95 type all is high special; The position of these genes in nucleotide sequence is shown in Table 1.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O95 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4350 to 4365 bases among the SEQ ID NO:1 and the Nucleotide of 4888 to 4903 bases; The Nucleotide of 5246 to 5263 bases among the SEQ ID NO:1 and the Nucleotide of 5690 to 5707 bases.The Nucleotide of 9347 to 9364 bases among the SEQ ID NO:1 and the Nucleotide of 9743 to 9758 bases; The Nucleotide of 9160 to 9175 bases among the SEQ ID NO:1 and the Nucleotide of 9650 to 9665 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O95 in the pork filling when using aforesaid method.

By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O antigen gene bunch.O antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology 1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O antigen-specific gene order of O95 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O95 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O95 type.In table, list the glycosyltransferase gene of the O antigen gene bunch of intestinal bacteria O95 type, the gene of abc transport system, comprised wzm, wzt gene and their function corresponding and size.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

The genomic dna that contains intestinal bacteria O95 type in the 13rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzm, orf5 gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd group after the correct band of expection size, the correct band of any expection size that in other groups, all do not increase, that is to say, not obtaining any PCR product band in the array mostly, so wzm, orf5 gene pairs intestinal bacteria O95 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O95 type, screen gene by PCR: wzm, orf5 to the O-antigen high special of intestinal bacteria O95 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O95 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 O95 type.These all oligonucleotide all can be used for the intestinal bacteria O95 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 O95 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O95 type, altogether by 13 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 O95 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O95 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Bioarray Solutions Ltd.

＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O95 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O95 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>13932

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcgtc ctggttacgc actcgtccaa gaatgcggta 60

gaaaaccact tcgacacctc ttacgaactc gaagcgttgc ttgagcagcg cgttaagcgt 120

cagctgctgg cggaagtgca gtctatttgt ccgccgggcg tcaccatcat gaacgtgcgt 180

caggcacagc cgctgggtct gggacactct attctttgtg cccgtccggt ggtgggtgat 240

aacccgttca tcgttgtgct gccggatatc attattgata acgcgtctgc cgatccgctg 300

cgatacaacc tggcggcaat ggtggcacgt ttcaacgaaa cgggccgtag ccaggtgctg 360

gcgaagcgca tgaaaggcga tctctccgag tactccgtaa ttcagaccaa agaagcgctg 420

gaaacggaag ggcaggttag ccgcatcgtt gagttcatcg aaaaaccgga tcagccgcag 480

acgctggact cggacctgat ggcggttggt cgctatgttc tgaacgccga catctgggcg 540

gaactggaaa aaaccgagcc aggcgcctgg gatcgtatcc agctcaccga tgcgattgcc 600

gagctggcga agaagcagtc tgttgacgcg atgctcatga ccggcgagag ctatgactgc 660

ggtaagaaaa tgggctatat gcaggcgttc gtgaactacg ggctgcgcaa cctgaaggaa 720

ggggcgaagt tccgtaagag aattgagaaa cttcttacac aataagtcgt taaaccggat 780

atttcggttg gaacaatatg caggtagctg agggggattc aaagctgttc gcagctttaa 840

aatcaacctc gctacctttt ttttgtgtta aaaatcagta cctaaagaga acgatgcgcc 900

gttgtgactc cttattttac ggtgagattt ttccttgttt tcagcgccgt aaaaggggag 960

aataaccaat tagcattttg tgccaacccc ttctaaaata gctagccaac ttcggtttga 1020

gggctaaaaa aactgtcatt gttttcagta tactggtagc tgtagagcca ggggcggtag 1080

cgtgttttaa tttctgctaa ctacagtcct ccggctctat gaattaagtt cgaatctaaa 1140

cattacgacg caatacggaa tagtcttgtg aaaatacttg tcactggtgg tgcaggtttt 1200

attggatctg cagtagttcg ttatattatt aataatactg aagattctgt ggtaaacgta 1260

gataagttga cctacgctgg caacctggaa tcgttggcaa aaattgccaa tcatccgcgc 1320

tacgcatttg agcgagcaga tatctgtgat gcgagcgttt tggcacgcat tttcgcggaa 1380

catcagcccg atgcggtaat gcacctggcg gcagaaagcc atgt tgaccg ttctatcacc 1440

ggtcccgctg cgtttataga aacgaacatc gttggtacct atgtatcgct ggaagccgcg 1500

cgccaatatt ggtctcacct agacgaggcg cgcaaatcgg catttcgttt ccatcatatt 1560

tctactgatg aagtctacgg cgatctgccg catccagatg aattgccagc gggaagcacg 1620

ctcccactgt tcaccgaaac gacgccttac tctccaagta gtccatattc gtcttctaaa 1680

gcatcaagcg atcatttagt gcgtgcctgg cgtcgtacct atggtcttcc gacattggtg 1740

acaaactgtt cgaataacta cggtccttat cacttcccgg aaaaactgat cccactcatt 1800

attctgaacg ccctcgcagg caaaccgctt ccggtatacg gtaacggcga gcagatacga 1860

gattggctct ttgtcgaaga tcatgcgcgc gcgctataca aagtggtaac tgaaggtact 1920

gcgggtgaaa cctacaatat cggtggccac aacgagcgta aaaacatcga ggttgtgaag 1980

acaatctgca ccattctgga cgaaatcatt gccgataagc ccgcaggcat ttctcacttc 2040

gccgaactga ttacctatgt aaccgaccgc ccggggcatg acctgcgtta cgccattgat 2100

gccagcaaaa ttaaaaatga tctgggatgg gtaccacagg agacgtttga gtccggtatc 2160

cgtaaaacgg ttgagtggta tttaaataac gaaagctggt ggaagcgggt aatggatggc 2220

tcctatgcgg gcgaacgtct gggactggcg gaataaaaaa ggaaaatatg atgaaaggta 2280

ttgtacttgc tggtggttcc ggaacgcgtc tctatcctat aactcagggt gtatcaaaac 2340

agttgttgcc tgtctatgat aaaccgatga tttattatcc tatttctgta ctgatgttgg 2400

cgggtatccg cgatatatta atcattacaa ctccagatga tatcgatgcg tttaagcgtt 2460

tattaggcga tggcagtcaa tttggcataa atctcagctt tgcagttcaa ccttcccctg 2520

atggcctggc gcaggcattc atcattggtg aagaatttat taatggcgaa ccatgtgcgc 2580

tcgtgttagg tgatactgtc ttttttggcc aaagctttag taaaaaactt gaagctgcat 2640

tccaactaac ctctggagcg atggtctttg gttatcaagt tcaagatcct gaacggtttg 2700

gtgtagttga atttgatagt gaattcaaag cgttatctat tgaagagaaa ccagccaaac 2760

ctaaatctaa ttgggctgta acgggtctat atttctacga taaaaatgtt gtcgaaatgg 2820

caaaacagat taaaccctcg gtgcggggtg agcttgagat tacaacatta aatcagatgt 2880

atcttgagcg tggaggattg caagtcgagt tgttaggtcg cggttttgca tggctagata 2940

caggaactca tgacagtctg atagaagcat ctcaattcat tcatacaatt gaaaagcgcc 3000

agggtttgaa agtcgcttgt cttgaagaaa tagctttccg taaaggctgg atgaataaag 3060

aacaactatc ggcacaagcg aaaaaattct caaaaaatga ctatggccgc tacttacaga 3120

tgctgattag tgagtaatat tacgatgtgt tcttttttat taaaagcaat gtgaggtaac 3180

gtggaatact ccaacttaga ttcgatggta cgaggctggt ttgtcggtgg ctttgagcca 3240

accttatata agactactga cgttgaggtt gccgtacaac attttactgc cggagatcac 3300

gaggccgctc attgccataa aattgctact gaaataacag ttattatttc gggtcgagcc 3360

aaaatggcgg gcagaattgt tgtagcaggt gacatagtta agatttcgcc aggggaatat 3420

actgattttg aagctttaga agatacagtt actgctgttg taaaattgcc tggcgcttta 3480

aatgataaat atctcaaagg agatgagtga tgttaaatat tgttgttcca atggctggtc 3540

gcggaagtcg ttttgccaaa gaaggttatg tggatccaaa gccactgatt aagttaaaag 3600

acaagcgaat gattcaggtt gtaatcaata atttaactcc ttctattcct catcgtttta 3660

tatttatatg ccagcggcaa catgttatcg attatagttt ggaccggtat ttaaaagagt 3720

gggcaccgaa ttcagaagta atttgtattg atggaattac tgaaggcgca gcatgtaccg 3780

taatgtgtgc tgaggaatta atcaataacg atgaacctct tatgattgca aactcggatc 3840

aatggattga tattgatatt aatgattatc taaattacat gaagcatgag gatttagacg 3900

ggcttatcat gacaatgaaa gccgatgacc ctaaatggtc ttatgcggaa atgaatgaaa 3960

ataaccatgt tactcgagtt gttgaaaaag aagttgtatc cgatgaagcg actgtaggga 4020

tttataatta taaaaaaggc gctgattttt gtagaagtgt caaaagaatg attgctttag 4080

atcaacgctc taatggtgaa ttttatgttg cacctgctta tacactcatg tatgagaatg 4140

aaggcgcaaa aattggaatt tataatattg gggaagaagc aaacgggatg tatggtttgg 4200

gtatccctgc tgatcttgaa ttgttccttt cccttgatgt atcaagaaaa gcgttggatt 4260

tctaatcatg cacgtagcaa aaaagatctg tttgattggg catgagtgcg ggtcatataa 4320

agggcaagga ggtatagcta catacattga actcaccgcg aaaggattcg caaaacttgg 4380

tttagaagtt catgttattt acatccatgg atccggggta gactgcgatg atataaagtc 4440

atggaagata aaaaatgatc ctagccttta taaaatatca cttgaagtag acaaggtttt 4500

agaagaaata aaaccggact ttgtagagtg tacagacttt atagggatga tgagttatac 4560

gctttcaaag cgagcgatca gaggtttaga ttataaatgc atttttataa ctaatcatca 4620

cacggggatt aaagaagtct gggaatgggg aacacagctt aaatttaatg agacagcaaa 4680

gtcatggatg aagtctttat atatagctga aagaacacaa tctcttttat cagatgcaaa 4740

cttctcgaca tctaattttc tagccagtta tttagatgca aaccatattg aatcatatca 4800

agtatcgccg tcttattatg aaatgaatga taattttggt gaaacaaata aaaatcacta 4860

tgattctaat ttattacgta tcatatcgct tggcagattc gagttaagaa aaaaacaaga 4920

gcttcttatt aaagctactt gtgaacttct tgatgaaggc tatgatattg aaacaacttt 4980

gattgggaat agtggcggtg atttatatac gcatcaggac tatatggaat attgctatag 5040

cctgattccc acagagtata agcataagtt tcatttttat gacttcatgc catataagtt 5100

gcttcagaag aaatataccg aatatgattt gtttgtaatc ccatctccat atgagaattt 5160

tccaaataca gctcttgaag ctatcaacta tggagttaca gttgtcggaa gtaaatccag 5220

tggcgtagct gatatgatag gggaggattt atccgattac tgcttcttgc ctgattcagt 5280

ttctgatatt aaaagagtga ttttaaaata taaccagtta gatgctgaag aaagggcgac 5340

tctacgtttg aaacaacgtc tgtccttaaa aaatcttact tgctttgaaa agagtataca 5400

agaacgcttt gaaaagtatc aagctgtaga tgagctacgt agtgaaaaaa atattgatga 5460

taaagatata ttgattgtat atcaggacaa aaatgggctc atcaataaag tagagtattg 5520

ctcagaaatt tattactccc ttgggagcaa atggaaagac tttatcagag aaattaaata 5580

tattgtcttg acctccgagg aatatcaatt taacaagata gagaactact atccggcacc 5640

cggaatcata tcctgttttt catcatatga tccttatgga acggttagag aaatacaaca 5700

ggctgaaaaa ggatattcat ctttaacatt atgtgtagag acaatcccat ataatgaagg 5760

tgattcaata tctgaatata tcgctcaagt tttactttca acaagcgatg tcatattctt 5820

cattgatgaa gaaaagaaag tagaacaagg catcagtatc gatatcaaat acgcgattga 5880

taaaattaag tttaattatt ctggtgaaaa atgaatataa taatccatgg gccgctagaa 5940

aaagatttct tatttaaaaa cttaaataaa attcggcacg cttcagaaga cgatcacata 6000

attatttctg tttataacga agaacttgaa aaaacaaagg ttattataga cagcgtccca 6060

gtcagaaata aggttacatt aatcggttct gatgatgttt ttaatcctgg cttttttaac 6120

attaacagac aaattaatct ggttagtgcc gcattaaacg tcattgaaga tgaaagtgac 6180

tttattctaa aagttcgaat ggatcagact attaactttc gttgggttag aaaagttgta 6240

actgagaagc atgaagaact gaaggataaa ttgatcacca ctaactgcta taccagaaaa 6300

gatcgtcttt atcatccatc tgatatgttc ctcgccggca cgaaaaaaac acttcgtgca 6360

tattatccag aagagttttt ccaagagact cacatggata atttattgaa aataaaagaa 6420

ctagtgctct ccggatataa aggcggatac cataaatatt ggccagaatc acgactgttt 6480

atgaattact tatccatatt aggtgaggaa ttacaggata cagagatctg tagtcagtca 6540

caattgaaaa agcatgttta tatttttaat tcatgggata ttggattaaa atggaaaaaa 6600

ttccttaaag gcaaagttaa cgttttacca tatttctttg ttttaagacc tttcgaaaga 6660

gggcctcttg agaacgcaga gaatttttta gcaagtgact taacaggtta tacaaaaaag 6720

cctttgaggg aaatgatata taatagttta gcaaaattat attttaaatc tggagcttat 6780

ttagcgaacc caatattcat agataacaaa tcacttatac caaaattact acgaaaaatg 6840

tttgatcatt catataaatt catccctccc attttgcata atcttatgta taaagtcgca 6900

aagagagttt attatgcatt caaataagat atatataatt gggtggtcag gctttattgg 6960

tcggcaatat attcattatc ttgataaaac gggctttaag ggtgaggttt ttcttaactc 7020

acgtgccgac gatgagcaaa aatacacttc ttctacagat aacatcacaa taaaatatgt 7080

taattatgag gagatgatta ctgggcttaa ttgttcaagg tcaactgtat tataccttgc 7140

aaatatgtac tcaccgggtg agtcgaatat ttaccctttg aatagtgtca aagaaaacat 7200

tattccattt atcacttttt tagaggatat caagaataat gcaagcaata tcaggttcat 7260

atttgcttct tcaggaggga ctatatatgg cgatagtcaa gaaggggaat gttgcagcga 7320

gaaccactta ttagctccga aaaccattta tgcagctaac aaaatagcgc aagagaacta 7380

tctcaacgtt tatcatgtta attatggtct tgattatcgg atagccagaa ttgcaaaccc 7440

ttatggcccg gggcagatat tgaaaggtgg tcaaggatta ataccagcaa tacttcgttc 7500

gtcattaaat aatgaaagcc ttcctgtgag tggtgatgga aatggtacaa gagattatat 7560

atatatagat gatctttgtt cacttttttt cagtctaagt agctatagcg gagcttgtag 7620

aacatttaat gcgggctctg gtaaagagta tagtataatg gaaatcataa gttgctttaa 7680

cgcagtgcat gacagaccaa taaaatacca tcatgttaaa accgaatctt cagttgtaag 7740

ccgtattgtc cttgatatca gtcgtgctaa aaatgagctg ggatggagcc ctaaaacttc 7800

cctatctgat gggattcgtg cctttatcaa ctggtcgatt caagtgatgt agttttccat 7860

atttcaataa tctaacctca catgacatac accactcaaa tctcggctca agatgtctcg 7920

attgttatcc aaggggcgat tttcgataca aaaaaacaaa aagttgatgc tcaatttatt 7980

gaatacttag aaaaagttat ttcgatattt ataggttgcg aatttattgt ttcgacgtgg 8040

gaatttcctc gcgaaatata cttcggtctt tgcgaaaaat atccgcaagt aaattttgtt 8100

ctgaatgctg atgttgggcc aataataaaa atagtagatg atgtaccgat tgtcaccaat 8160

gttaatagga tgattttttc gacaataagt ggactcaggg aagttaatcg caaatatgct 8220

attaaattga gaacagattc tttcttatac aatgattcga tactccaatc gctttattat 8280

gttatgaata aacgcgaatt tttcggatta gacttggaaa agagaaatga gcgatatcgg 8340

atctttgatc atcatgtaat taactgtaat ctatttgccc gcaatccgcg cagtcattta 8400

cctttcttat atcatcctgg tgatattttc ctggcaggat taactaaaga tttaattaag 8460

cttttcaata taccattggc gacggaagac ctaataaaaa aatgcaatag tgtgagaaat 8520

acttgcttta tgaaacttgt acctgaacaa tatatttggg ttcagtgtat taaaatgtca 8580

agaagtacag atacgttcga ttattcaaat ttcacatatg atgaaaaaga aatcgaaaaa 8640

tcagagcagt attatttaaa taattttgta ccatacactg cagaagaatt gggtttttgt 8700

tggccaaagc atcgagaggt ttattttaac aaagggagtt caagtattta tgatcatgaa 8760

gattggcatc atttgtatca taaatacatt gacggttttg aacaacctac aagttatgca 8820

cataaaaaaa gaagtgtggt tatattcttt atgaagatgt attttttcat tcgtagctct 8880

ctgcttaaaa taaaattcat tcgcaaattt gcttacaaag tttttgtaaa aagaggatga 8940

cttatgaagg gatttgtttc aacgccctcc agtacaacgc gctttagtga agtttttgaa 9000

tcacgtaatt taattgcaga acttattcgg cgagatttta atgtccgcta taagcaaaca 9060

gggcttggag tattatgggc aataattaat ccaggcgtca atattcttct ttatctcttt 9120

gtatttggtt tgttagttag gttaccaacc ccagaatata atgctcctta tgctgcggtg 9180

ttaatctcag cgattttatt ttggaattta ttttctacta gcatgctcac agcaagtgat 9240

gcgctgatta ataacatgca tcttgttact aaagtttatt tcccaaggtt atctttatgt 9300

atagcaagtg tatttgttgc attgattgat ttcttgatag cttttatcgt gtttgtcccg 9360

ttagcattat tgtatcatgt ggacataagt ctaattagat tgcttttatt aatcccatgt 9420

ataattataa ctctgatgct tggatgtggt tttggctgtg caatggctat attgaaattg 9480

aagtatagag atattagaca tatacttcct ctaataaccc aggttatttt ctttgcgtca 9540

cctattgtat atactctatc cattgttcca gaacaatata aaatattcta ttcttttaac 9600

cccatcactg ggatcgtaat gatgtctcgc tgggctatct taggcggagc acccattgac 9660

ttgacaatgc ttacttatag cctaattagt gcatttgtcg ttatgtgttt aggtgtcatt 9720

tattttgtta aaaatgatcg atctgtggcg gattacgaat gattgagttt gattcggtaa 9780

gtaagcagta tgaaatcaac catcgacggg ccaaaagctt acgcgatgtt ttttcattta 9840

acaaaaagcc agatgcaaat gcagaaatat ttaaagcggt tgatgatgtc agctttagta 9900

ttgatagcgg taaatcagta ggtgtcttgg gacgaaatgg tgcgggaaaa tcaacgcttt 9960

taaaattatt aacgcgtatc atttccccga catcaggcaa aataactgta gatggctcca 10020

tttcttgtct gctggaagtt ggcgctggtt ttcaccatga actcagtggc tacgaaaata 10080

tttttctatg tggctcaatc ttgggaatga gtcgtaaaca agtcaaatct aaaatagaat 10140

ctattattga tttttctgag gtcgagaaat ttattgacga acctgttaag tattattcat 10200

caggtatgta tatgcgctta gcttttgcga taggtgctca tcttgactcg gatattctag 10260

tgatcgacga agctttggct gttggtgact ctcgttttca aaataaatgt ttaaataaaa 10320

tcgacgagat tagaaaaagt ggaaagacta tattttttgt tagtcatagc attgaacaag 10380

taaaaaaagt gtgtgatacc agtattgtta tggatcatgg gaaattattg ttccagggca 10440

atactgatga agctgaaaga atttacaata gtcttactaa tggtgcgtga gtatgatcat 10500

tatttctcat agagggtatt ggaagttgag cagtgaaaaa aatactgctt tggctttcaa 10560

acgctcattt tctttaggtt atggtactga aacagatatt cgtgactata agggtagttt 10620

agtcatcagt catgatatac cagatgagag ttcattaagc atagactctt tttttgaaat 10680

ttataatcaa tatgatgtca aaggcccttt ggcattaaac gtaaaatcag atgggctgca 10740

aaaactaata aaagaaaaat taaacgagta caaggttagt aattattttt tctttgatat 10800

gtctatcccg gatactcttg ggtatttatc agaaaaacta aatacgttcg tgcgtgttag 10860

tgaatatgaa acactaaacg atctctatga agatgctgat ggtgtttgga ttgacggttt 10920

caaagaaaat attataacag aatctttact cgaaaaaatt atttctgatg ggaagcgtgc 10980

ttgtattgta tccgctgatc tgcataaaaa cgatccagct acgcaatgga aacttttaaa 11040

aagttttaaa acaaatattc ttcagtcgtc aaatattatt ctttgtactg atttacctga 11100

aaaagcgtcg gagtttttct atggataata aaattaaagc cgtaatcttc gatatggacg 11160

gagttcttat cgaagctaaa gactggcatt acgaagctct aaatcgcgct ttgggactct 11220

tcggtctgga gataaccaag catgaacatc ttacaaccta tgatggtcta cctactaaag 11280

ataagttagt catgttgtct cgtgataaag gtttgtcaaa tgcgctgcat acctttatta 11340

atgaaatgaa gcaacagtat actatggata tggttcataa attttgtaaa cctatgttcc 11400

atcatgaatt cgccctatca atgcttaaac aagatgggta caaattagct gtagcatcta 11460

attcaatacg gaattccgta gaggtgatgc ttgataaagc taaactatta gcttatttgg 11520

agttttttat ttctaatcag gatgtgaaaa aaggtaagcc ggatcctgag atgtatactc 11580

ttgctatttc aaagttggga ttaagacctt ctgaatgctt aattgttgaa gataatgaga 11640

atgggataaa agcagctaaa gcaagtggtg ctcacgttct tgaagttgaa actgtatacg 11700

atgtaaatta tcaaaacatc aaacaacgta ttaaaaaaat tgaaaacgat gagggtggaa 11760

aatgattaat atcctaattc caatgagtgg agagaatctt tatgaaacct ccagtaattt 11820

catctaccca aaaattttaa ctgaagttgc aaataaaact ctcttggaat attcgcaatt 11880

aatatttgat aatctgcgag acgaaaaaaa tcttatttac attgctccag aagataaact 11940

taacactctt ggtttgaaat caatcattca aactattaca gatggtaaag gtaaaattgt 12000

tgccctccaa gggcaaacca agggtgctgt ctgtagttgt cttatggcga tcgatgatct 12060

ctcacttgat gacgaattga ttatttcttc tgcggatcat tatattgacg ataatttgca 12120

aaggatcgtt aatgatttta gggtcatgga cgctgacgct ggggttctta cctttgaatc 12180

tgtgcatccc aaatggtcgt ttgtaacact aaatgagcat ggtgcagttg ttcaggccgc 12240

agagaaaaaa gcgattagca gaacagctgt agctgggctg tattatttta aaaaggcgcg 12300

tgattttgtc gaggctgcga tgaatctcat tagaaaggat ggcgatataa atggtaattt 12360

ctatttgtct tcctgcctta atgagctcgt tttgttgggt aaggttatca aatgtcgccc 12420

gctacaagat gctatctatc ataactttta tgatgcccac gcagttaaat cttttgagct 12480

tgctcagaca tccctttcta atagcgttaa gcaactcacg aattcctaca ttgaagcatt 12540

taactcgaaa gatatagaag aatgtctttt tcttttttca gacgatgcgg ttcttgttga 12600

gcccaacaaa acatttactg ggaaagatga aataaaggaa ttgcttaacg atatttttgt 12660

gaacaattct aaactccaat tccgtgccga taatattatt gctgatgata gtaaatctgc 12720

aatcaagttt acacttgagc ttactaacga gactgttcat ggcgttgatt tcattgaatg 12780

gaatagtgcg ggtaaaatcc ataaactgac tgctttttta aatgattaaa atagttacca 12840

gttttggaat gtgaactgtg aacatttatg gatgacttta attgatgaaa aatcgtgtga 12900

tttgatatct ttttttttaa ttaatgattt atcgaaattt tttaaaatcg cacaaattca 12960

cgctattcgc tgcgaagagc ttgagttaac ataatgttca cttctcccag caggataatg 13020

ggcttgctgg taaaccacac tagacaggag tatgtaatgt ctaagcaaca aatcggcgtt 13080

gtcggtatgg cagtgatggg ggcgcaacct ggcgctcaac atcgaaagcc gtggttatac 13140

cgtctccgtt ttcaaccgct cccgtgataa gaccggagaa gtgattgccg agaacccagg 13200

caagaaactg gtccctttct atacggttaa agaattcgtc gaatctctgg aaacccctcg 13260

tcgtatcctg ttaatggtga aagcaggtgc aggtaccgat gctgctatcg actccctgaa 13320

gccttacctc gaaaaaggcg acatcatcat tgatggcggt aacaccttct tccaggacac 13380

cattcgtcgt aatcgtgaac tgtctgctga aggcttcaac ttcattggca ctggtgtttc 13440

cggtggtgaa gagggcgctc tgaaaggccc atccatcatg cctgggggcc agaaagaagc 13500

atacgaactg gttgcgccta ttctgaccaa aatcgcagct gttgcagaag atggcgagcc 13560

gtgtgtgacc tatatcggtc cggacggtgc agggcactat gttaaaatgg tgcacaacgg 13620

cattgaatac ggcgatatgc agctgatcgc ggaagcatac tctctcctga aaggcggcct 13680

gaatctcacc aatgaagagc tggcggaaac cttcaccgag tggaacaaag gcgagctgaa 13740

cagctacctg atcgacatca ccaaagatat cttcacgaag aaagatgaag agggtaaata 13800

cctggttgat gtgattctgg atgaagcagc gaacaaaggc accggcaaat ggaccagcca 13860

gagttctctg gatctgggcg aaccgctgtc tctgatcacc gaatctgtct tcgcgcgtta 13920

catctcttct ct 13932

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O95 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 (℃)
								Wzm	Abc transport	8944-9762	9347-9364	9743-9758	412bp	0 *	63.3
			9160-9175	9650-9665	506bp	0 *	61.3
								Orf5	Glycosyltransferase	4268-5914	4350-4365	4888-4903	554bp	0 *	62.8
			5246-5263	5690-5707	462bp	0 *	57

^*Only in intestinal bacteria O95 type, obtain a correct band

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

Group number 1, wild-type e. coli	The bacterium source O1 that contains in this group, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, IMVS a O19ab，O20，O21，O22，O23，O24
		2, wild-type e. coli	O4，O10，O25，O26，O27，O28，O29，O30，O32，O33，O34，O35，IMVS a O36，O37，O38，O40，O41，O42，O43
3, wild-type e. coli	O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56，IMVS a O57，O58，O60，O61，O62，O53
		4, wild-type e. coli	O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78，IMVS a O79，O80，O81，O82，O83，O68
5, wild-type e. coli	O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99，IMVS a O101，O102，O103，O104，O105，O106，O97
		6, wild-type e. coli	O107，O108，O109，O110，O111，O112ab，O112ac，O113， IMVS a

	O115，O116，O118，O120，O123，O125，O126，O128，O117
			7, wild-type e. coli	O129，O130，O131，O132，O133，O134，O135，O136，O137， IMVS a O138，O139，O141，O142，O143，O144，O145，O140
8, wild-type e. coli	O146，O147，O148，O150，O152，O154，O156，O157，O158， IMVS a O159，O160，O161，O163，O164，O165，O166，O153 b
			9, wild-type e. coli shigella dysenteriae	O168，O169，O170，O171，O172，O173， c D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12，D13 d
10, Shigella bogdii	B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， d B16，B17，B18
			11, shigella flexneri	F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v：4)，F5(v：7)，F6， d DS，DR
12, wild-type e. coli	O3，O11，O39，O59，O64，O73，O96，O100，O114，O151，O155， IMVS a O124，O167，O162，O121，O127，O149，O119
			13, the 12nd group of bacterial strain adds the intestinal bacteria reference culture		O95 IMVS a

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

a.Institute of Medical and Veterinary Science(IMVS)，Anelaide，Australia

b.Statens Serum Institut，Copenhagen，Denmark

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

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

Table 3 intestinal bacteria O95 type O antigen gene structure iron

E.coli O95 O-antigen gene cluster←

Table 4 intestinal bacteria O95 type O antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCGTC CTGGTTACGC ACTCGTCCAA GAATGCGGTA 60

GAAAACCACT TCGACACCTC TTACGAACTC GAAGCGTTGC TTGAGCAGCG CGTTAAGCGT 120

CAGCTGCTGG CGGAAGTGCA GTCTATTTGT CCGCCGGGCG TCACCATCAT GAACGTGCGT 180

CAGGCACAGC CGCTGGGTCT GGGACACTCT ATTCTTTGTG CCCGTCCGGT GGTGGGTGAT 240

AACCCGTTCA TCGTTGTGCT GCCGGATATC ATTATTGATA ACGCGTCTGC CGATCCGCTG 300

CGATACAACC TGGCGGCAAT GGTGGCACGT TTCAACGAAA CGGGCCGTAG CCAGGTGCTG 360

GCGAAGCGCA TGAAAGGCGA TCTCTCCGAG TACTCCGTAA TTCAGACCAA AGAAGCGCTG 420

GAAACGGAAG GGCAGGTTAG CCGCATCGTT GAGTTCATCG AAAAACCGGA TCAGCCGCAG 480

ACGCTGGACT CGGACCTGAT GGCGGTTGGT CGCTATGTTC TGAACGCCGA CATCTGGGCG 540

GAACTGGAAA AAACCGAGCC AGGCGCCTGG GATCGTATCC AGCTCACCGA TGCGATTGCC 600

GAGCTGGCGA AGAAGCAGTC TGTTGACGCG ATGCTCATGA CCGGCGAGAG CTATGACTGC 660

GGTAAGAAAA TGGGCTATAT GCAGGCGTTC GTGAACTACG GGCTGCGCAA CCTGAAGGAA 720

GGGGCGAAGT TCCGTAAGAG AATTGAGAAA CTTCTTACAC AATAAGTCGT TAAACCGGAT 780

ATTTCGGTTG GAACAATATG CAGGTAGCTG AGGGGGATTC AAAGCTGTTC GCAGCTTTAA 840

AATCAACCTC GCTACCTTTT TTTTGTGTTA AAAATCAGTA CCTAAAGAGA ACGATGCGCC 900

GTTGTGACTC CTTATTTTAC GGTGAGATTT TTCCTTGTTT TCAGCGCCGT AAAAGGGGAG 960

AATAACCAAT TAGCATTTTG TGCCAACCCC TTCTAAAATA GCTAGCCAAC TTCGGTTTGA 1020

GGGCTAAAAA AACTGTCATT GTTTTCAGTA TACTGGTAGC TGTAGAGCCA GGGGCGGTAG 1080

CGTGTTTTAA TTTCTGCTAA CTACAGTCCT CCGGCTCTAT GAATTAAGTT CGAATCTAAA 1140

Orf1's is initial

CATTACGACG CAATACGGAA TAGTCTT GTG AAAATACTTG TCACTGGTGG TGCAGGTTTT 1200

ATTGGATCTG CAGTAGTTCG TTATATTATT AATAATACTG AAGATTCTGT GGTAAACGTA 1260

GATAAGTTGA CCTACGCTGG CAACCTGGAA TCGTTGGCAA AAATTGCCAA TCATCCGCGC 1320

TACGCATTTG AGCGAGCAGA TATCTGTGAT GCGAGCGTTT TGGCACGCAT TTTCGCGGAA 1380

CATCAGCCCG ATGCGGTAAT GCACCTGGCG GCAGAAAGCC ATGTTGACCG TTCTATCACC 1440

GGTCCCGCTG CGTTTATAGA AACGAACATC GTTGGTACCT ATGTATCGCT GGAAGCCGCG 1500

CGCCAATATT GGTCTCACCT AGACGAGGCG CGCAAATCGG CATTTCGTTT CCATCATATT 1560

TCTACTGATG AAGTCTACGG CGATCTGCCG CATCCAGATG AATTGCCAGC GGGAAGCACG 1620

CTCCCACTGT TCACCGAAAC GACGCCTTAC TCTCCAAGTA GTCCATATTC GTCTTCTAAA 1680

GCATCAAGCG ATCATTTAGT GCGTGCCTGG CGTCGTACCT ATGGTCTTCC GACATTGGTG 1740

ACAAACTGTT CGAATAACTA CGGTCCTTAT CACTTCCCGG AAAAACTGAT CCCACTCATT 1800

ATTCTGAACG CCCTCGCAGG CAAACCGCTT CCGGTATACG GTAACGGCGA GCAGATACGA 1860

GATTGGCTCT TTGTCGAAGA TCATGCGCGC GCGCTATACA AAGTGGTAAC TGAAGGTACT 1920

GCGGGTGAAA CCTACAATAT CGGTGGCCAC AACGAGCGTA AAAACATCGA GGTTGTGAAG 1980

ACAATCTGCA CCATTCTGGA CGAAATCATT GCCGATAAGC CCGCAGGCAT TTCTCACTTC 2040

GCCGAACTGA TTACCTATGT AACCGACCGC CCGGGGCATG ACCTGCGTTA CGCCATTGAT 2100

GCCAGC AAA TTAAAA TGA TCTGGGATGG GTACCACAGG AGACGTTTGA GTCCGGTATC 2160

CGTAAAACGG TTGAGTGGTA TTTAAATAAC GAAAGCTGGT GGAAGCGGGT AATGGATGGC 2220

The termination orf2's of Orf1 is initial

TCCTATGCGG GCGAACGTCT GGGACTGGCG GAA TAAAAAA GGAAAAT ATGATGAAAGGTA 2280

TTGTACTTGC TGGTGGTTCC GGAACGCGTC TCTATCCTAT AACTCAGGGT GTATCAAAAC 2340

AGTTGTTGCC TGTCTATGAT AAACCGATGA TTTATTATCC TATTTCTGTA CTGATGTTGG 2400

CGGGTATCCG CGATATATTA ATCATTACAA CTCCAGATGA TATCGATGCG TTTAAGCGTT 2460

TATTAGGCGA TGGCAGTCAA TTTGGCATAA ATCTCAGCTT TGCAGTTCAA CCTTCCCCTG 2520

ATGGCCTGGC GCAGGCATTC ATCATTGGTG AAGAATTTAT TAATGGCGAA CCATGTGCGC 2580

TCGTGTTAGG TGATACTGTC TTTTTTGGCC AAAGCTTTAG TAAAAAACTT GAAGCTGCAT 2640

TCCAACTAAC CTCTGGAGCG ATGGTCTTTG GTTATCAAGT TCAAGATCCT GAACGGTTTG 2700

GTGTAGTTGA ATTTGATAGT GAATTCAAAG CGTTATCTAT TGAAGAGAAA CCAGCCAAAC 2760

CTAAATCTAA TTGGGCTGTA ACGGGTCTAT ATTTCTACGA TAAAAATGTT GTCGAAATGG 2820

CAAAACAGAT TAAACCCTCG GTGCGGGGTG AGCTTGAGAT TACAACATTA AATCAGATGT 2880

ATCTTGAGCG TGGAGGATTG CAAGTCGAGT TGTTAGGTCG CGGTTTTGCA TGGCTAGATA 2940

CAGGAACTCA TGACAGTCTG ATAGAAGCAT CTCAATTCAT TCATACAATT GAAAAGCGCC 3000

AGGGTTTGAA AGTCGCTTGT CTTGAAGAAA TAGCTTTCCG TAAAGGCTGG ATGAATAAAG 3060

AACAACTATC GGCACAAGCG AAAAAATTCT CAAAAAATGA CTATGGCCGC TACTTACAGA 3120

The termination of orf2

TGCTGATTAG TGAG TAATAT TACGATGTGT TCTTTTTTAT TAAAAGCAAT GTGAGGTAAC 3180

Orf3's is initial

GTGGAATACT CCAACTTAGA TTCGATGGTA CGAGGCTGGT TTGTCGGTGG CTTTGAGCCA 3240

ACCTTATATA AGACTACTGA CGTTGAGGTT GCCGTACAAC ATTTTACTGC CGGAGATCAC 3300

GAGGCCGCTC ATTGCCATAA AATTGCTACT GAAATAACAG TTATTATTTC GGGTCGAGCC 3360

AAAATGGCGG GCAGAATTGT TGTAGCAGGT GACATAGTTA AGATTTCGCC AGGGGAATAT 3420

ACTGATTTTG AAGCTTTAGA AGATACAGTT ACTGCTGTTG TAAAATTGCC TGGCGCTTTA 3480

The termination orf4's of orf3 is initial

AATGATAAAT ATCTCAAAGG AGATGAG TGA TGTTAAATAT TGTTGTTCCA ATGGCTGGT 3540

GCGGAAGTCG TTTTGCCAAA GAAGGTTATG TGGATCCAAA GCCACTGATT AAGTTAAAAG 3600

ACAAGCGAAT GATTCAGGTT GTAATCAATA ATTTAACTCC TTCTATTCCT CATCGTTTTA 3660

TATTTATATG CCAGCGGCAA CATGTTATCG ATTATAGTTT GGACCGGTAT TTAAAAGAGT 3720

GGGCACCGAA TTCAGAAGTA ATTTGTATTG ATGGAATTAC TGAAGGCGCA GCATGTACCG 3780

TAATGTGTGC TGAGGAATTA ATCAATAACG ATGAACCTCT TATGATTGCA AACTCGGATC 3840

AATGGATTGA TATTGATATT AATGATTATC TAAATTACAT GAAGCATGAG GATTTAGACG 3900

GGCTTATCAT GACAATGAAA GCCGATGACC CTAAATGGTC TTATGCGGAA ATGAATGAAA 3960

ATAACCATGT TACTCGAGTT GTTGAAAAAG AAGTTGTATC CGATGAAGCG ACTGTAGGGA 4020

TTTATAATTA TAAAAAAGGC GCTGATTTTT GTAGAAGTGT CAAAAGAATG ATTGCTTTAG 4080

ATCAACGCTC TAATGGTGAA TTTTATGTTG CACCTGCTTA TACACTCATG TATGAGAATG 4140

AAGGCGCAAA AATTGGAATT TATAATATTG GGGAAGAAGC AAACGGGATG TATGGTTTGG 4200

GTATCCCTGC TGATCTTGAA TTGTTCCTTT CCCTTGATGT ATCAAGAAAA GCGTTGGATT 4260

The termination orf5's of orf4 is initial

TC TAATC ATGACGTAGCAA AAAAGATCTG TTTGATTGGG CATGAGTGCG GGTCATATAA 4320

AGGGCAAGGA GGTATAGCTA CATACATTGA ACTCACCGCG AAAGGATTCG CAAAACTTGG 4380

TTTAGAAGTT CATGTTATTT ACATCCATGG ATCCGGGGTA GACTGCGATG ATATAAAGTC 4440

ATGGAAGATA AAAAATGATC CTAGCCTTTA TAAAATATCA CTTGAAGTAG ACAAGGTTTT 4500

AGAAGAAATA AAACCGGACT TTGTAGAGTG TACAGACTTT ATAGGGATGA TGAGTTATAC 4560

GCTTTCAAAG CGAGCGATCA GAGGTTTAGA TTATAAATGC ATTTTTATAA CTAATCATCA 4620

CACGGGGATT AAAGAAGTCT GGGAATGGGG AACACAGCTT AAATTTAATG AGACAGCAAA 4680

GTCATGGATG AAGTCTTTAT ATATAGCTGA AAGAACACAA TCTCTTTTAT CAGATGCAAA 4740

CTTCTCGACA TCTAATTTTC TAGCCAGTTA TTTAGATGCA AACCATATTG AATCATATCA 4800

AGTATCGCCG TCTTATTATG AAATGAATGA TAATTTTGGT GAAACAAATA AAAATCACTA 4860

TGATTCTAAT TTATTACGTA TCATATCGCT TGGCAGATTC GAGTTAAGAA AAAAACAAGA 4920

GCTTCTTATT AAAGCTACTT GTGAACTTCT TGATGAAGGC TATGATATTG AAACAACTTT 4980

GATTGGGAAT AGTGGCGGTG ATTTATATAC GCATCAGGAC TATATGGAAT ATTGCTATAG 5040

CCTGATTCCC ACAGAGTATA AGCATAAGTT TCATTTTTAT GACTTCATGC CATATAAGTT 5100

GCTTCAGAAG AAATATACCG AATATGATTT GTTTGTAATC CCATCTCCAT ATGAGAATTT 5160

TCCAAATACA GCTCTTGAAG CTATCAACTA TGGAGTTACA GTTGTCGGAA GTAAATCCAG 5220

TGGCGTAGCT GATATGATAG GGGAGGATTT ATCCGATTAC TGCTTCTTGC CTGATTCAGT 5280

TTCTGATATT AAAAGAGTGA TTTTAAAATA TAACCAGTTA GATGCTGAAG AAAGGGCGAC 5340

TCTACGTTTG AAACAACGTC TGTCCTTAAA AAATCTTACT TGCTTTGAAA AGAGTATACA 5400

AGAACGCTTT GAAAAGTATC AAGCTGTAGA TGAGCTACGT AGTGAAAAAA ATATTGATGA 5460

TAAAGATATA TTGATTGTAT ATCAGGACAA AAATGGGCTC ATCAATAAAG TAGAGTATTG 5520

CTCAGAAATT TATTACTCCC TTGGGAGCAA ATGGAAAGAC TTTATCAGAG AAATTAAATA 5580

TATTGTCTTG ACCTCCGAGG AATATCAATT TAACAAGATA GAGAACTACT ATCCGGCACC 5640

CGGAATCATA TCCTGTTTTT CATCATATGA TCCTTATGGA ACGGTTAGAG AAATACAACA 5700

GGCTGAAAAA GGATATTCAT CTTTAACATT ATGTGTAGAG ACAATCCCAT ATAATGAAGG 5760

TGATTCAATA TCTGAATATA TCGCTCAAGT TTTACTTTCA ACAAGCGATG TCATATTCTT 5820

CATTGATGAA GAAAAGAAAG TAGAACAAGG CATCAGTATC GATATCAAAT ACGCGATTGA 5880

The termination of the initial orf5 of orf6

TAAAATTAAG TTTAATTATT CTGGTGAAAA ATGAATATAA TAATCCATGG GCCGCTAGAA 5940

AAAGATTTCT TATTTAAAAA CTTAAATAAA ATTCGGCACG CTTCAGAAGA CGATCACATA 6000

ATTATTTCTG TTTATAACGA AGAACTTGAA AAAACAAAGG TTATTATAGA CAGCGTCCCA 6060

GTCAGAAATA AGGTTACATT AATCGGTTCT GATGATGTTT TTAATCCTGG CTTTTTTAAC 6120

ATTAACAGAC AAATTAATCT GGTTAGTGCC GCATTAAACG TCATTGAAGA TGAAAGTGAC 6180

TTTATTCTAA AAGTTCGAAT GGATCAGACT ATTAACTTTC GTTGGGTTAG AAAAGTTGTA 6240

ACTGAGAAGC ATGAAGAACT GAAGGATAAA TTGATCACCA CTAACTGCTA TACCAGAAAA 6300

GATCGTCTTT ATCATCCATC TGATATGTTC CTCGCCGGCA CGAAAAAAAC ACTTCGTGCA 6360

TATTATCCAG AAGAGTTTTT CCAAGAGACT CACATGGATA ATTTATTGAA AATAAAAGAA 6420

CTAGTGCTCT CCGGATATAA AGGCGGATAC CATAAATATT GGCCAGAATC ACGACTGTTT 6480

ATGAATTACT TATCCATATT AGGTGAGGAA TTACAGGATA CAGAGATCTG TAGTCAGTCA 6540

CAATTGAAAA AGCATGTTTA TATTTTTAAT TCATGGGATA TTGGATTAAA ATGGAAAAAA 6600

TTCCTTAAAG GCAAAGTTAA CGTTTTACCA TATTTCTTTG TTTTAAGACC TTTCGAAAGA 6660

GGGCCTCTTG AGAACGCAGA GAATTTTTTA GCAAGTGACT TAACAGGTTA TACAAAAAAG 6720

CCTTTGAGGG AAATGATATA TAATAGTTTA GCAAAATTAT ATTTTAAATC TGGAGCTTAT 6780

TTAGCGAACC CAATATTCAT AGATAACAAA TCACTTATAC CAAAATTACT ACGAAAAATG 6840

TTTGATCATT CATATAAATT CATCCCTCCC ATTTTGCATA ATCTTATGTA TAAAGTCGCA 6900

The termination of the initial orf6 of orf7

AAGAGAGTTT ATT ATGCATT CAAA TAAGAT ATATATAATT GGGTGGTCAG GCTTTATTGG 6960

TCGGCAATAT ATTCATTATC TTGATAAAAC GGGCTTTAAG GGTGAGGTTT TTCTTAACTC 7020

ACGTGCCGAC GATGAGCAAA AATACACTTC TTCTACAGAT AACATCACAA TAAAATATGT 7080

TAATTATGAG GAGATGATTA CTGGGCTTAA TTGTTCAAGG TCAACTGTAT TATACCTTGC 7140

AAATATGTAC TCACCGGGTG AGTCGAATAT TTACCCTTTG AATAGTGTCA AAGAAAACAT 7200

TATTCCATTT ATCACTTTTT TAGAGGATAT CAAGAATAAT GCAAGCAATA TCAGGTTCAT 7260

ATTTGCTTCT TCAGGAGGGA CTATATATGG CGATAGTCAA GAAGGGGAAT GTTGCAGCGA 7320

GAACCACTTA TTAGCTCCGA AAACCATTTA TGCAGCTAAC AAAATAGCGC AAGAGAACTA 7380

TCTCAACGTT TATCATGTTA ATTATGGTCT TGATTATCGG ATAGCCAGAA TTGCAAACCC 7440

TTATGGCCCG GGGCAGATAT TGAAAGGTGG TCAAGGATTA ATACCAGCAA TACTTCGTTC 7500

GTCATTAAAT AATGAAAGCC TTCCTGTGAG TGGTGATGGA AATGGTACAA GAGATTATAT 7560

ATATATAGAT GATCTTTGTT CACTTTTTTT CAGTCTAAGT AGCTATAGCG GAGCTTGTAG 7620

AACATTTAAT GCGGGCTCTG GTAAAGAGTA TAGTATAATG GAAATCATAA GTTGCTTTAA 7680

CGCAGTGCAT GACAGACCAA TAAAATACCA TCATGTTAAA ACCGAATCTT CAGTTGTAAG 7740

CCGTATTGTC CTTGATATCA GTCGTGCTAA AAATGAGCTG GGATGGAGCC CTAAAACTTC 7800

The termination of orf7

CCTATCTGAT GGGATTCGTG CCTTTATCAA CTGGTCGATT CAAGTGATG T AGTTTTCCAT 7860

Orf8's is initial

ATTTCAATAA TCTAACCTCA C ATGACATAC ACCACTCAAA TCTCGGCTCA AGATGTCTCG 7920

ATTGTTATCC AAGGGGCGAT TTTCGATACA AAAAAACAAA AAGTTGATGC TCAATTTATT 7980

GAATACTTAG AAAAAGTTAT TTCGATATTT ATAGGTTGCG AATTTATTGT TTCGACGTGG 8040

GAATTTCCTC GCGAAATATA CTTCGGTCTT TGCGAAAAAT ATCCGCAAGT AAATTTTGTT 8100

CTGAATGCTG ATGTTGGGCC AATAATAAAA ATAGTAGATG ATGTACCGAT TGTCACCAAT 8160

GTTAATAGGA TGATTTTTTC GACAATAAGT GGACTCAGGG AAGTTAATCG CAAATATGCT 8220

ATTAAATTGA GAACAGATTC TTTCTTATAC AATGATTCGA TACTCCAATC GCTTTATTAT 8280

GTTATGAATA AACGCGAATT TTTCGGATTA GACTTGGAAA AGAGAAATGA GCGATATCGG 8340

ATCTTTGATC ATCATGTAAT TAACTGTAAT CTATTTGCCC GCAATCCGCG CAGTCATTTA 8400

CCTTTCTTAT ATCATCCTGG TGATATTTTC CTGGCAGGAT TAACTAAAGA TTTAATTAAG 8460

CTTTTCAATA TACCATTGGC GACGGAAGAC CTAATAAAAA AATGCAATAG TGTGAGAAAT 8520

ACTTGCTTTA TGAAACTTGT ACCTGAACAA TATATTTGGG TTCAGTGTAT TAAAATGTCA 8580

AGAAGTACAG ATACGTTCGA TTATTCAAAT TTCACATATG ATGAAAAAGA AATCGAAAAA 8640

TCAGAGCAGT ATTATTTAAA TAATTTTGTA CCATACACTG CAGAAGAATT GGGTTTTTGT 8700

TGGCCAAAGC ATCGAGAGGT TTATTTTAAC AAAGGGAGTT CAAGTATTTA TGATCATGAA 8760

GATTGGCATC ATTTGTATCA TAAATACATT GACGGTTTTG AACAACCTAC AAGTTATGCA 8820

CATAAAAAAA GAAGTGTGGT TATATTCTTT ATGAAGATGT ATTTTTTCAT TCGTAGCTCT 8880

The termination of orf8

CTGCTTAAAA TAAAATTCAT TCGCAAATTT GCTTACAAAG TTTTTGTAAA AAGAGGA TGA 8940

Orf9's is initial

CTT ATGAAGG GATTTGTTTC AACGCCCTCC AGTACAACGC GCTTTAGTGA AGTTTTTGAA 9000

TCACGTAATT TAATTGCAGA ACTTATTCGG CGAGATTTTA ATGTCCGCTA TAAGCAAACA 9060

GGGCTTGGAG TATTATGGGC AATAATTAAT CCAGGCGTCA ATATTCTTCT TTATCTCTTT 9120

GTATTTGGTT TGTTAGTTAG GTTACCAACC CCAGAATATA ATGCTCCTTA TGCTGCGGTG 9180

TTAATCTCAG CGATTTTATT TTGGAATTTA TTTTCTACTA GCATGCTCAC AGCAAGTGAT 9240

GCGCTGATTA ATAACATGCA TCTTGTTACT AAAGTTTATT TCCCAAGGTT ATCTTTATGT 9300

ATAGCAAGTG TATTTGTTGC ATTGATTGAT TTCTTGATAG CTTTTATCGT GTTTGTCCCG 9360

TTAGCATTAT TGTATCATGT GGACATAAGT CTAATTAGAT TGCTTTTATT AATCCCATGT 9420

ATAATTATAA CTCTGATGCT TGGATGTGGT TTTGGCTGTG CAATGGCTAT ATTGAAATTG 9480

AAGTATAGAG ATATTAGACA TATACTTCCT CTAATAACCC AGGTTATTTT CTTTGCGTCA 9540

CCTATTGTAT ATACTCTATC CATTGTTCCA GAACAATATA AAATATTCTA TTCTTTTAAC 9600

CCCATCACTG GGATCGTAAT GATGTCTCGC TGGGCTATCT TAGGCGGAGC ACCCATTGAC 9660

TTGACAATGC TTACTTATAG CCTAATTAGT GCATTTGTCG TTATGTGTTT AGGTGTCATT 9720

The termination of the initial orf9 of orf10

TATTTTGTTA AAAATGATCG ATCTGTGGCG GATTACGA AT GATTGAGTTT GATTCGGTAA 9780

GTAAGCAGTA TGAAATCAAC CATCGACGGG CCAAAAGCTT ACGCGATGTT TTTTCATTTA 9840

ACAAAAAGCC AGATGCAAAT GCAGAAATAT TTAAAGCGGT TGATGATGTC AGCTTTAGTA 9900

TTGATAGCGG TAAATCAGTA GGTGTCTTGG GACGAAATGG TGCGGGAAAA TCAACGCTTT 9960

TAAA TTATT AACGCGTATC ATTTCCCCGA CATCAGGCAA AATAACTGTA GATGGCTCCA 10020

TTTCTTGTCT GCTGGAAGTT GGCGCTGGTT TTCACCATGA ACTCAGTGGC TACGAAAATA 10080

TTTTTCTATG TGGCTCAATC TTGGGAATGA GTCGTAAACA AGTCAAATCT AAAATAGAAT 10140

CTATTATTGA TTTTTCTGAG GTCGAGAAAT TTATTGACGA ACCTGTTAAG TATTATTCAT 10200

CAGGTATGTA TATGCGCTTA GCTTTTGCGA TAGGTGCTCA TCTTGACTCG GATATTCTAG 10260

TGATCGACGA AGCTTTGGCT GTTGGTGACT CTCGTTTTCA AAATAAATGT TTAAATAAAA 10320

TCGACGAGAT TAGAAAAAGT GGAAAGACTA TATTTTTTGT TAGTCATAGC ATTGAACAAG 10380

TAAAAAAAGT GTGTGATACC AGTATTGTTA TGGATCATGG GAAATTATTG TTCCAGGGCA 10440

The termination orf11's of orf10 is initial

ATACTGATGA AGCTGAAAGA ATTTACAATA GTCTTACTAA TGGTGCG TGA GT ATGATCAT 10500

TATTTCTCAT AGAGGGTATT GGAAGTTGAG CAGTGAAAAA AATACTGCTT TGGCTTTCAA 10560

ACGCTCATTT TCTTTAGGTT ATGGTACTGA AACAGATATT CGTGACTATA AGGGTAGTTT 10620

AGTCATCAGT CATGATATAC CAGATGAGAG TTCATTAAGC ATAGACTCTT TTTTTGAAAT 10680

TTATAATCAA TATGATGTCA AAGGCCCTTT GGCATTAAAC GTAAAATCAG ATGGGCTGCA 10740

AAAACTAATA AAAGAAAAAT TAAACGAGTA CAAGGTTAGT AATTATTTTT TCTTTGATAT 10800

GTCTATCCCG GATACTCTTG GGTATTTATC AGAAAAACTA AATACGTTCG TGCGTGTTAG 10860

TGAATATGAA ACACTAAACG ATCTCTATGA AGATGCTGAT GGTGTTTGGA TTGACGGTTT 10920

CAAAGAAAAT ATTATAACAG AATCTTTACT CGAAAAAATT ATTTCTGATG GGAAGCGTGC 10980

TTGTATTGTA TCCGCTGATC TGCATAAAAA CGATCCAGCT ACGCAATGGA AACTTTTAAA 11040

AAGTTTTAAA ACAAATATTC TTCAGTCGTC AAATATTATT CTTTGTACTG ATTTACCTGA 11100

The termination of the initial orf11 of orf12

AAAAGCGTCG GAGTTTTTCT ATGGA TAATA AAATTAAAGC CGTAATCTTC GATATGGACG 11160

GAGTTCTTAT CGAAGCTAAA GACTGGCATT ACGAAGCTCT AAATCGCGCT TTGGGACTCT 11220

TCGGTCTGGA GATAACCAAG CATGAACATC TTACAACCTA TGATGGTCTA CCTACTAAAG 11280

ATAAGTTAGT CATGTTGTCT CGTGATAAAG GTTTGTCAAA TGCGCTGCAT ACCTTTATTA 11340

ATGAAATGAA GCAACAGTAT ACTATGGATA TGGTTCATAA ATTTTGTAAA CCTATGTTCC 11400

ATCATGAATT CGCCCTATCA ATGCTTAAAC AAGATGGGTA CAAATTAGCT GTAGCATCTA 11460

ATTCAATACG GAATTCCGTA GAGGTGATGC TTGATAAAGC TAAACTATTA GCTTATTTGG 11520

AGTTTTTTAT TTCTAATCAG GATGTGAAAA AAGGTAAGCC GGATCCTGAG ATGTATACTC 11580

TTGCTATTTC AAAGTTGGGA TTAAGACCTT CTGAATGCTT AATTGTTGAA GATAATGAGA 11640

ATGGGATAAA AGCAGCTAAA GCAAGTGGTG CTCACGTTCT TGAAGTTGAA ACTGTATACG 11700

ATGTAAATTA TCAAAACATC AAACAACGTA TTAAAAAAAT TGAAAACGAT GAGGGTGGAA 11760

The termination of the initial orf12 of orf13

A ATGATTAAT ATCCTAATTC CAATGAGTGG AGAGAATCTT TATGAAACCT CCAGTAATTT 11820

CATCTACCCA AAAATTTTAA CTGAAGTTGC AAATAAAACT CTCTTGGAAT ATTCGCAATT 11880

AATATTTGAT AATCTGCGAG ACGAAAAAAA TCTTATTTAC ATTGCTCCAG AAGATAAACT 11940

TAACACTCTT GGTTTGAAAT CAATCATTCA AACTATTACA GATGGTAAAG GTAAAATTGT 12000

TGCCCTCCAA GGGCAAACCA AGGGTGCTGT CTGTAGTTGT CTTATGGCGA TCGATGATCT 12060

CTCACTTGAT GACGAATTGA TTATTTCTTC TGCGGATCAT TATATTGACG ATAATTTGCA 12120

AAGGATCGTT AATGATTTTA GGGTCATGGA CGCTGACGCT GGGGTTCTTA CCTTTGAATC 12180

TGTGCATCCC AAATGGTCGT TTGTAACACT AAATGAGCAT GGTGCAGTTG TTCAGGCCGC 12240

AGAGAAAAAA GCGATTAGCA GAACAGCTGT AGCTGGGCTG TATTATTTTA AAAAGGCGCG 12300

TGATTTTGTC GAGGCTGCGA TGAATCTCAT TAGAAAGGAT GGCGATATAA ATGGTAATTT 12360

CTATTTGTCT TCCTGCCTTA ATGAGCTCGT TTTGTTGGGT AAGGTTATCA AATGTCGCCC 12420

GCTACAAGAT GCTATCTATC ATAACTTTTA TGATGCCCAC GCAGTTAAAT CTTTTGAGCT 12480

TGCTCAGACA TCCCTTTCTA ATAGCGTTAA GCAACTCACG AATTCCTACA TTGAAGCATT 12540

TAACTCGAAA GATATAGAAG AATGTCTTTT TCTTTTTTCA GACGATGCGG TTCTTGTTGA 12600

GCCCAACAAA ACATTTACTG GGAAAGATGA AATAAAGGAA TTGCTTAACG ATATTTTTGT 12660

GAACAATTCT AAACTCCAAT TCCGTGCCGA TAATATTATT GCTGATGATA GTAAATCTGC 12720

AATCAAGTTT ACACTTGAGC TTACTAACGA GACTGTTCAT GGCGTTGATT TCATTGAATG 12780

The termination of orf13

GAATAGTGCG GGTAAAATCC ATAAACTGAC TGCTTTTTTA AATGAT TAAA ATAGTTACCA 12840

GTTTTGGAAT GTGAACTGTG AACATTTATG GATGACTTTA ATTGATGAAA AATCGTGTGA 12900

TTTGATATCT TTTTTTTTAA TTAATGATTT ATCGAAATTT TTTAAAATCG CACAAATTCA 12960

CGCTATTCGC TGCGAAGAGC TTGAGTTAAC ATAATGTTCA CTTCTCCCAG CAGGATAATG 13020

GGCTTGCTGG TAAACCACAC TAGACAGGAG TATGTAATGT CTAAGCAACA AATCGGCGTT 13080

GTCGGTATGG CAGTGATGGG GGCGCAACCT GGCGCTCAAC ATCGAAAGCC GTGGTTATAC 13140

CGTCTCCGTT TTCAACCGCT CCCGTGATAA GACCGGAGAA GTGATTGCCG AGAACCCAGG 13200

CAAGAAACTG GTCCCTTTCT ATACGGTTAA AGAATTCGTC GAATCTCTGG AAACCCCTCG 13260

TCGTATCCTG TTAATGGTGA AAGCAGGTGC AGGTACCGAT GCTGCTATCG ACTCCCTGAA 13320

GCCTTACCTC GAAAAAGGCG ACATCATCAT TGATGGCGGT AACACCTTCT TCCAGGACAC 13380

CATTCGTCGT AATCGTGAAC TGTCTGCTGA AGGCTTCAAC TTCATTGGCA CTGGTGTTTC 13440

CGGTGGTGAA GAGGGCGCTC TGAAAGGCCC ATCCATCATG CCTGGGGGCC AGAAAGAAGC 13500

ATACGAACTG GTTGCGCCTA TTCTGACCAA AATCGCAGCT GTTGCAGAAG ATGGCGAGCC 13560

GTGTGTGACC TATATCGGTC CGGACGGTGC AGGGCACTAT GTTAAAATGG TGCACAACGG 13620

CATTGAATAC GGCGATATGC AGCTGATCGC GGAAGCATAC TCTCTCCTGA AAGGCGGCCT 13680

GAATCTCACC AATGAAGAGC TGGCGGAAAC CTTCACCGAG TGGAACAAAG GCGAGCTGAA 13740

CAGCTACCTG ATCGACATCA CCAAAGATAT CTTCACGAAG AAAGATGAAG AGGGTAAATA 13800

CCTGGTTGAT GTGATTCTGG ATGAAGCAGC GAACAAAGGC ACCGGCAAAT GGACCAGCCA 13860

GAGTTCTCTG GATCTGGGCG AACCGCTGTC TCTGATCACC GAATCTGTCT TCGCGCGTTA 13920

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

Claims (3)

1, a kind of oligonucleotide is characterized in that, is selected from the Nucleotide of 4350 to 4365 bases among the SEQ ID NO:1; The Nucleotide of 4888 to 4903 bases among the SEQ ID NO:1; The Nucleotide of 5246 to 5263 bases among the SEQ ID NO:1; The Nucleotide of 5690 to 5707 bases among the SEQ ID NO:1; The Nucleotide of 9347 to 9364 bases among the SEQ ID NO:1; The Nucleotide of 9743 to 9758 bases among the SEQ ID NO:1; The Nucleotide of 9160 to 9175 bases among the SEQ ID NO:1; The Nucleotide of 9650 to 9665 bases among the SEQ IDNO:1.

2, a kind of oligonucleotide is right, it is characterized in that, is selected from the Nucleotide of 4350 to 4365 bases among the SEQ ID NO:1 and the Nucleotide of 4888 to 4903 bases among the SEQ ID NO:1; The Nucleotide of 5690 to 5707 bases among the Nucleotide of 5246 to 5263 bases among the SEQ IDNO:1 and the SEQ ID NO:1; The Nucleotide of 9743 to 9758 bases among the Nucleotide of 9347 to 9364 bases among the SEQ ID NO:1 and the SEQ ID NO:1; The Nucleotide of 9160 to 9175 bases among the SEQ ID NO:1 and the Nucleotide of 9650 to 9665 bases among the SEQ IDNO:1.

3, the application of the nucleotide pair of the Nucleotide of claim 1 or claim 2 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray as probe, for detecting intestinal bacteria O95 type.