CN1271206C - Nucleotide specific for escherichia coli 012 O-antigen - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O12. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O12 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 11951 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 O12. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O12 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O12 by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O12 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O12 type (Escherichia coli O12), particularly relate in the intestinal bacteria O12 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O12 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 of lipopolysaccharidebiosynthesis in entericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and gene nomenclature " Trends inMicrobiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydiiO-anfigen loci: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 coli O111 andSalmonella enterica O35 gene clusters:gene clusters encoding the same colitose-containing Oantigen are highly conserved " .Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose and paratosesynthase genes (rfb) by polymerase chain reaction for identification of S.enterica majorserogroups (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 microbiologicalinvestigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausagecontaminated with Shiga-like toxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.and Reeves of a supposition, P.R. (1995) Sequence and analysis of theO antigen gene (rfb) cluster of Escherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet.

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specific selection andbacterial 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 ' sidentification of the Enterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasiveEscherichia coli antigens O28ac; O112ac; O124; O136, O143, O144; O152 and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O12 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O12 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 O12 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O12 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf1, orf4, orf8 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O12 type respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are high specials to the O-antigen of intestinal bacteria O12 type; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O12 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect and identification of escherichia coli O12 type O-antigen and detection and identification of escherichia coli O12 type by these methods.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O12 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

The objective of the invention is to realize by following technical scheme.

The present invention is characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O12 type: it is the isolating Nucleotide shown in SEQ ID NO:1,11951 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O12 type, comprising called after orf1, wzx, wzy, orf4, fnl1, fnl2, fnl3, orf8,9 genomic constitutions of orf9 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O12 type, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf1, off4, orf8 gene; Wherein said gene: wzx is the Nucleotide of 2147 to 3406 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 3419 to 4522 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1115 to 2074 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4523 to 5617 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8893 to 10101 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O12 type wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O12 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 2743 to 2759 bases among the SEQ ID NO:1 and the Nucleotide of 3190 to 3205 bases; The Nucleotide of 2594 to 2613 bases among the SEQ ID NO:1 and the Nucleotide of 3051 to 3076 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 3815 to 3834 bases among the SEQ IDNO:1 and the Nucleotide of 4405 to 4420 bases; The Nucleotide of 3664 to 3679 bases among the SEQ IDNO:1 and the Nucleotide of 4034 to 4049 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O12 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 O12 type is providing the O-antigen of expressing intestinal bacteria O12 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 O12 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 O12 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O12 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 O12 type bunch: with the genome of intestinal bacteria O12 type is template by increase its O-antigen gene bunch of LongPCR, 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 O12 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O12 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O12 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O12, 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 O12.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O12 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O12 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O12 type bunch: with the genome of intestinal bacteria O12 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, 55 annealing 15 seconds, 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, 0.9 μ l 0.1MMnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mMDTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O12 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 O12 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O12 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 O12 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 9 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 O12 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O12 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O12 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O12 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 O12 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 l supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 2743 to 2759 bases among the SEQ ID NO:1 and the Nucleotide of 3190 to 3205 bases; The Nucleotide of 2594 to 2613 bases among the SEQ ID NO:1 and the Nucleotide of 3051 to 3076 bases; The Nucleotide of 3815 to 3834 bases among the SEQ ID NO:1 and the Nucleotide of 4405 to 4420 bases; The Nucleotide of 3664 to 3679 bases among the SEQ ID NO:1 and the Nucleotide of 4034 to 4049 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 O12 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 O12 type, its complete sequence shown in SEQ ID NO:1,11951 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 O12 type by method of the present invention, as shown in table 3, it comprises called after orf1, wzx, wzy, orf4, fnl1, fnl2, fnl3, orf8,9 genomic constitutions of orf9 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 O12 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf1, orf4, orf8 gene).Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria O12 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O12 type is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O12 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O12 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O12 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As used herein, " oligonucleotide " mainly refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 2147 to 3406 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 3419 to 4522 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O12 type.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function are arranged or with wzy the gene of identity function is arranged with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from the wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O12 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 coming from coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O12 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 encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase 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 O12 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

General a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of is used to detect the many to oligonucleotide of the inventive method, many here is that the gene that comes from coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O12 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from the wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.

The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, method of the present invention and molecule also are applied to these other polysaccharide antigen.

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O12 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 O12 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 O12 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30 μ l TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O12 type bunch:

With the genome of intestinal bacteria O12 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 TTAAGT 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, 55 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 WizardPCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9 μ l 0.1M MnCl ₂, 1 μ l1: the DNaseI of the 1mg/mL of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5mL substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2mL culture is transferred to 200mL, and 37 ℃ of 250rpm thermal agitations are cultivated 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 50 μ l, one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains 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 O12 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 O12 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O12 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 O12 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffmder finds gene, find the reading frame of 9 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 O12 type at last, as shown in table 3.

By retrieving and comparing, the aminoacid sequence of finding orf1, off4 encoded protein and other known glycosyltransferases has the consistence of 34-35% and the sequence similarity of 52-57%, and of encoding in orf8 encoded protein and the Escherichia coli O-antigen gene bunch is speculated as L-fucosaminetransferase (AAN60461) 94% sequence identity and 97% sequence similarity are more arranged.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the albumen of these three genes encodings and the consensus sequence of known glycosyltransferase is very high, so we infer this three genes encoding glycosyltransferases.Because the definite function of these three genes can't be determined, so we are with the temporary called after orf1 of these two genes, orf4 and orf8.

Orf5, orf6, orf7 encoded protein respectively with Escherichia coli O-antigen gene bunch in Fnl1 (AAN60461), Fnl2 (AAN60462), Fnl3 (AAN60463) albumen of coding 94～96% consensus amino acid sequence is arranged, by search to Pfam protein-based order sequenced data storehouse, the homology desired value of consensus sequence of finding the albumen of these three genes encodings and known L-fucosamine systhetase is very high, so we are fnl1, fnl2, fnl3 with these three unnamed genes.

Orf9 encoded protein and WbuC albumen have 94% sequence identity and 98% sequence similarity.But the proteic function of WbuC is also unknown.Because the definite function of this gene can't be determined, so we are with the temporary called after orf9 of this gene.

Orf2 and orf3 are the proteic genes that there is transmembrane segment in only two codings among the intestinal bacteria O12.The aminoacid sequence of the O-antigen transferring enzyme (AAL25627) of orf2 encoded protein and Edwardsiella ictaluri has 43% consistence and 64% similarity, it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf2 is wzx.The O-antigen polysaccharase (CAD63717) of orf3 encoded protein and Lactobacillus plantarum WCFS1 has 24% consistence and 44% similarity, it contains 10 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf3 is wzy.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O12 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 O12 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O12 type all is high special; The position of these genes in nucleotide sequence sees Table 1.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O12 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 is done 106 and 107 times dilution, remaining bacterium liquid is put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, 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 l supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 2743 to 2759 bases among the SEQ ID NO:1 and the Nucleotide of 3190 to 3205 bases; The Nucleotide of 2594 to 2613 bases among the SEQ ID NO:1 and the Nucleotide of 3051 to 3076 bases; The Nucleotide of 3815 to 3834 bases among the SEQ ID NO:1 and the Nucleotide of 4405 to 4420 bases; The Nucleotide of 3664 to 3679 bases among the SEQ ID NO:1 and the Nucleotide of 4034 to 4049 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 O12 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 intestinal bacteria O12 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 O12 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O-antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O12 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O12 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

The genomic dna that contains intestinal bacteria O12 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 25 μ l.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get 10 μ lPCR products and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O12 type and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O12 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 O12 type.These all oligonucleotide all can be used for the intestinal bacteria O12 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 O12 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O12 type, altogether by 9 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 O12 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O12 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O12 type

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O12 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>11951

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaacagcg cgtgaagcgt 120

cagctgctgg cggaagtgca gtccatctgt ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggtcactcc attttatgtg cacgacccgc cattggtgac 240

aatccatttg tcgtggtgct gccagacgtt gtgatcgacg acgccagcgc cgacccgctg 300

cgctacaacc ttgctgccat gattgcacga ttcaacgaaa cgggtcgtag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactcggtca tccagaccaa agaaccactg 420

gaccgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgttggagt cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcacgcagcc aggtgcatgg gggcgtattc agctgactga tgccattgcc 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tgggttatat gcaggcattt gtgaagtatg gactacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg cattgagaag ctgttaagcg tataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgt 840

tgcgaatttt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgt tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgggttaatt ctaaaattag 1080

aattttacga cactataagt aaaataggtt tataatgaag agtaaattag tttccataat 1140

cataccagca tacaatgctc aggattggat tgaaaggtgt ttagtatctt gccttaatca 1200

gagctatcaa aatatagaaa ttattgtaat aaatgatgga agtacagata atactgctaa 1260

tattctcgag aagttcaatg taaataaact gaaaatattt catgaaaaaa actctggtgt 1320

agcaatagca agacgaaaag gtttggatta ttctagagga gagtatatct tttttcttga 1380

tagtgatgat tttataaatg agtctgcttt agaaatattg gtaaccaaga ttgataatta 1440

taatgctgac gtggttatag cgcaagctaa acatcttaaa aagaaaagca cttttacatc 1500

atcttttaaa attgaaattg atccactata ttcatttttg aatggatttt taccaagaac 1560

tttatggcca ttattattta aaagatctgt tattgagaaa aatctaatac catctcaata 1620

tacagttagt gaagactatg taataatgtc aaaaatatat accttgccaa ttaaaatcgt 1680

tgtcgttaat gattgtctat acaattatgt aaaacacacg ggtagcgtaa cggcgaatat 1740

aacgccacag aaatataatg accactataa agctcaccat tatattgaaa aacagttatt 1800

cgaaacgtta tcagataaat ataaaataag ttttataaga aacaatttag attttcttta 1860

tagtttaatt attatcaatt caccatacgt cagtcatcaa atggaatata tttgggggaa 1920

atatattaaa gaagatatta ataaagctac aaggaggctt tctctgaaga aaaaaataat 1980

acttaaacct aatcacgtag ccttgatgag acttgggccg ttaagaatga taatattatt 2040

aatgaataaa ctaagaagat acaatgagaa ttaaaactag taatataaaa atgataatga 2100

taatattttg ataatgacat agttaagtca taaaaggtta ttttctgtga atatagtatt 2160

gaaaaattca atgtatcttg tttttatgca gatgtgcaat tatgctattc ccttaattac 2220

gctaccttat ttaacaagat gcttggggtt ttatcaatat ggtgtattga atactgccat 2280

caatattata ctatacatca tcttggttat tgattttgga tttaatttaa gcgccacaag 2340

agaaatatca cagaaaaaaa ataactttgt agcggtatct aaaatattta cagatgtgat 2400

ttttgcaaaa attatacttt ttttcggtgt gcttgttttt actttcctca tacttaaact 2460

aattcccgac tatcaagatg taacaagatt ggtttatttt atgttacctc aagtcctatc 2520

ttcgatatta ttcccactgt ggttatatca agggtatgaa aaaattggat atgctgcatt 2580

tataaacgtt cttggaaaat gtataacaat cccattgcta ttaatctttg tacaaaatag 2640

caaagatgta acaaatgctg caattatatt aagttggtct tctttagttc ccttattaat 2700

ttattatata cgtagtaatc ttccgcgaac aaagttttat acctcaaggt gtagattcaa 2760

gtacttaaat agaacattag ctaatgcact ggtcgttttt attggttcca ttgctgtaaa 2820

cctgtatact ttaagcacac cattagtatt aagtatagtt agtgattttg atcaagttgg 2880

ttattttgtt gcagctgata aaataagggc tgcatttatt ggcgtttttt taattgtagg 2940

gcaggcgatt tatcctagag caagtgccct gtataatgaa gatataattg cgtataataa 3000

atttgtcata agcattatat tttaccaaat tatagtatgt acgttctctg ctatagcctt 3060

ttattacttc atgccagagg tggcaccaat tattttgagt caaaacaaat taaacgtaga 3120

agagttatct gttataatta aattgatgtc gccaatgatt attttgatac ctctttcggt 3180

gatattagct aactgcgtac ttttgcctca aaggaaaaat aaacaatacg ctttagtgcc 3240

tattattaca gtcataattc atatgagtta ctctataatt ttgtgtgaaa agttaggtgc 3300

gctaggggca agttacgcaa tacttctcac agaaattatt agttgtgctt tactagcata 3360

ttttacattc aaagatagac taattcagct caatccagcc agataatatt ggataatcat 3420

gaatcattct tatatctatg tttatatatt ttttgtgttt ctttcatttt ttgagcttaa 3480

tgatactaat agagcaagat gtgatttata ttatgccata caagttattg tattattact 3540

aacaatgggg ttgtcgtatc aaataggtgt tgattgggtt gaatatgaac gtgtttataa 3600

tgggaattcg gaatcattaa ataattacga attcggttat atagtattga ataatattgc 3660

aaactattta ggggctagtt tttgggggtt cgtgatatta ataaaatata cgttttttat 3720

atcattgttt cttatggtaa gaaaattcag tagattacct gttctgacta caacattaat 3780

actagggttg ttatttccat ttattaatga ccctttacga caaataatat ctgcaagtat 3840

attgttcttt acgatttttt tctataacag gccccctaaa atattgggga ttataggggg 3900

tggggtattt cattctacat ttattataat tataattcgt tatcttaaaa tttttacaaa 3960

aagatctatt gtgcttattt tcgtttttgt gggggtggca attcttttgt tcttaaagta 4020

tggttctttt ttaggtgatg gaatgttatt taggaaatta aatttttaca tggagtattc 4080

atcattatca aatttgtatg gctttgccct aaggtttttg ctattgtcat ttattgtttt 4140

taatattgac gtttacaaag ctaattgcaa attcatcaga ggagatatct gttattattt 4200

ctggattttt agtataacat atttatggct tgaaataata tttgtaacat tccctttaat 4260

ccctcagcgg atgcggcttt atttgttacc atttgctttt atactattaa gtaactacct 4320

ttattatagt cgattaactt tcacaaaaaa tattcttgct tttatatgtg taatcatttc 4380

attttcttca ttgtgtcttt ttttagatgg tcctatgggg catttttata gcattactga 4440

taatatagta ttaaaatata ttcagggttt tactaacgat aaaacatatg atgcaaacca 4500

gttctggatt aatggaattt aaatgaaaaa aaatgtcttg ataatagctt ctgcagcaaa 4560

agaaggtggg gctttaagca tacttacaga aagtttaaat aaccttaaaa tattaaatat 4620

tcattttaca gttgtggtca atccatctgt ggttaaatat ttgcctgttg ctaataatat 4680

aaaatatcat atggtagata catcggcgtg gattaaaaga atagcatatg atttctatgg 4740

ctatcctggt ctcaagtatg aagaatatat agcgtgcata aattttcaaa atatacctat 4800

aagaactaaa ataaagcaaa tagtatatta tcatcagtca ttacctcttt cggatacatt 4860

attctcaccg tttaataaag aaacgcgagt tttatattta tatcaaaaat tctatggttt 4920

atttttcaag ttaaacaaaa ggtatatatc gcatctagta gttcaggcaa attggattag 4980

agaaaaatat ctgaaatttg gtgttgaaaa taataaaatt gaagttattg cacctcaagt 5040

cgaaattttt aaatatataa gtttagagtt taataaaata agacctgtag agaatgtatt 5100

tttttatcct gcaaatgcgt atacttataa aaatcatttg attataatca aagctttaaa 5160

ttatttaggg agtgtttata ttaaaaaaca tcgtattaag gttgttttta ctataaataa 5220

tagtgagtca aattatcttc aatcaatggt tttgaattat aatttgcagg atgttgttag 5280

ttttattggt cgaattccga gagccgatgt ttattctctc ttgataaaat caagagctct 5340

tcttttccca agtaaattag aaacttatgg tttaccatta aaagaggcgc ggcaactagg 5400

tgtatttatt gtcgcatcga agttagatta cgcaactgaa gtattggagg gatacgaata 5460

taaaagatta tgctcgccag atagtgctaa agaatgggct tcggctataa aggaaattat 5520

agaaaataag attgatgtcg gaaatacagt cactagagat aatattaaag agagcgattt 5580

gttttctgat tttattaaaa gtaaaataat ggagtgatta tttatgtttg cagaaaaaac 5640

attgcttatt actggtggta cggggtcttt tggtaatgct gttcttagac gtttccttga 5700

cactgatatc aaagaaatac gcattttttc tcgagatgag aaaaagcagg acgatatgag 5760

gaaaaagtataataatcaga aaattaag tt ctatataggt gatgttcgcg attattcgag 5820

tatccttaat gcttctcgag gtgttgattt tatttatcat gcagcagctc tgaagcaagt 5880

gccttcctgc gaattccacc ccatggaagc tgtaaaaacg aatattttag gtactgaaaa 5940

cgttctagaa gctgcaatag ctaatcgcgt taggcgaatt gtatgtctga gtacagataa 6000

agccgtatat cctattaatg caatgggcat atctaaagca atgatggaaa aagtcattgt 6060

tgcaaaatca cgtaatcttg atagttcaaa aacagttatc tgcggaacac gttatgggaa 6120

tgtaatggct tcacgtggat cggtcatccc attgtttgtt gatctaatca aatctggtaa 6180

accattgacc attaccgatc ccaatatgac tcgtttcatg atgacgcttg aggatgctgt 6240

tgatctggtc ctttatgcct tcgagcatgg aaataacggt gatattttcg ttcagaaagc 6300

tcctgcggca acaattcaaa cattagccat tgcacttaag gaattgctaa atgcccatga 6360

acatccaatc aatattattg gaactcgaca cggggaaaaa ctttacgaag cgttattgag 6420

ccgagaggaa atgatagcag cggaagatat gggtgattat tatcgtgttc caccagatct 6480

ccgcgacttg aactatggta aatatgtgga acatggtgac cgtcgtatct cggaagtgga 6540

agattataac tctcataata ctgagagatt agatgttgag ggtatgaaaa aattactgct 6600

aaaacttcct tttatccggg caattcgttc tggtgaagat tatgagttgg attcataata 6660

tgaaaatttt agttactggt gcttcagggt ttatcggccg taatttggtt ttccgcctta 6720

aggaggctgg ttataacgaa cttattacga tagatcgtaa ctcttctttg gcggatttag 6780

agcagggact taagcaggca gatttcattt ttcaccttgc aggagtaaat cgtcctgtga 6840

aggagagtga atttgaagag ggaaatagca acgtaactca acagattgtt gatattctga 6900

aaaaaaatag taaaaatact cctatcatgc tgagttcttc catccaggct gaatgtgata 6960

acgcttatgg aaagagtaaa gcgactgcgg agaaaatcat tcagcagtat ggggaaacga 7020

caaatgccaa atattatatt tatcgtttgc cgaatgtatt cggtaagtgg tgtcgcccaa 7080

attataactc ctttatagca actttctgcc atcgcattgc aaatgatgaa actattacaa 7140

ttaatgatcc ttcagcagtt gttgatctgg tgtatataga tgacttttgt tctgacatat 7200

taaagctatt ggaaggagcg aacgaaactg gttacaggac attcggtcca atttattctg 7260

ttactgttgg tgaagtggca caattaattt accgatttaa agaaagtcgc caaacattaa 7320

tcaccgaaga tgtaggtaat ggatttacac gtgcattgta ctcaacatgg ttaagttatc 7380

tgtctcctga acagtttgct tatacggttc cttcttatag tgatgataga ggggtattct 7440

gtgaagtatt gaaaacgaaa aacgcgggcc agttttcgtt ctttactgcg catccaggaa 7500

ttactcgggg ggggcattat catcattcca aaaatgagaa atttattgtc atccgaggaa 7560

gtgcttgttt caaatttgaa aatattgtca cgggtgagcg atatgaattt aatgtctcct 7620

cagatgattt taaaattgtt gaaacagttc cggggtggac gcatgacatc actaataatg 7680

gctcggatga gctagttgtt atgctttggg caaatgaaat atttaatcgt tctgaaccag 7740

atactatagc gagagtttta tcgtgaaaaa attgaaagtc atgtcggttg ttgggactcg 7800

tccagaaatt attcgactct cgcgtgtcct tgcaaaatta gatgaatatt gtgaccacct 7860

tattattcat actggccaaa actacgatta tgaattgaat gaagtttttt tcaaagattt 7920

gggtgttcgc aaaccagatt attttcttaa tgccgcaggt aaaaatgcag cagagactat 7980

tggacaagtt atcattaaag ttgatgatgt ccttgaacag gaaaaaccag aagctatgtt 8040

agttcttggc gatactaact cctgtatttc agcaatacca gcaaagcgtc gaaaaattcc 8100

gatcttccat atggaggcgg ggaatcgttg ttttgaccaa cgcgtaccgg aagaaactaa 8160

cagaaaaata gttgaccaca ctgctgatat caatatgaca tatagcgata tcgctcgtga 8220

atatcttctg gctgaaggtg taccagccga tagaattatt aaaaccggta gcccaatgtt 8280

tgaagtactc actcattaca tgccgcagat tgatggtccc gatgtacttt ctcgcctgaa 8340

tttaacacct gggaatttct ttgtggtaag tgcccacaga gaagaaaatg ttgatacccc 8400

taaacagctc gcgaaactgg cgaatatact taataccgcg gctgaaagat ataatatccc 8460

ggtagttgtt tctactcatc cgcgcactcg caaccgcatc aatgaaaacg gtattcaatt 8520

ccataaaaat atcttgctac ttaagccatt aggatttcac gattacaacc atctgcaaaa 8580

aaattcacgt gctgttttat cggacagcgg gactattaca gaagagtcct ccattatgaa 8640

tttccctgca ctcaatatac gagaagcgca cgaacgcccg gaaggcttcg aagaaggggc 8700

agtaatgatg gttggtcttg agtctgagcg cgtgttacag gcattagaaa ttattgcaac 8760

acaacctcgt ggtgaattac gcttacttcg tcaggtcagt gactatagta tgccaaatgt 8820

ttcagataaa gttgtgcgta ttatccattc atatactgac tacgttaaac gggttgtctg 8880

gaagcaatac taatgaaact tgcattaatc attgatgatt atttgcccca tagcacacga 8940

gttggggcta aaatgtttca tgagttaggc ctggaattgc tgggcaaagg ccatgatgta 9000

actgtaatta cgcctgacat cactttacaa gcaatctatt ctgttagtat gattgatggt 9060

ataaaggttt ggcgtttcaa aagtggacct ttaaaggata taggtaaggc taagcgtgcc 9120

ataaatgaaa ctcttttatc ttttcgtgca tggcgtgcat taaagcacct cattcaacat 9180

gatacatttg atggtatcgt ttattattcc ccctctattt tttggggaga cttggttaaa 9240

aaaataaaac agcgatgcca gtgcccaagc tatctggtcc taagggatat gtttccacag 9300

tgggtcattg atgcaggtat gttgaaagcc ggttcgccaa ttgaaaaata tttcaggtat 9360

tttgaaaaaa aatcatatca acaggctgac cggattgggt taatgtctga taagaatctt 9420

gagatatttc gtcaggctaa taaaggttat ccatgtgaag ttttacgtaa ttgggcctca 9480

atgactcctg tgtgtgccag cgatgattat aattcccttc gccaaaaata cgatctaaaa 9540

gataaagtca ttttttttta tggaggtaat attgggcatg ctcaggatat gacaaactta 9600

ttgcgccttg cgcgcaatat gatgcgttat catgatgctc atttcctgtt tatagggcag 9660

ggtgatgaag ttgacctgat taattctctt gctgcagaat ggaatttaac taatttcact 9720

catctacctt cagtgaacca ggaagagttt aaattaattt tatctgaagt tgatgttggc 9780

ctgttctccc tttcgtctcg ccattcttca cataatttcc cggggaaatt actagggtat 9840

atggttcaat caatcccgat ccttgggagc gtgaatggcg gtaatgatct gatggatgta 9900

attaataagc acagggccgg gttcattcat gttaatggtg aagatgataa actgttcgaa 9960

tctgcacaat tgcttcttgc agattcggct ttaagaaagc agttaggtca gaacgctaat 10020

gtgttgttaa aatctcaatt ttcggttgaa tcggcggcac atactatcga agtccgactg 10080

gaggcaggag aatgcgttta gttgatgaca atattctgga tgagcttttt cgcacagcag 10140

taaattctga acgtttgcgc gctcattatt tattgcacgc atctcatcag gagaaggttc 10200

aacgtttact tattgcattt gtacgcgaca gctatgttga gccccattgg catgagttac 10260

cgcatcagtg ggaaatgttt gtcgtcatgc aagggcaatt agaagtttgt ttgtatgaga 10320

aaaatggtga gatacaaaaa cagtttattg ttggagacgg tactggaata agcgtcgtgg 10380

aattttcccc aggtgatata catagtgtca aatgcctgtc accgaatgcc cttatgttgg 10440

agataaagga aggaccattt gacccactga aagctaaggc tttttctaag tggttatagg 10500

gcgatacacc accctttatt cttctatctt attctataca tgctgggtta ccatcttagc 10560

ttcttcaagc cgcgcaaccc cgcggtgaac accccctgac aggagtaaac aatgtcaaag 10620

caacagatcg gcgtcgtcgg tatggcagtg atggggcgca accttgcgct caacatcgaa 10680

agccgtggtt ataccgtctc tattttcaac cgttcccgtg aaaagacgga agaagtgatt 10740

gccgaaaatc caggcaagaa actggttcct tactatacgg tgaaagagtt tgttgaatct 10800

ctggaaacgc ctcgtcgcat cctgttaatg gtgaaagcag gtgcaggcac ggatgctgct 10860

attgattctc tcaagccata cctcgataaa ggtgacatca tcattgatgg tggtaacacc 10920

ttcttccagg acacaattcg tcgtaactgt gagctgtctg cagaaggttt taactttatc 10980

ggtaccggtg tttccggtgg tgaagaaggt gcgctgaaag gaccttccat catgcctggt 11040

gggcagaaag aagcctatga actggttgct ccgatcctga ccaaaatcgc cgccgttgct 11100

gaagatggcg aaccgtgcgt tacctatatt ggtgccgatg gcgcaggtca ctatgtgaag 11160

atggttcaca acggtattga atacggtgat atgcagctga ttgctgaagc ctattctctg 11220

cttaaaggtg gcctgaatct ctctaacgaa gaactggcgc agacgtttac cgagtggaat 11280

aacggtgaac taagcagcta cctgatcgac atcaccaaag acatcttcac taaaaaagat 11340

gaagacggta actacctggt tgatgtgatc ctggatgaag cggctaacaa aggtaccggt 11400

aaatggacca gccagagcgc gctggatctc ggcgaaccgc tgtcgctgat taccgagtct 11460

gtgtttgcac gttatatctc ttctctgaaa gagcagcgtg ttgccgcatc taaagttctc 11520

tctggcccgc aagcgcagcc agctggcgac aaaggtgagt tcatcgaaaa agttcgccgt 11580

gcgctgtatc ttggcaaaat cgtttcttac gctcagggct tctctcagct gcgtgcagcg 11640

tctgaagagt acaactggga tctgaactac ggcgaaatcg cgaagatttt ccgtgctggt 11700

tgcatcattc gtgcgcagtt cttgcagaaa atcaccgatg cttatgccga aaatccgcag 11760

atcgctaacc tgctgctggc tccgtacttc aagcaaattg ccgatgacta ccagcaggcg 11820

ctgcgtgatg tcgttgctta tgcagtacag aacggtatcc cggttccgac cttcgctgct 11880

gcggttgcct attacgatag ctaccgtgcc gctgttctgc ctgcgaacct aatccaggca 11940

cagcgcgact a 11951

Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O12 type bunch and primer and PCR data wherein

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzx	The transhipment enzyme	2147-3406	2743-2759	3190-3205	463bp	0 *	55
			2594-2613	3051-3076	483bp	0 *	60
								wzy	Polysaccharase	3419-4522	3815-3834	4405-4420	606bp	0 *	55
			3664-3679	4034-4049	386bp	0 *	50

^*Only in intestinal bacteria O12 type, obtain a correct band

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

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli	O1，O2，O5，O7，O8，O9，O13，O14，O15，O16，O17，O18，O19ab， O20，O21，O22，O23，O24	IMVS a
2, wild-type e. coli	O4，O10，O25，O26，O27，O28，O29，O30，O32，O33，O34，O35， O36，O37，O38，O40，O41，O42，O43	IMVS a
			3, wild-type e. coli	O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O53	IMVS a
4, wild-type e. coli	O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O68	IMVS a
			5, wild-type e. coli	O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99， O101，O102，O103，O104，O105，O106，O97	IMVS a
6, wild-type e. coli	O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128，O117	IMVS a
			7, wild-type e. coli	O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O141，O142，O143，O144，O145，O140	IMVS a
8, wild-type e. coli	O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166，O153	IMVS a b
			9, wild-type e. coli shigella dysenteriae	O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12，D13	c d
10, Shigella bogdii	B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18	d
			11, shigella flexneri	F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v:4)，F5(v:7)，F6， DS，DR	d
12, wild-type e. coli	O3，O11，O39，O59，O64，O73，O96，O95，O100，O114，O151，O155， O124，O167，O162，O121，O127，O149，O119	IMVS a
			13, the 1st group of bacterial strain adds the intestinal bacteria reference culture	O12	IMVS a

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

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

b.Statens Serum Institut，Copenhagen，Denmark

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

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

Table 3 intestinal bacteria O12 type O-antigen gene structure iron

Escherichia coli O12 O antigen gene cluster

orf# galF orf1 wzx wzy orf4 fnl1 fnl2 fnl3 orf8 orf9 gnd

G+C％ 52.2 28.2 31.1 27.8 28.9 39.3 38.8 41.2 39.7 41.4 50.6

content

Table 4 intestinal bacteria O12 type O-antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTTC TTGAACAGCG CGTGAAGCGT 120

CAGCTGCTGG CGGAAGTGCA GTCCATCTGT CCGCCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT GGGTCACTCC ATTTTATGTG CACGACCCGC CATTGGTGAC 240

AATCCATTTG TCGTGGTGCT GCCAGACGTT GTGATCGACG ACGCCAGCGC CGACCCGCTG 300

CGCTACAACC TTGCTGCCAT GATTGCACGA TTCAACGAAA CGGGTCGTAG CCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCGGTCA TCCAGACCAA AGAACCACTG 420

GACCGTGAAG GTAAAGTCAG CCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGTTGGAGT CAGACATCAT GGCCGTTGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACGCAGCC AGGTGCATGG GGGCGTATTC AGCTGACTGA TGCCATTGCC 600

]GAACTGGCGA AAAAACAGTC CGTTGATGCC ATGCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGTTATAT GCAGGCATTT GTGAAGTATG GACTACGCAA CCTGAAAGAA 720

GGGGCGAAGT TCCGTAAAGG CATTGAGAAG CTGTTAAGCG TATAATGAAA ATCTGACCGG 780

ATGTAACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TCGTGGCGAA AGTAATTTGT 840

TGCGAATTTT CCTGCCGTTG TTTTATATAA ACAATCAGAA TAACAACGAG TTAGCAATAG 900

GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG AGCGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TGGGTAACGT TCGTCACATC GTAGACATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGGGTTAATT CTAAAATTAG 1080

Orf1's is initial

AATTTTACGA CACTATAAGT AAAATAGGTT TATA ATGAAG AGTAAATTAG TTTCCATAAT 1140

CATACCAGCA TACAATGCTC AGGATTGGAT TGAAAGGTGT TTAGTATCTT GCCTTAATCA 1200

GAGCTATCAA AATATAGAAA TTATTGTAAT AAATGATGGA AGTACAGATA ATACTGCTAA 1260

TATTCTCGAG AAGTTCAATG TAAATAAACT GAAAATATTT CATGAAAAAA ACTCTGGTGT 1320

AGCAATAGCA AGACGAAAAG GTTTGGATTA TTCTAGAGGA GAGTATATCT TTTTTCTTGA 1380

TAGTGATGAT TTTATAAATG AGTCTGCTTT AGAAATATTG GTAACCAAGA TTGATAATTA 1440

TAATGCTGAC GTGGTTATAG CGCAAGCTAA ACATCTTAAA AAGAAAAGCA CTTTTACATC 1500

ATCTTTTAAA ATTGAAATTG ATCCACTATA TTCATTTTTG AATGGATTTT TACCAAGAAC 1560

TTTATGGCCA TTATTATTTA AAAGATCTGT TATTGAGAAA AATCTAATAC CATCTCAATA 1620

TACAGTTAGT GAAGACTATG TAATAATGTC AAAAATATAT ACCTTGCCAA TTAAAATCGT 1680

TGTCGTTAAT GATTGTCTAT ACAATTATGT AAAACACACG GGTAGCGTAA CGGCGAATAT 1740

AACGCCACAG AAATATAATG ACCACTATAA AGCTCACCAT TATATTGAAA AACAGTTATT 1800

CGAAACGTTA TCAGATAAAT ATAAAATAAG TTTTATAAGA AACAATTTAG ATTTTCTTTA 1860

TAGTTTAATT ATTATCAATT CACCATACGT CAGTCATCAA ATGGAATATA TTTGGGGGAA 1920

ATATATTAAA GAAGATATTA ATAAAGCTAC AAGGAGGCTT TCTCTGAAGA AAAAAATAAT 1980

ACTTAAACCT AATCACGTAG CCTTGATGAG ACTTGGGCCG TTAAGAATGA TAATATTATT 2040

The termination of orf1

AATGAATAAA CTAAGAAGAT ACAATGAGAA T TAAAACTAG TAATATAAAA ATGATAATGA 2100

Orf2's is initial

TAATATTTTG ATAATGACAT AGTTAAGTCA TAAAAGGTTA TTTTCT GTGA ATATAGTATT 2160

GAAAAATTCA ATGTATCTTG TTTTTATGCA GATGTGCAAT TATGCTATTC CCTTAATTAC 2220

GCTACCTTAT TTAACAAGAT GCTTGGGGTT TTATCAATAT GGTGTATTGA ATACTGCCAT 2280

CAATATTATA CTATACATCA TCTTGGTTAT TGATTTTGGA TTTAATTTAA GCGCCACAAG 2340

AGAAATATCA CAGAAAAAAA ATAACTTTGT AGCGGTATCT AAAATATTTA CAGATGTGAT 2400

TTTTGCAAAA ATTATACTTT TTTTCGGTGT GCTTGTTTTT ACTTTCCTCA TACTTAAACT 2460

AATTCCCGAC TATCAAGATG TAACAAGATT GGTTTATTTT ATGTTACCTC AAGTCCTATC 2520

TTCGATATTA TTCCCACTGT GGTTATATCA AGGGTATGAA AAAATTGGAT ATGCTGCATT 2580

TATAAACGTT CTTGGAAAAT GTATAACAAT CCCATTGCTA TTAATCTTTG TACAAAATAG 2640

CAAAGATGTA ACAAATGCTG CAATTATATT AAGTTGGTCT TCTTTAGTTC CCTTATTAAT 2700

TTATTATATA CGTAGTAATC TTCCGCGAAC AAAGTTTTAT ACCTCAAGGT GTAGATTCAA 2760

GTACTTAAAT AGAACATTAG CTAATGCACT GGTCGTTTTT ATTGGTTCCA TTGCTGTAAA 2820

CCTGTATACT TTAAGCACAC CATTAGTATT AAGTATAGTT AGTGATTTTG ATCAAGTTGG 2880

TTATTTTGTT GCAGCTGATA AAATAAGGGC TGCATTTATT GGCGTTTTTT TAATTGTAGG 2940

GCAGGCGATT TATCCTAGAG CAAGTGCCCT GTATAATGAA GATATAATTG CGTATAATAA 3000

ATTTGTCATA AGCATTATAT TTTACCAAAT TATAGTATGT ACGTTCTCTG CTATAGCCTT 3060

TTATTACTTC ATGCCAGAGG TGGCACCAAT TATTTTGAGT CAAAACAAAT TAAACGTAGA 3120

AGAGTTATCT GTTATAATTA AATTGATGTC GCCAATGATT ATTTTGATAC CTCTTTCGGT 3180

GATATTAGCT AACTGCGTAC TTTTGCCTCA AAGGAAAAAT AAACAATACG CTTTAGTGCC 3240

TATTATTACA GTCATAATTC ATATGAGTTA CTCTATAATT TTGTGTGAAA AGTTAGGTGC 3300

GCTAGGGGCA AGTTACGCAA TACTTCTCAC AGAAATTATT AGTTGTGCTT TACTAGCATA 3360

The termination orf3's of orf2 is initial

TTTTACATTC AAAGATAGAC TAATTCAGCT CAATCCAGCC AGA TAATATT GGATAATC AT 3420

GAATCATTCT TATATCTATG TTTATATATT TTTTGTGTTT CTTTCATTTT TTGAGCTTAA 3480

TGATACTAAT AGAGCAAGAT GTGATTTATA TTATGCCATA CAAGTTATTG TATTATTACT 3540

AACAATGGGG TTGTCGTATC AAATAGGTGT TGATTGGGTT GAATATGAAC GTGTTTATAA 3600

TGGGAATTCG GAATCATTAA ATAATTACGA ATTCGGTTAT ATAGTATTGA ATAATATTGC 3660

AAACTATTTA GGGGCTAGTT TTTGGGGGTT CGTGATATTA ATAAAATATA CGTTTTTTAT 3720

ATCATTGTTT CTTATGGTAA GAAAATTCAG TAGATTACCT GTTCTGACTA CAACATTAAT 3780

ACTAGGGTTG TTATTTCCAT TTATTAATGA CCCTTTACGA CAAATAATAT CTGCAAGTAT 3840

ATTGTTCTTT ACGATTTTTT TCTTAAACAG GCCCCCTAAA ATATTGGGGA TTATAGGGGG 3900

TGGGGTATTT CATTCTACAT TTATTATAAT TATAATTCGT TATCTTAAAA TTTTTACAAA 3960

AAGATCTATT GTGCTTATTT TCGTTTTTGT GGGGGTGGCA ATTCTTTTGT TCTTAAAGTA 4020

TGGTTCTTTT TTAGGTGATG GAATGTTATT TAGGAAATTA AATTTTTACA TGGAGTATTC 4080

ATCATTATCA AATTTGTATG GCTTTGCCCT AAGGTTTTTG CTATTGTCAT TTATTGTTTT 4140

TAATATTGAC GTTTACAAAG CTAATTGCAA ATTCATCAGA GGAGATATCT GTTATTATTT 4200

CTGGATTTTT AGTATAACAT ATTTATGGCT TGAAATAATA TTTGTAACAT TCCCTTTAAT 4260

CCCTCAGCGG ATGCGGCTTT ATTTGTTACC ATTTGCTTTT ATACTATTAA GTAACTACCT 4320

TTATTATAGT CGATTAACTT TCACAAAAAA TATTCTTGCT TTTATATGTG TAATCATTTC 4380

ATTTTCTTCA TTGTGTCTTT TTTTAGATGG TCCTATGGGG CATTTTTATA GCATTACTGA 4440

TAATATAGTA TTAAAATATA TTCAGGGTTT TACTAACGAT AAAACATATG ATGCAAACCA 4500

The termination orf4's of orf3 is initial

GTTCTGGATT AATGGAATT T AAATGAAAAA AAATGTCTTG ATAATAGCTT CTGCAGCAAA 4560

AGAAGGTGGG GCTTTAAGCA TACTTACAGA AAGTTTAAAT AACCTTAAAA TATTAAATAT 4620

TCATTTTACA GTTGTGGTCA ATCCATCTGT GGTTAAATAT TTGCCTGTTG CTAATAATAT 4680

AAAATATCAT ATGGTAGATA CATCGGCGTG GATTAAAAGA ATAGCATATG ATTTCTATGG 4740

CTATCCTGGT CTCAAGTATG AAGAATATAT AGCGTGCATA AATTTTCAAA ATATACCTAT 4800

AAGAACTAAA ATAAAGCAAA TAGTATATTA TCATCAGTCA TTACCTCTTT CGGATACATT 4860

ATTCTCACCG TTTAATAAAG AAACGCGAGT TTTATATTTA TATCAAAAAT TCTATGGTTT 4920

ATTTTTCAAG TTAAACAAAA GGTATATATC GCATCTAGTA GTTCAGGCAA ATTGGATTAG 4980

AGAAAAATAT CTGAAATTTG GTGTTGAAAA TAATAAAATT GAAGTTATTG CACCTCAAGT 5040

CGAAATTTTT AAATATATAA GTTTAGAGTT TAATAAAATA AGACCTGTAG AGAATGTATT 5100

TTTTTATCCT GCAAATGCGT ATACTTATAA AAATCATTTG ATTATAATCA AAGCTTTAAA 5160

TTATTTAGGG AGTGTTTATA TTAAAAAACA TCGTATTAAG GTTGTTTTTA CTATAAATAA 5220

TAGTGAGTCA AATTATCTTC AATCAATGGT TTTGAATTAT AATTTGCAGG ATGTTGTTAG 5280

TTTTATTTGT CGAATTCCGA GAGCCGATGT TTATTCTCTC TTGATAAAAT CAAGAGCTCT 5340

TCTTTTCCCA AGTAAATTAG AAACTTATGG TTTACCATTA AAAGAGGCGC GGCAACTAGG 5400

TGTATTTATT GTCGCATCGA AGTTAGATTA CGCAACTGAA GTATTGGAGG GATACGAATA 5460

TAAAAGATTA TGCTCGCCAG ATAGTGCTAA AGAATGGGCT TCGGCTATAA AGGAAATTAT 5520

AGAAAATAAG ATTGATGTCG GAAATACAGT CACTAGAGAT AATATTAAAG AGAGCGATTT 5580

The termination orf5's of orf4 is initial

GTTTTCTGAT TTTATTAAAA GTAAAATAAT GGAG TGATTA TTT ATGTTTG CAGAAAAAAC 5640

ATTGCTTATT ACTGGTGGTA CGGGGTCTTT TGGTAATGCT GTTCTTAGAC GTTTCCTTGA 5700

CACTGATATC AAAGAAATAC GCATTTTTTC TCGAGATGAG AAAAAGCAGG ACGATATGAG 5760

GAAAAAGTAT AATAATCAGA AAATTAAGTT CTATATAGGT GATGTTCGCG ATTATTCGAG 5820

TATCCTTAAT GCTTCTCGAG GTGTTGATTT TATTTATCAT GCAGCAGCTC TGAAGCAAGT 5880

GCCTTCCTGC GAATTCCACC CCATGGAAGC TGTAAAAACG AATATTTTAG GTACTGAAAA 5940

CGTTCTAGAA GCTGCAATAG CTAATCGCGT TAGGCGAATT GTATGTCTGA GTACAGATAA 6000

AGCCGTATAT CCTATTAATG CAATGGGCAT ATCTAAAGCA ATGATGGAAA AAGTCATTGT 6060

TGCAAAATCA CGTAATCTTG ATAGTTCAAA AACAGTTATC TGCGGAACAC GTTATGGGAA 6120

TGTAATGGCT TCACGTGGAT CGGTCATCCC ATTGTTTGTT GATCTAATCA AATCTGGTAA 6180

ACCATTGACC ATTACCGATC CCAATATGAC TCGTTTCATG ATGACGCTTG AGGATGCTGT 6240

TGATCTGGTC CTTTATGCCT TCGAGCATGG AAATAACGGT GATATTTTCG TTCAGAAAGC 6300

TCCTGCGGCA ACAATTCAAA CATTAGCCAT TGCACTTAAG GAATTGCTAA ATGCCCATGA 6360

ACATCCAATC AATATTATTG GAACTCGACA CGGGGAAAAA CTTTACGAAG CGTTATTGAG 6420

CCGAGAGGAA ATGATAGCAG CGGAAGATAT GGGTGATTAT TATCGTGTTC CACCAGATCT 6480

CCGCGACTTG AACTATGGTA AATATGTGGA ACATGGTGAC CGTCGTATCT CGGAAGTGGA 6540

AGATTATAAC TCTCATAATA CTGAGAGATT AGATGTTGAG GGTATGAAAA AATTACTGCT 6600

The termination of the initial orf5 of orf6

AAAACTTCCT TTTATCCGGG CAATTCGTTC TGGTGAAGAT T ATGAGTTGG ATTCA TAATA 6660

TGAAAATTTT AGTTACTGGT GCTTCAGGGT TTATCGGCCG TAATTTGGTT TTCCGCCTTA 6720

AGGAGGCTGG TTATAACGAA CTTATTACGA TAGATCGTAA CTCTTCTTTG GCGGATTTAG 6780

AGCAGGGACT TAAGCAGGCA GATTTCATTT TTCACCTTGC AGGAGTAAAT CGTCCTGTGA 6840

AGGAGAGTGA ATTTGAAGAG GGAAATAGCA ACGTAACTCA ACAGATTGTT GATATTCTGA 6900

AAAAAAATAG TAAAAATACT CCTATCATGC TGAGTTCTTC CATCCAGGCT GAATGTGATA 6960

AGGCTTATGG AAAGAGTAAA GCGACTGCGG AGAAAATCAT TCAGCAGTAT GGGGAAACGA 7020

CAAATGCCAA ATATTATATT TATCGTTTGC CGAATGTATT CGGTAAGTGG TGTCGCCCAA 7080

ATTATAACTC CTTTATAGCA ACTTTCTGCC ATCGCATTGC AAATGATGAA ACTATTACAA 7140

TTAATGATCC TTCAGCAGTT GTTGATCTGG TGTATATAGA TGACTTTTGT TCTGACATAT 7200

TAAAGCTATT GGAAGGAGCG AACGAAACTG GTTACAGGAC ATTCGGTCCA ATTTATTCTG 7260

TTACTGTTGG TGAAGTGGCA CAATTAATTT ACCGATTTAA AGAAAGTCGC CAAACATTAA 7320

TCACCGAAGA TGTAGGTAAT GGATTTACAC GTGCATTGTA CTCAACATGG TTAAGTTATC 7380

TGTCTCCTGA ACAGTTTGCT TATACGGTTC CTTCTTATAG TGATGATAGA GGGGTATTCT 7440

GTGAAGTATT GAAAACGAAA AACGCGGGCC AGTTTTCGTT CTTTACTGCG CATCCAGGAA 7500

TTACTCGGGG GGGGCATTAT CATCATTCCA AAAATGAGAA ATTTATTGTC ATCCGAGGAA 7560

GTGCTTGTTT CAAATTTGAA AATATTGTCA CGGGTGAGCG ATATGAATTT AATGTCTCCT 7620

CAGATGATTT TAAAATTGTT GAAACAGTTC CGGGGTGGAC GCATGACATC ACTAATAATG 7680

GCTCGGATGA GCTAGTTGTT ATGCTTTGGG CAAATGAAAT ATTTAATCGT TCTGAACCAG 7740

The termination orf7's of orf6 is initial

ATACTATAGC GAGAGTTTTA TCG TGAAAAA ATTGAAAGTC ATGTCGGTTG TTGGGACTCG 7800

TCCAGAAATT ATTCGACTCT CGCGTGTCCT TGCAAAATTA GATGAATATT GTGACCACCT 7860

TATTATTCAT ACTGGCCAAA ACTACGATTA TGAATTGAAT GAAGTTTTTT TCAAAGATTT 7920

GGGTGTTCGC AAACCAGATT ATTTTCTTAA TGCCGCAGGT AAAAATGCAG CAGAGACTAT 7980

TGGACAAGTT ATCATTAAAG TTGATGATGT CCTTGAACAG GAAAAACCAG AAGCTATGTT 8040

AGTTCTTGGC GATACTAACT CCTGTATTTC AGCAATACCA GCAAAGCGTC GAAAAATTCC 8100

GATCTTCCAT ATGGAGGCGG GGAATCGTTG TTTTGACCAA CGCGTACCGG AAGAAACTAA 8160

CAGAAAAATA GTTGACCACA CTGCTGATAT CAATATGACA TATAGCGATA TCGCTCGTGA 8220

ATATCTTCTG GCTGAAGGTG TACCAGCCGA TAGAATTATT AAAACCGGTA GCCCAATGTT 8280

TGAAGTACTC ACTCATTACA TGCCGCAGAT TGATGGTCCC GATGTACTTT CTCGCCTGAA 8340

TTTAACACCT GGGAATTTCT TTGTGGTAAG TGCCCACAGA GAAGAAAATG TTGATACCCC 8400

TAAACAGCTC GCGAAACTGG CGAATATACT TAATACCGCG GCTGAAAGAT ATAATATCCC 8460

GGTAGTTGTT TCTACTCATC CGCGCACTCG CAACCGCATC AATGAAAACG GTATTCAATT 8520

CCATAAAAAT ATCTTGCTAC TTAAGCCATT AGGATTTCAC GATTACAACC ATCTGCAAAA 8580

AAATTCACGT GCTGTTTTAT CGGACAGCGG GACTATTACA GAAGAGTCCT CCATTATGAA 8640

TTTCCCTGCA CTCAATATAC GAGAAGCGCA CGAACGCCCG GAAGGCTTCG AAGAAGGGGC 8700

AGTAATGATG GTTGGTCTTG AGTCTGAGCG CGTGTTACAG GCATTAGAAA TTATTGCAAC 8760

ACAACCTCGT GGTGAATTAC GCTTACTTCG TCAGGTCAGT GACTATAGTA TGCCAAATGT 8820

TTCAGATAAA GTTGTGCGTA TTATCCATT CATATACTGAC TACGTTAAAC GGGTTGTCTG 8880

The termination orf8's of orf7 is initial

GAAGCAATAC TAATGAAACT TGCATTAATC ATTGATGATT ATTTGCCCCA TAGCACACGA 8940

GTTGGGGCTA AAATGTTTCA TGAGTTAGGC CTGGAATTGC TGGGCAAAGG CCATGATGTA 9000

ACTGTAATTA CGCCTGACAT CACTTTACAA GCAATCTATT CTGTTAGTAT GATTGATGGT 9060

ATAAAGGTTT GGCGTTTCAA AAGTGGACCT TTAAAGGATA TAGGTAAGGC TAAGCGTGCC 9120

ATAAATGAAA CTCTTTTATC TTTTCGTGCA TGGCGTGCAT TAAAGCACCT CATTCAACAT 9180

GATACATTTG ATGGTATCGT TTATTATTCC CCCTCTATTT TTTGGGGAGA CTTGGTTAAA 9240

AAAATAAAAC AGCGATGCCA GTGCCCAAGC TATCTGGTCC TAAGGGATAT GTTTCCACAG 9300

TGGGTCATTG ATGCAGGTAT GTTGAAAGCC GGTTCGCCAA TTGAAAAATA TTTCAGGTAT 9360

TTTGAAAAAA AATCATATCA ACAGGCTGAC CGGATTGGGT TAATGTCTGA TAAGAATCTT 9420

GAGATATTTC GTCAGGCTAA TAAAGGTTAT CCATGTGAAG TTTTACGTAA TTGGGCCTCA 9480

ATGACTCCTG TGTGTGCCAG CGATGATTAT AATTCCCTTC GCCAAAAATA CGATCTAAAA 9540

GATAAAGTCA TTTTTTTTTA TGGAGGTAAT ATTGGGCATG CTCAGGATAT GACAAACTTA 9600

TTGCGCCTTG CGCGCAATAT GATGCGTTAT CATGATGCTC ATTTCCTGTT TATAGGGCAG 9660

GGTGATGAAG TTGACCTGAT TAATTCTCTT GCTGCAGAAT GGAATTTAAC TAATTTCACT 9720

CATCTACCTT CAGTGAACCA GGAAGAGTTT AAATTAATTT TATCTGAAGT TGATGTTGGC 9780

CTGTTCTCCC TTTCGTCTCG CCATTCTTCA CATAATTTCC CGGGGAAATT ACTAGGGTAT 9840

ATGGTTCAAT CAATCCCGAT CCTTGGGAGC GTGAATGGCG GTAATGATCT GATGGATGTA 9900

ATTAATAAGC ACAGGGCCGG GTTCATTCAT GTTAATGGTG AAGATGATAA ACTGTTCGAA 9960

TCTGCACAAT TGCTTCTTGC AGATTCGGCT TTAAGAAAGC AGTTAGGTCA GAACGCTAAT 10020

GTGTTGTTAA AATCTCAATT TTCGGTTGAA TCGGCGGCAC ATACTATCGA AGTCCGACTG 10080

The termination of the initial orf8 of orf9

GAGGCAGGAG A ATGCGTT TA GTTGATGACA ATATTCTGGA TGAGCTTTTT CGCACAGCAG 10140

TAAATTCTGA ACGTTTGCGC GCTCATTATT TATTGCACGC ATCTCATCAG GAGAAGGTTC 10200

AACGTTTACT TATTGCATTT GTACGCGACA GCTATGTTGA GCCCCATTGG CATGAGTTAC 10260

CGCATCAGTG GGAAATGTTT GTCGTCATGC AAGGGCAATT AGAAGTTTGT TTGTATGAGA 10320

AAAATGGTGA GATACAAAAA CAGTTTATTG TTGGAGACGG TACTGGAATA AGCGTCGTGG 10380

AATTTTCCCC AGGTGATATA CATAGTGTCA AATGCCTGTC ACCGAATGCC CTTATGTTGG 10440

The termination of orf9

AGATAAAGGA AGGACCATTT GACCCACTGA AAGCTAAGGC TTTTTCTAAG TGGTTA TAGG 10500

GCGATACACC ACCCTTTATT CTTCTATCTT ATTCTATACA TGCTGGGTTA CCATCTTAGC 10560

TTCTTCAAGC CGCGCAACCC CGCGGTGAAC ACCCCCTGAC AGGAGTAAAC AATGTCAAAG 10620

CAACAGATCG GCGTCGTCGG TATGGCAGTG ATGGGGCGCA ACCTTGCGCT CAACATCGAA 10680

AGCCGTGGTT ATACCGTCTC TATTTTCAAC CGTTCCCGTG AAAAGACGGA AGAAGTGATT 10740

GCCGAAAATC CAGGCAAGAA ACTGGTTCCT TACTATACGG TGAAAGAGTT TGTTGAATCT 10800

CTGGAAACGC CTCGTCGCAT CCTGTTAATG GTGAAAGCAG GTGCAGGCAC GGATGCTGCT 10860

ATTGATTCTC TCAAGCCATA CCTCGATAAA GGTGACATCA TCATTGATGG TGGTAACACC 10920

TTCTTCCAGG ACACAATTCG TCGTAACTGT GAGCTGTCTG CAGAAGGTTT TAACTTTATC 10980

GGTACCGGTG TTTCCGGTGG TGAAGAAGGT GCGCTGAAAG GACCTTCCAT CATGCCTGGT 11040

GGGCAGAAAG AAGCCTATGA ACTGGTTGCT CCGATCCTGA CCAAAATCGC CGCCGTTGCT 11100

GAAGATGGCG AACCGTGCGT TACCTATATT GGTGCCGATG GCGCAGGTCA CTATGTGAAG 11160

ATGGTTCACA ACGGTATTGA ATACGGTGAT ATGCAGCTGA TTGCTGAAGC CTATTCTCTG 11220

CTTAAAGGTG GCCTGAATCT CTCTAACGAA GAACTGGCGC AGACGTTTAC CGAGTGGAAT 11280

AACGGTGAAC TAAGCAGCTA CCTGATCGAC ATCACCAAAG ACATCTTCAC TAAAAAAGAT 11340

GAAGACGGTA ACTACCTGGT TGATGTGATC CTGGATGAAG CGGCTAACAA AGGTACCGGT 11400

AAATGGACCA GCCAGAGCGC GCTGGATCTC GGCGAACCGC TGTCGCTGAT TACCGAGTCT 11460

GTGTTTGCAC GTTATATCTC TTCTCTGAAA GAGCAGCGTG TTGCCGCATC TAAAGTTCTC 11520

TCTGGCCCGC AAGCGCAGCC AGCTGGCGAC AAAGGTGAGT TCATCGAAAA AGTTCGCCGT 11580

GCGCTGTATC TTGGCAAAAT CGTTTCTTAC GCTCAGGGCT TCTCTCAGCT GCGTGCAGCG 11640

TCTGAAGAGT ACAACTGGGA TCTGAACTAC GGCGAAATCG CGAAGATTTT CCGTGCTGGT 11700

TGCATCATTC GTGCGCAGTT CTTGCAGAAA ATCACCGATG CTTATGCCGA AAATCCGCAG 11760

ATCGCTAACC TGCTGCTGGC TCCGTACTTC AAGCAAATTG CCGATGACTA CCAGCAGGCG 11820

CTGCGTGATG TCGTTGCTTA TGCAGTACAG AACGGTATCC CGGTTCCGAC CTTCGCTGCT 11880

GCGGTTGCCT ATTACGATAG CTACCGTGCC GCTGTTCTGC CTGCGAACCT AATCCAGGCA 11940

CAGCGCGACT A 11951

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

Claims (4)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O12 type, it is characterized in that: its wzx sequence is the Nucleotide of 2147 to 3406 bases among the SEQ ID NO:1 or Nucleotide that the wzy sequence is 3419 to 4522 bases among the SEQ IDNO:1 or above-mentioned sequence for inserting, lack or replace one or more Nucleotide, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O12 type.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O12 type, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 2743 to 2759 bases among the SEQ ID NO:1 and the Nucleotide of 3190 to 3205 bases, the Nucleotide of 2594 to 2613 bases among the SEQ ID NO:1 and the Nucleotide of 3051 to 3076 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 3815 to 3834 bases among the SEQ IDNO:1 and the Nucleotide of 4405 to 4420 bases; The Nucleotide of 3664 to 3679 bases among the SEQ IDNO:1 and the Nucleotide of 4034 to 4049 bases.

3, the application of oligonucleotide in detecting the O-antigen of expressing the antigenic bacterium of O-, identifying bacterium of claim 1, any described O-antigen-specific to intestinal bacteria O12 type of 2.

4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O12 type of 2, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, and the confession detection bodies reaches the bacterium in the environment outward.