CN1261569C - Nucleotide specific for escherichia coli 0149 O-antigen - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O145. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O145 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 16932 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotide from glycosyltransferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O145. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O145 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O145 by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O145 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O145 type (Escherichia coli O145), particularly relate in the intestinal bacteria O145 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O145 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, all there is the conserved sequence of one section 39bp O-antigen gene bunch upstream, be called JUMPStart sequence [Reeves, P.R., et al. (1996) " Bacterial polysaccharidesynthesis and gene nomenclature " Trends in Microbiology, 4:495-503].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

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

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specificselection and bacterial populations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' s identification of theEnterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between theenteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152and and SHigella O antigens " J.clinMicrobiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O145 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O145 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 O145 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O145 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf7, orf14 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of intestinal bacteria O145 type respectively comprises orf7, orf14 gene; The gene that coming from coding transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O145 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O145 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O145 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O145 type of these methods detections and identification of escherichia coli O145 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O145 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 O145 type: it is the isolating Nucleotide shown in SEQ ID NO:1,16932 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 O145 type is comprising called after orf1, nnaB, nnaC nnaA, wzx, wzy, orf7, orf8, orf9, orf10, fn11, fn12, fn13, orf14,15 genomic constitutions of orf15 are all between JUMPStart sequence and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O145 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 orf7, orf14 gene; Wherein said gene: wzx is the Nucleotide of 4369 to 5607 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 5615 to 6802 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 6805 to 8061 bases among the SEQ ID NO:1; Orf14 is the Nucleotide of 13879 to 15090 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O145 type wherein also comprises coming from described wzx gene, wzy gene or glycosyltransferase gene orf7, orf14 gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O145 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 4468 to 4488 bases among the SEQ ID NO:1 and the Nucleotide of 5059 to 5077 bases; The Nucleotide of 5054 to 5074 bases among the SEQ ID NO:1 and the Nucleotide of 5532 to 5552 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6061 to 6078 bases among the SEQ IDNO:1 and the Nucleotide of 6687 to 6706 bases; The Nucleotide of 5849 to 5866 bases among the SEQ IDNO:1 and the Nucleotide of 6280 to 6297 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O145 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 O145 type is providing the O-antigen of expressing intestinal bacteria O145 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 O145 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 bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O145 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O145 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 O145 type bunch: with the genome of intestinal bacteria O145 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 O145 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O145 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 former high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O145 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O145, 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 O145.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O145 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O145 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O145 type bunch: with the genome of intestinal bacteria O145 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JUMPStart sequences Design upstream primer wl-1098 (5 '-ATT GGTAGC TGT AAG CCA AGG GCG GTA GCG T-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer wl-913 (5 '-TAG TCG CGT GNG CCT GGA TTA AGTTCG C-3 ') in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the WizardPCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 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 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of BiO-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of BiO-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-protogene bunch library of intestinal bacteria O145 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 O145 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O145 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 O145 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 15 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 O145 type at last;

(6) specific gene screening: at wzx, the wzy gene gene design primer in the O-antigen gene of dysentery intestinal bacteria O145 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 O145 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 O145 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 O145 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4468 to 4488 bases among the SEQ ID NO:1 and the Nucleotide of 5059 to 5077 bases; The Nucleotide of 5054 to 5074 bases among the SEQ ID NO:1 and the Nucleotide of 5532 to 5552 bases; The Nucleotide of 6061 to 6078 bases among the SEQ ID NO:1 and the Nucleotide of 6687 to 6706 bases; The Nucleotide of 5849 to 5866 bases among the SEQ ID NO:1 and the Nucleotide of 6280 to 6297 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 O145 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 O145 type, its complete sequence shown in SEQ ID NO:1,16932 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 O145 type by method of the present invention, as shown in table 3, it comprises 15 genomic constitutions of called after orf1, nnaB, nnaC, nnaA, wzx, wzy, orf7, orf8, orf9, orf10, fn11, fn12, fn13, orf14, orf15, all between JUMPStart sequence and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O145 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 (orf7, orf14 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 O145 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O145 type is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf7, orf14 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, also listed these oligonucleotide to the position in O-antigen gene bunch and with the size of these oligonucleotide to the product of the PCR reaction done for primer in table 1, 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 O145 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O145 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O145 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As used herein, " oligonucleotide " mainly refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 4369 to 5607 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 5615 to 6802 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide is high special to intestinal bacteria O145 type.

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

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O145 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O145 type.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O145 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O145 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O145 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 O145 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 O145 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O145 type bunch:

With the genome of intestinal bacteria O145 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JUMPStart sequences Design upstream primer wl-1098 (5 '-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer wl-913 (5 '-TAG TCG CGT GNGCCT GGA TTA AGT TCG C-3 ') in O-antigen gene bunch downstream; With the Expand LongTemplate PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mMDTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O145 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 O145 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O145 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O145 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 15 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 O145 type at last, as shown in table 3.

By retrieving and comparing, find that the WckD that encodes in orf1 encoded protein and intestinal bacteria (AAK64367) the O-antigen gene bunch has 74% consensus amino acid sequence and 88% similarity, by search, find that the homology desired value of the consensus sequence of orf1 encoded protein and known Bacterialtransferase hexapeptide is 0.028 to Pfam protein-based order sequenced data storehouse; Because the definite function of this gene can't be determined, so we are with the temporary called after orf1 of this gene.The NeuNAc condensing enzyme of coding has 81% consensus amino acid sequence and 91% similarity in orf2 encoded protein and intestinal bacteria (AAK64368) the O-antigen gene bunch, by search, find that the homology desired value of the consensus sequence of orf2 encoded protein and known NeuB family is 2.3e to Pfam protein-based order sequenced data storehouse ^-122Therefore we are nnaB with this unnamed gene.The CMP-NeuNAc synthetase of coding has 56% consensus amino acid sequence and 75% similarity in orf3 encoded protein and intestinal bacteria (AAK64369) the O-antigen gene bunch, by search, find that the homology desired value of the consensus sequence of orf3 encoded protein and known Cytidylyltransferase is 2.1e to Pfam protein-based order sequenced data storehouse ^-46Therefore we are nnaC with this unnamed gene.The GlcNAc-2-epimerase of coding has 63% consensus amino acid sequence and 79% similarity in orf4 encoded protein intestinal bacteria (AAK64370) the O-antigen gene bunch, by search, find that the homology desired value of the consensus sequence of orf4 encoded protein and known UDP-N-acetylglucosamine 2-epimerase is 1e to Pfam protein-based order sequenced data storehouse ^-110Therefore we are nnaA with this unnamed gene.The ORF_16 of coding has 29% consensus amino acid sequence and 48% similarity in Orf7 encoded protein and big Pseudomonasaeruginosa (AAM27597) the O-antigen gene bunch, because the definite function of this gene can't be determined, so we are with the temporary called after orf7 of this gene.The Orf8 encoded protein respectively with Bdellovibrio bacteriovorus (CAE79560) O-antigen gene bunch in the LPS biosynthesis protein WbpG of coding very high 68% consensus amino acid sequence and 84% similarity are arranged, by search, find that the homology desired value of the consensus sequence of orf8 encoded protein and known PP-loop family is 0.012 to Pfam protein-based order sequenced data storehouse.Because the definite function of this gene can't be determined, so we are with the temporary called after orf8 of this gene.The Orf9 encoded protein respectively with the O-antigen gene of Vibrio vulnificus YJ016 (BAC93119) bunch in the glutamine amidotransferase of coding 58% consensus amino acid sequence and 76% similarity are arranged, by search, find that the homology desired value of the consensus sequence of orf9 encoded protein and known Glutamine amidotransferase class-I is 1e to Pfam protein-based order sequenced data storehouse ^-24Because the definite function of this gene can't be determined, so we are with the temporary called after orf9 of this gene.The Orf10 encoded protein respectively with the O-antigen gene of Bdellovibrio bacteriovorus (CAE79562) bunch in the Imidazole glycerol phosphate synthase subunit hisF of coding 63% consensus amino acid sequence and 78% similarity are arranged, by search, find that the homology desired value of the consensus sequence of orf10 encoded protein and known Histidine biosynthesis protein is 1.6e to Pfam protein-based order sequenced data storehouse ^-55Because the definite function of this gene can't be determined, so we are with the temporary called after orf10 of this gene.The Orf11 encoded protein respectively with the O-antigen gene of intestinal bacteria (AAN60461) bunch in coding 4,6-dehydratase, 3-and 5-epimerizase has 88% consensus amino acid sequence and 94% similarity, by search, find that the homology desired value of the consensus sequence of orf11 encoded protein and known Polysaccharide biosynthesis protein is 1.4e to Pfam protein-based order sequenced data storehouse ^-3Therefore we are with the temporary called after fn11 of this gene.The Orf12 encoded protein respectively with the O-antigen gene of intestinal bacteria (AAN60462) bunch in the reductase of coding 71% consensus amino acid sequence and 83% similarity are arranged, by search, find that the homology desired value of the consensus sequence of orf12 encoded protein and known NAD dependent epimerase/dehydratase family is 0.00053 to Pfam protein-based order sequenced data storehouse.Therefore we are with the temporary called after fn12 of this gene.The Orf13 encoded protein respectively with the O-antigen gene of intestinal bacteria (AAN60463) bunch in the C-2 epimerizase of coding 89% consensus amino acid sequence and 95% similarity are arranged, by search, find that the homology desired value of the consensus sequence of orf13 encoded protein and known UDP-N-acetylglucosamine 2-epimerase is 1.9e to Pfam protein-based order sequenced data storehouse ^-121Therefore we are fnl3 with this unnamed gene.The Orf14 encoded protein respectively with the O-antigen gene of intestinal bacteria (AAN60464) bunch in the L-fucosamine transferase of coding 71% consensus amino acid sequence and 84% similarity are arranged, because the definite function of this gene can't be determined, so we are with the temporary called after orf14 of this gene.The Orf15 encoded protein respectively with the O-antigen gene of intestinal bacteria (AAN60465) bunch in the WbuC protein of coding 68% consensus amino acid sequence and 87% similarity are arranged, because the definite function of this gene can't be determined, so we are with the temporary called after orf15 of this gene.

Orf5 and orf6 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O145 kind.The O-antigen transferring enzyme of Orf5 encoded protein and intestinal bacteria (AAO37697) has 22% sequence identity, 45% 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 orf5 is wzx.The O-antigen polysaccharase of Orf6 encoded protein and Vibrio cholerae (BAA33635) has 26% consistence, 45% 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 orf6 is wzy.

Embodiment 6: the screening of specific gene:

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O145 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 O145 group, has obtained the expection size, other the group in all do not increase the expection clip size correct product, so the O-of wzx, wzy gene pairs intestinal bacteria O145 type former all be 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 O145 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4468 to 4488 bases among the SEQ ID NO:1 and the Nucleotide of 5059 to 5077 bases; The Nucleotide of 5054 to 5074 bases among the SEQ ID NO:1 and the Nucleotide of 5532 to 5552 bases; The Nucleotide of 6061 to 6078 bases among the SEQ ID NO:1 and the Nucleotide of 6687 to 6706 bases; The Nucleotide of 5849 to 5866 bases among the SEQ ID NO:1 and the Nucleotide of 6280 to 6297 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 O145 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 O145 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 O145 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O145 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O145 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 IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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 O145 type in the 13rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O145 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O145 type, screen gene by PCR: two glycosyltransferase genes (orf7, orf14 gene) to the O-antigen high special of intestinal bacteria O145 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O145 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 O145 type.These all oligonucleotide all can be used for the intestinal bacteria O145 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 O145 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O145 type, altogether by 15 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 JUMPStart sequence 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 O145 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O145 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 O145 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O145 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>16932

<212>DNA

<213>Escherichia coli

<400>1

attggtagct gtaagccaag ggcggtagcg tgtgtttttg agatatttat gtattatcgt 60

tgtcatgaat acaagtaaca acaaagttaa tgtgatctaa tcaaaccatt agatggtatg 120

aatatgaagc aaaaattaat aattattggt gctggtggtt ttgctaaaac tgtaattgac 180

agtttggatc atgaaaagta tgaaatagaa gggttcattg atacatttaa aacaggtgag 240

catcaaggat accctatttt aggtgatact ttaggtgtca tagacgaacc taaccaatac 300

ctatacttta ttgcaattgg agatcccgat tacagagcct tatggatgaa gttaatagaa 360

gaaatgaaac tttcaactat caatgtaatt gacagaacat ctataatttc tgaacattct 420

aggcttggta cctgcattta tatagggaaa atggctatca ttaactgtga ttcagaacta 480

gaagatggag tggtgataaa cacaagagca ttagtagagc atgggaatta tatatcatat 540

tgtacaaata tctctactaa tgttgttctt aatggtgatg tattcgttgg ggaaaaatcc 600

tttataggaa gttgtaccgt tgttaatggg caactaaaga taggtaattc atcaattatt 660

ggttctggtt ctgtagttat tcgtgatgtt cctgacaatg tagtagtggc cggggctcct 720

acaaagttta ttagagcaag gtgattttta ttatgtcgag aatttatatt gttgctgaaa 780

ttggttgtaa tcataacgga gatttcgagt tagctaagaa aatggtaaaa gaggcaaaat 840

ctgccggggt agatgcagta aaattccaaa cttttaaagc tgagcaactt atctcaaaat 900

atgctcctaa agcagaatat caaataaaag ttaccggtaa tgaagagact caattagaga 960

tgactcgtaa gttagagttg ccatatgatg agtttatcaa gctggaagaa tatgcaaaag 1020

aacttgggtt ggatgtcttc tcaacaccat ttgattttga ttcaatcgat tttcttgcat 1080

ecagaaatca aaaagtttgg aagattccat caggcgaatt attaaattta ccatatttag 1140

agaaaatagc caaattgcca atcgaagata aaaaaattgt actatcaact ggtatggcaa 1200

cagttgatga aatcaatttg gcattaaagg tttttattga taatggcatt acacatagaa 1260

atataacaat attacattgt aatacagagt atccaacgcc atttgaagat gtaaatctaa 1320

atacaatttc tggattcaaa aaaatattta gtcaatataa tattggattt tctgatcatt 1380

ctccaggtta ttttgctggt attgcatcag tgccttatgg gatcactttt attgagaaac 1440

attttacact ggataaaaat tttgaaggtc cagatcataa agcatcagtt acacctgagg 1500

aacttaaatt actatgcgaa ggaatacgtg cagtagaaat ttcacttggt agctatgaaa 1560

agttagtaac aaattcagaa cgaaagaata aagtagtagc tcgaaaatca attgttgcta 1620

aatgtgcaat aaaaaaagga gatattttta cgacggaaaa tattacgaca aagcgaccag 1680

gaaacggaat tagtccaatg tactggtatg aggtcttagg taaaatagct gaaaaggatt 1740

ttgatgagga tcaactaatt gagcactctg attttagtaa acaagaggtt tgagttgtgt 1800

cattaaaaaa aattgccatt atacctgctc ggtctggatc taagggctta ccaaataaaa 1860

atatactaat gttattggat cgcccattaa ttgcttatac tatcgaagca gcgattagct 1920

ctaatatatt tgataaaatt attgtttcaa cagattcatt agaatataaa tatattgcag 1980

agaaatatgg cgcggaagta atattacgca caaaagaatt atcgttggat tcggcaacat 2040

cttttatggt agttcaagat gtgcttgaaa aatgtccagg atatgattat tttgtgcttc 2100

tacaaccaac atcaccattt cgtaattata agcatattaa gaatgctgtt gagcaatttg 2160

aaaataatca cgaagcaaaa tttttggtgt ctgtggtaga aagtgataaa agttcggcgt 2220

taattaaacc aattgataat tcactgtcat taaggaactt tgactgtgat tttagtacat 2280

atcgtagaca aaataaaaaa gaatactgtc caaatggtgc gatttttatt ggttatgttt 2340

ctaattattt aagacaaaaa catttctttg gtgctgacag tatagcctat attatgaata 2400

aagaagattc tattgatatc gatgatcagt tagattttga attggcaatt ctaatccaaa 2460

caaaaaaaaa taaaaaaaat ttattggata atgctattat caaacgaatt gtcgataaaa 2520

aagatttatt taacaaagtt gagcaaatta cactgattgg acattcaatt tttgattatt 2580

gggatctcag caacatatgt ggaattaagg ttaacaattt aggcattgct ggcattgata 2640

gcgaaaaata ttacaaatac atcattgaaa aaaacatgct caccaatata ggtaagtatg 2700

tgctattaat ttcaggcaca aacgacattg tgaacgatgg ttggactatt gaatacacta 2760

taaaatggac aaaaaatctg atcaatagag taaaaattat aaatcctgat gttactatca 2820

ttctgctagc agtgccgcct gttcgaggaa gagttgatag ggataataat actatcaata 2880

aattaaatct ggcaatgaaa caatatttta gtaaattaga caatgttatt tggatgccgt 2940

tatctccatc tttttatgat gagttcggca atctgaatga aaattatact tatgatggtt 3000

tacatttcac gttacaagct tacaaacagt tagaaaatga tatttcgagt atcttaaaat 3060

gaaaagaaaa ttgctctatg tcaccggctc aagagctgag tacggtatta tgaaacgact 3120

attaaaaagt ctaaaagatg atcctgatat tgatttatca attattgcaa caggtatgca 3180

ttgcgattct gaatatggtt atacatataa aacaatcgag aatgatggtt tcttaattga 3240

aaaattaatt gatttgcagc tgaaaaataa aaccaacgca gatgttttga aaacgatgtc 3300

gatatgccaa caagcatttg gtgagcattt ccaagagaaa aaatatgatg ctgtcattat 3360

attaggtgat cgatacgaaa tattttcagt ggcagtagca gcttctatgc ataacctacc 3420

tattattcat cttcatggtg gagagaaaac gttagggaat tatgatgaat ttatacgaca 3480

ttcaataacg aaaatgagtc gtctacatct agtatcaact gatgaatatc ggaaaagggt 3540

tatccaactt ggtgaagatc ctaattgtgt atttaatgta ggtgcgctag gggctgaaaa 3600

cagtttgcaa ctagaattac cctctaaaaa ggagcttgaa aataagtacg gtgggttaaa 3660

tagaaaatat ttcgtagtcg tatttcatcc tgaaacgtta tcaactttac ctgttagaga 3720

gcagtattta gaattactag aagcattagc tagttttagc gaagaatacg attatatctt 3780

tattggctca aatgctgata ctggttcaga acaaataaga gaaatgactc tcgatttttg 3840

tagagtatat ggctgtcgat atatgatttc tgtgagacct gaagaatatc tagctttgat 3900

aaaatattca tatggcttaa taggcaattc gtcatctggg ttaattgagg ttccaagctt 3960

aaaggttccg acgattaata taggcgatcg ccaaaaagga agggttagag gtgattcggt 4020

aatagatacc atatgtaaaa aagaaagtat agaatgtgcc attgcttatt cacaacagaa 4080

aagtttcatt gaacgtgtaa aaaatgctca taatccttac tataataatg atgtaatgaa 4140

tagtattgtt aaaatcatta aagatttttt aacttatgaa aagttgggtt ataaagactt 4200

ctatgatgtt gcatatgact atagtcattt aaaataaaca acatgggtat tgtttttaat 4260

aagttatttg tatgatattt taaatttaaa aacctcatat taatatttaa attattgcaa 4320

atttaataat cacagattaa aaacttccga ttttaacatt ggccttggat gtttaatact 4380

atgcttaagt attactcaag tgttggatta agagggatta ctctacttac taaatttatt 4440

ttcattgttt tgcttgctcg acttttacca tcaacagatt taggagtgta tggattaatt 4500

aatgcagctg taggatatgg tattttcgtt gtaggttttg agttttatac gtattcaacg 4560

agagaaataa ttaactcgca aaaaaatagg ctttttttta tactaaaaaa tcaagctcta 4620

tttactgtta tatcttatat actatgtata ccggcattta tttttttatt atatttagaa 4680

atattaccat ctggaagtga atactggttt atcctacttt tattttttga gcacttatca 4740

caagagatta atagagttct aataacaata gaaagtcaat cgattgcaag ttttattctt 4800

tttgtaagac aaggtgtatg gtgttggtta gctatagctg tgatgctagt gtatccgaac 4860

ttaagaaata taacagttgt atttattttt tggtttggtg gtactgtgtc cgcgagtgtg 4920

cttggagtgg cttatatttt aaataaaaaa aaacaaagcg atattacaaa ctgggattgg 4980

acgtggataa aaaaaggtat aaagctgtct gtaccaatgc taattgcagc ccttgcacta 5040

cgaggctttt tcacgtttga tagattcgcg gtagaaaaaa tatcgggcct agaagttttg 5100

ggaggatata cattatttgt tagtatgact tcagctattc aatcattttt ggatactatt 5160

ttgatatctt tttcatttcc aaagcttgcc ttgttatatt cagggaaaaa atatataaaa 5220

tttaaatctg agttaagaaa attcacttat aaattaattt tactactatc tttcttgagc 5280

atctgttgct tttttactgg gattatattg gttaagtggt tggataaacg agattacata 5340

caattatttc ctgtatttat attattaata gcagcgactt atatctattg tataagtctt 5400

attccacata ttgctttata cgcgatgaga gaagatcgtt acatattagt aagtcaactg 5460

atatcatttt tatctttttt actatttgtt ttttttagcg tatatcaaag tgatatctat 5520

tacttgctaa ttggtatgat agctagtttt gtattacttt tgatcttaaa aatgatcccg 5580

ttatataaaa ttctaaaaaa ggtttaaaat aaacgtgaat ataaagaaag ataagtttat 5640

aaatggagtg attttttttt ggttaattat ttcttcgtta tattacttaa atgctatttt 5700

ttctggtgtt gacacattaa aatataatga agatttaacg caaaaaatta taaaatatat 5760

agtttgctta gttataagtc taagtatctt atttatttac aagaaattta attatttttt 5820

tgtattgttt tttttcttgt tcctgtctgt tgcttcagcc cttttcagtg gtgcggtaac 5880

aatttacgca acaacaatgt tgattattgc aactatgatc agcttttgcc tgattattcc 5940

tctattttct tataatatgg tgaaagttaa tagagttctt ttatggacag gagttattgt 6000

aggcacgatt tctgtattag aattaacggt attttataat tatatggttt catattgggc 6060

tgccactgat gggattaggt caatatcttc tcttctgaat cctacgaata gtggtgctta 6120

ttcagcgatt attattttaa tcgccttggt gacaaatata aaaagtcttt ttaaaagagc 6180

tttatttctt ataatgccga tgataacgtt aattagcagt ggttcgcgca cagcatggtt 6240

atcacttggt atgacacttt tattaacagt agtattgaga gacagtgcca gcattcgctt 6300

gcgaaaaaaa atatttactc ttgcaagcat tggcactgtt tgcggtgcat tgtacgccat 6360

attttatatg ggcagtatct ctggtattga atcacaatat cgaggtctta atacgtatac 6420

tgcatcaatt cgagttgaaa actttctgac atatttaaat ttagttgatc tgaatatgtt 6480

gctacctgat tttttagata aaaatataaa tctcatttca gataactttt atctcgtaat 6540

gtttaattat gccggtctaa tcggcttttt tattgtttta ttaattttat tgctgcttat 6600

cttctggaac atacaattta aaatatttaa tgagttaatg gctgaagata tagccatttg 6660

gagagttgtt tttatttatt tcctaatatc cgggctttca aattcattta taaattcttt 6720

tcctgtaaat caattgttct ttatctcatg cggatattat atatataaat ataaattagt 6780

taaaagctct ataggaagat aaatatgcaa ttatcaatgt cgcaaataat agaatgttat 6840

ttacacgcaa cacaaaatga aatagatatc gataagtctt tatcaaaaag cattacaaaa 6900

aaaatgccac cacttcccag gacaagtaaa tgggctgggg aaaggtggag ttcatcgagt 6960

ttgcaaatta tttatgcagt gattatgttt gcctttattt ttggggggtt tttattatat 7020

gttatattat tttttgctaa atatatattg gctaaattta aaaattccat ttcagaaata 7080

aaggttgcta ataataagga aggtaaattt tactattttt cgttttcaga tttagaaatg 7140

aggcaaactt gctatttttt taaagataat gatagttaca aattatctcg agatgaactt 7200

attataataa aattaccatg ggtaaattat acaccaacga gttgtgactt taatgctatt 7260

aatttatatg aattaacatc gtttctagat gtggcaagag cattcttgtt atctattttt 7320

agttatatct attatttaaa accatcacgt ataaaatggc ttcttcatat ttatacagca 7380

ccttcttggt ttttggttgc tatgggaatg aataatatta aaggcaatct cgcaagtagt 7440

gaacattatg atcgttgggc tgttttaact gattttatat gcagaataaa aaggaaaaga 7500

tatatattaa ttcaacatgg ctcgttactg gcgttaaaga ctaagggtta tgaatttttc 7560

tctctctcat ataaactgaa agctgtatca gagctcgcta tctttaacga aattgagctt 7620

gaattatttt tagagcatat aatctcacag gctaatgatt acaatataaa aattcatttc 7680

tatcaacagc cattttatgt ttcatcaatt aataataaag gattatcaat tctaattatt 7740

gggcattcat tgtgtgaacg aggacaacta agtcttgggt cacaactttc aacgttatca 7800

gataatattg ttctttatta taaagaacat ccaaaagcta gagcatctga aaaagccaaa 7860

aagacaaaat ggaatttcat tacagatgat gattatttcc ctgatgtaga tattgtcatt 7920

tcgtatcctt ccactttggc atatcaatac aaagagttga ataaaatagt tattcttcat 7980

gaattagata atattgatca aaataaaatt gacgagatac ttatgaccat tagaaaaaat 8040

aagggcgtgt atggaaaata aaaattatca aatttgtaat cattgtatta tggatacatc 8100

tgatcctcat attacttttg atgatagcgg agtatgcaat tactgcgcca attataaaaa 8160

caatatattg ccgacatgga gaaaaggttt gttttcagat gtttctttat ctgaaatggc 8220

aagtaaaata aaggcggacg caaagaatag tgattttgat tgtattattg gactatcagg 8280

ggggcttgat agttcatatg ctgcatatat tgcaaaagaa aagatggggc tacgcccatt 8340

attattccat gttgatgccg gctggaatac agatcaggct gttggtaata ttgaaaaact 8400

tattgaaggc cttggtttag aactttatac agaggtagta aactgggagg aaatgaaaga 8460

tcttcaactg tctttcttaa aatcaggaat tccagatcaa gatctagttc aggacgcttc 8520

ctttttttca tcattatata agtttgctcg acaacataga attaaacatg tcattacggg 8580

gtccaatttt tctactgagt gttgtcgtga accagaagaa tggggaggat atttaggcat 8640

tgatactctg ttatttaatg atatccatac gaaatttgga gaaaaaccgt taaaaacttt 8700

tcctttagtg gatatattgg tttataaaat ttactatcag aaaattcttg gtatgaaagt 8760

gcatcatcca ctaaatttag ttccttttaa taagaaggat gcagaaaatg aacttaataa 8820

aaaatttggc tggcaaccct tccagcataa acatcatgag tcccgtttca cccggtttta 8880

tgaagattat tggctgccac gcagatttgg ttatgagaaa cgaagagctc acttttcgag 8940

tttaattatg acgggacaaa tgtctcgtga acaagctctt gagaggattt caaaaccaga 9000

aatggatgag cactttctta aacaagagtt tgagtatgta gcgcacaaac ttggtattag 9060

tgttgaaaat ttgcaagagc tattttttat gcccaaaaaa acatataagg attataagaa 9120

taagcgttgg ttgattggtt taggtgcgaa tgtcttacgt gcacttggat tagaaaaaag 9180

gtatttcaga tgattacaat cattgattat ggtattggaa atatacaagc atttttgaat 9240

gtatataagc gactaggcat taatgcgggt gttgcccgtt ctgttgatga tatttttagt 9300

gctacacatt taataccgcc tggagttggg gcttttgatc aagctatgac tctttttaat 9360

aattcaggtt tacgagatag tattgaaaaa cgtgtatatg aagaaaagat tccaattatt 9420

ggtatctgtg tgggtatgca aatgcttgcg acctctagtg aggaggggag catgccaggt 9480

ttaggatgga ttccaggtac ggttcgagca tttagtagca atatctcttc acaaaacctc 9540

ccgatgcccc acatgggatg gaataattta attaaaagaa caagtacacc attgctgaaa 9600

gatttcgcag tagaaccttc attttatttt ttacactcat actattacga atgtgatgac 9660

acaggtgatg tattggcaac tgctaattat ggtcataatt ttcattgtat aattagcagg 9720

aataacatat atggtattca atgtcatcca gagaaaagcc attcatcggg atcccaacta 9780

ctaaaaaatt ttgcagagat ctaatagcat gcttagacca agaatcatac cctgtttact 9840

tatccacgat tctggcttag tcaaaacagt taattttaaa tcccctaagt atgttggtga 9900

tccaattaat gccgtcaaaa ttttcaatga gaaagaagct gatgaattga tggtcctaga 9960

tattgatgcg acatccaggg gactcgaacc taattatgat ttaataaaaa aactcgcagc 10020

ggaatgccgt atgcctttat gctatggagg aggcgtaacc agcgtagcac aggctacaaa 10080

aattatctct cttggtgttg aaaaggtatc aataagttca gcagctgttg aaaatcctaa 10140

tcttgttaga gaattggcag aagcggttgg aaaacaaagt gttgttgttg tgctggatat 10200

tatcaaacga aaaggtctat tctcaaaggg ctatgaactt tcgacaagga ataacactcg 10260

caagcataaa atcgatcccg tctcgtttgc aaaggaaatg gccgatctgg gagctggaga 10320

aattgtcatc aattttgtag ataatgatgg tgtcatggga ggatatgacg ttgcatattg 10380

ctcaactatc aaatcgcaaa taaatattcc tgttacgttt ctcgggggcg ctggtagtta 10440

tgaccatctt tctgcactga ttgaccaatg tggcatcgtt ggtgcggcag ctggtagtct 10500

ttttgtattt aaagggaagt atagggcagt attgataagc tatccaacac ctgaacagaa 10560

agatattatt tgtaatggtg taatgagtaa tcgtttataa ggggttatga tgttcaaaga 10620

taaaatactg ttaatcactg gtggtactgg ttcatttggg aatgctgtat taaatcgctt 10680

tctttccaca gatgtaaaag aaatacgcat atttagtcgt gatgaaaaaa agcaagatga 10740

tatgcgtaaa aattataaca ataaaaaatt gaaattctat attggtgatg ttagagatta 10800

tcgtagtatt ttgaatgcga ctcgcggagt tgattttata tatcacgcag cggcacttaa 10860

gcaagttcca tcatgtgagt ttcatcctgt ggaggctgtt aaaactaatg ttcttggtac 10920

agaaaatgtg cttgaagctg ctatagcgaa tgaagttaaa agggttgtgt gtcttagtac 10980

tgataaagct gtatatccga tcaacgctat gggaatttca aaagctatga tggaaaaagt 11040

catggttgcg aaatcgcgta atgttgactg caacaaaaca gtaatatgtg gtacccgtta 11100

tgggaatgtc atggcctctc gtggatctgt tattccatta tttgttgatc ttatcaaatc 11160

tggcaagcca ctaacaatta ctgaccctaa catgacccgt tttatgatga ctcttgagga 11220

tgctgtagat ttagtacttt atgcattcga acatggtaat aacggagata tttttgtaca 11280

aaaagctcca gctgcaacta tcgaaacatt agcaactgct ttaaaggatt tactaaatgc 11340

tgctcatcac ccagtaaata ttattggtac gcgtcatggt gagaagttat ttgaagcttt 11400

gctcagtcgt gaggaaatgc tctctgctga agatatgggg gattattatc gtataccacc 11460

agatcttcgt gaccttaatt atggaaaata tgttgaacaa ggtgatagtc gtatatccga 11520

ggttgaagat tacaactctc ataatacaca acggttgaac gttgaaggca tgaaaacgct 11580

tttgctcaaa ttggatttta ttcgtgcact tcatgcaggt taacaacatg gctaagactc 11640

atgatataaa aatattgatt actggtgcaa atggttttat tggccgtaat ttaagtttga 11700

ggcttgagga actaggttac aagaatctta ttagaattga tcgggattca acagaacaag 11760

atcttgaaca aggcttacag gacgctgatt tcatttatca tttggctgga gtcaatagac 11820

caaagactga tgatgaattt aaatccggaa acagtgattt aactaaatac atagttgagt 11880

atcttatttc tataggaaag aagataccaa ttatgctaag ttcttctata caagcagaac 11940

ttgacaatgc ttatggggta agcaaagctt tagctgaacg ttatattgaa aaatatgcta 12000

ctactagtgg ttctccatat tatattttca gatatccaaa cgtttttggc aaatggtgca 12060

aaccaaatta taattctttt gttgcgactt tttgctataa tattgcgaat gatcttgcca 12120

taactatcaa tgatgcatct gcgccagtta atttggttta tattgatgat gtttgtgcta 12180

aagcgataga gcttctctcg ggggcaactg aaagcggata taaagctgtt gcaccaattt 12240

attcaactac tgttggtgaa gttgcagaat taatttatag attcaagaat agccgttcca 12300

ccctgatcac agaggctgtc gggacgggat ttacccgtgc attgtattct acatggctga 12360

cttatttacc agcagagaag tttgcgtaca aggtaccttt ttatggggat gcccgcggag 12420

tcttttgtga gatgttgaaa acgccttcag cggggcagtt ttcatttttt actgctcacc 12480

ctggtattac gcgtggcgga cattaccatc acagtaaaaa tgagaagttt ttggtcattc 12540

gaggtcaggc atgctttaaa tttgaacatg tgattaccgg tgagcgatat gaactgaatg 12600

tttcatcgga tgagttcaag attgttgaaa cagttcctgg ttggacacat gacattacaa 12660

atattggaac tgaggaatta atagtcatgc tctgggcaaa tgaaattttc aaccgtgatg 12720

agcccgatac tattgcgaga cctctataat gaaaaaatta aaagttatgt ctgttgttgg 12780

aacccgtcct gagattatcc gtttgtcgag ggttcttgct aagtttgatg aatactgcga 12840

gcatattatt gtccatactg gtcaaaatta tgattacgaa ttaaatgaag tattcttcaa 12900

tgacttgggt gttcgaaaac ctgattattt tttaaatgca gcgggaaaaa atgcggcgga 12960

aaccattggc caagttatta ttaaggtaga tgaagtatta gaaatcgaaa aacctgaagc 13020

aatactggta ttgggcgata cgaattcatg tatttctgcc attccggcca aacgccataa 13080

agttcctata tttcatatgg aagcaggtaa ccgttgtttc gatcaacgcg tgcctgaaga 13140

aaccaacaga cgtattgttg accatacggc tgatatcaat atgacctaca gtgatattgc 13200

tcgtgaatat ctcttggctg aaggtatccc agctgatcgg atcataaaaa ctggtagccc 13260

tatgtttgag gttctttcat attatatgcc ccaaattgat ggttcagatg tgctatcgcg 13320

tttgaatcta cagtctggtg agttttttgt agtaagtgcg catcgtgaag agaatgttga 13380

ttctccaaaa cagctcgtaa agcttgcgaa cattctaaat actgttgctg aaaaatataa 13440

tcttccagtt attgtctcca cacacccaag gacacgtaac cgaatccgtg agcaaggaat 13500

tgaatttcat tcaaatataa atctactgaa accattgggt ttccatgatt ataaccactt 13560

gcagaagaac tcacgagctg tgctttcaga tagcggtact atcactgaag agtcatccat 13620

catgaatttc ccagcggtaa acatccggga agcgcatgag cgtccggaag gctttgagga 13680

agcatccgtc atgatggtgg ggttagagtg tgaacgcgta ttacaagcgc tggatattct 13740

ggcaacacaa ccgcgaggtg aagtccgtct tttacgtcag gttagtgatt acagcatgcc 13800

aaatgtgtcg gataaagttg tcagaattgt tcactcttac acagattatg ttaagagagt 13860

tgtctggaaa gaatattgat gaaacttgct ttaatcatag atgattacct gcccaacagt 13920

actcgtgttg gtgcaaaaat gtttcatgaa cttgctcaag aatttatcca gcgtgggcac 13980

gatgttacgg taattactcc tggtacgggc atgcaagaag agatttcttt tgataccttt 14040

cagggggtaa aaacatggcg ttttaaaagc gggccgctca aggatgtaag taaaattcag 14100

cgagtggtca atgaaacgct tttgtcctat cgggcgtgga aagccatcaa aaaatgggta 14160

aaaaaagaga cctttgaggg tgtgatttat tattcacctt ccatattctg ggggtattta 14220

gttaaaaaaa ttaaagctcg ttgccaatgt cctgcttatc ttattttaag agatatgttt 14280

ccacaatggg taattgatgc aggaatcctt aatgctggtt ccccaataga aegctacttt 14340

cgtctttttg aaaaaatatc ttgtcgtcag gcaaatcgta ttggacttat gtctgataag 14400

aatcttgatg tttttcggaa agataataaa ggctatccgt gcgaagtttt gcgtaattgg 14460

gcatccctaa caccaacgat catacccaag gattacatac cactacgtaa gcgacttggc 14520

ctagaggata aaaccatttt cttctatggt ggaaacatag gtcatgcaca ggacatgaca 14580

aacttgatgc gacttgtgag aaacatggca gcatatcctc aagctcattt cctatttatt 14640

ggccagggtg atgaagttga attaattaat tcattagcct ctgagtgggc attgacgaat 14700

ttcacctatt tgccctcggt taatcaggat gaatttaagt tcattttgtc ggaaatggat 14760

atcggcttgt tttctctttc cgctagacac tcttcccata attttcctgg taagttatta 14820

ggctatatgg ttcagtcgct acctatttta ggtagcgtaa atgccggaaa tgatttgctc 14880

gacattgtca atcaaaataa tgccggatta atccatgtca atggtgagga cgataaatta 14940

tgtcaatctg cgctattaat gttgcatgat attgatgtgc gccggcaact tggttcgggg 15000

gcgaatatat tgttgaaaga acaattctcc gttgagtctg cggcacagac gatagaaatg 15060

aggttggagg catgcaatgc gattaattga taatgaccaa ctcgacaaat tatatgatca 15120

agccgagcaa tcggaacgtt tacgttccca ccttattatg cacggctcgc atcaagaaaa 15180

ggtacagcgt ttacttattg cattagtaaa gggcagctat gttgaaccgc attatcacaa 15240

acttcctcat cagtgggaaa tgttcattgt tatggagggg caacttcagg tttgtttgta 15300

tggtagaaat ggtgaggtta taaagcaatt tatagcagga gataatattg gaataagcat 15360

tgtggagttt tctccgggcg atatacacag tgtcgaatgc ctatctccgc gtgctcttat 15420

ggtggaagtt aaagaggggc catttgaccc ttcttttgca aaatcgttcg tgtgatgctt 15480

gtctaaagta catcttctgc tatctactca agctaaacct gagttaacat ccataccata 15540

tttcaagctg cgcatatctt gcgcggtgac caccccctga caggagtatg taatgtccaa 15600

gcaacagatt ggcgtagtcg gtatggcagt gatggggcgc aatcttgcgc ttaacatcga 15660

aagccgtggt tataccgtct ctattttcaa ccgttcccgt gaaaagacgg aagaagtgat 15720

tgccgaaaat ccaggcaaaa aactggttcc ttactatacg gtgaaagagt tcgttgaatc 15780

tctggaaacg cctcgtcgca tcctgttaat ggttaaagca ggtgcaggca cggatgctgc 15840

tattgattcc ctcaagccat acctcgataa aggtgacatc attattgacg gtggtaatac 15900

cttcttccag gacactattc gtcgtaatcg tgagctttct gcagaaggct ttaatttcat 15960

tggtaccggt gtttccggtg gggaagaagg tgcactgaaa ggaccttcca ttatgcctgg 16020

tggccagaaa gaagcctatg aactggttgc accgatcctg accaaaatcg ccgcagtggc 16080

tgaagacggt gagccatgcg ttacctatat tggtgccgat ggcgcgggtc actatgtgaa 16140

gatggttcac aacggtattg aatacggtga tatgcagctg attgctgaag cttattctct 16200

gcttaaaggt ggcctgaacc tcaccaacga agaactggca cagaccttta ccgagtggaa 16260

taacggtgaa ctgagcagct acctgatcga catcaccaaa gatatcttca ctaaaaaaga 16320

tgaagacggt aactacctgg ttgatgtgat tctggatgaa gcggctaaca aaggtaccgg 16380

taaatggacc agccagagcg cgctggatct cggcgaaccg ctgtcgctga ttaccgagtc 16440

tgtgtttgca cgttatatct cttctctgaa agatcagcgt gttgccgcgt ctaaagttct 16500

ctctggcccg caagcgcagc cagctggcta caaagctgag ttcattgaaa aagttcgtcg 16560

tgcgctgtat ctgggcaaaa tcgtttctta cgctcagggc ttctctcagc tgcgtgctgc 16620

gtctgaagaa tacaactggg atctgaacta cggcgaaatc gcgaagattt tccgtgctgg 16680

ctgcatcatc cgtgcgcagt tcctgcagaa aatcaccgat gcctatgccg aaaatccgca 16740

gatcgctaac ctgctgctgg ctccgtactt caagcaaatt gccgatgact atcagcaggc 16800

gctgcgcgat gtcgttgctt atgcagtaca gaacggtatc ccggttccga ccttcgccgc 16860

tgcggttgcc tattatgaca gctaccgttc cgctgttctg cctgcgaacc taatccaggc 16920

gcagcgcgac ta 16932

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O145 type bunch and oligosaccharide unit treatment gene and wherein Primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzx	The transhipment enzyme	4369-5607	4468-4488	5059-5077	610bp	0 *	59
			5O54-5074	5532-5552	499bp	0 *	59
								wzy	Polysaccharase	5615-6802	6061-6078	6687-6706	646bp	0 *	59
			5849-5866	6280-6297	449bp	0 *	59

* only in intestinal bacteria O145 type, obtain a correct band

Table 2166 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 2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli shigella dysenteriae 10, Shigella bogdii 11, shigella flexneri 12, wild-type e. coli 13, the 7th group of bacterial strain adds the escherichia coli reference culture	O1，O2，O5，O7，O8，O9，O12，O13，O14，O15，O16，O17，O18， O19ab，O20，O21，O22，O23，O24 O4，O10，O25，O26，O27，O28，O29，O30，O32，O33，O34，O35， O36，O37，O38，O40，O41，O42，O43 O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O53 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O68 O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99， O101，O102，O103，O104，O105，O106，O97 O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128，O117 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O141，O142，O143，O144，O140 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166，O153 O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12，D13 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v：4)，F5(v：7)，F6， DS，DR O3，O11，O39，O59，O64，O73，O96，O95，O100，O114，O151，O155， O124，O167，O162，O121，O127，O149，O119 O145	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a IMVS a

For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group as negative 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 O145 type O antigen gene structure iron

E.coli O 145 O-antigen gene cluster

orf# orfl nnaB nnaC nnaA wzx wzy orf7 orf8 orf9 orf10 fnl1 fn12 fn13 orf14 orf15 gnd

G+C 34.4 32.6 30.1 32.8 29.3 29.3 29.0 34.7 36.4 39.4 37.0 38.1 40.6 40.2 41.4 50.3

content

Table 4 intestinal bacteria O145 type O antigen gene cluster gene position

ATTGGTAGCT GTAAGCCAAG GGCGGTAGCG TGTGTTTTTG AGATATTTAT GTATTATCGT 60

Initial from Orf1

TGTCATGAAT ACAAGTAACA ACAAAGTTAA TGTGATCTAA TCAAACCATT AGATGGT ATG 120

AATATGAAGC AAAAATTAAT AATTATTGGT GCTGGTGGTT TTGCTAAAAC TGTAATTGAC 180

AGTTTGGATC ATGAAAAGTA TGAAATAGAA GGGTTCATTG ATACATTTAA AACAGGTGAG 240

CATCAAGGAT ACCCTATTTT AGGTGATACT TTAGGTGTCA TAGACGAACC TAACCAATAC 300

CTATACTTTA TTGCAATTGG AGATCCCGAT TACAGAGCCT TATGGATGAA GTTAATAGAA 360

GAAATGAAAC TTTCAACTAT CAATGTAATT GACAGAACAT CTATAATTTC TGAACATTCT 420

AGGCTTGGTA CCTGCATTTA TATAGGGAAA ATGGCTATCA TTAACTGTGA TTCAGAACTA 480

GAAGATGGAG TGGTGATAAA CACAAGAGCA TTAGTAGAGC ATGGGAATTA TATATCATAT 540

TGTACAAATA TCTCTACTAA TGTTGTTCTT AATGGTGATG TATTCGTTGG GGAAAAATCC 600

TTTATAGGAA GTTGTACCGT TGTTAATGGG CAACTAAAGA TAGGTAATTC ATCAATTATT 660

GGTTCTGGTT CTGTAGTTAT TCGTGATGTT CCTGACAATG TAGTAGTGGC CGGGGCTCCT 720

Initial from the termination orf2 of Orf1

ACAAAGTTTA TTAGAGCAAG G TGATTTTTA TT ATGTCGAG AATTTATATT GTTGCTGAAA780

TTGGTTGTAA TCATAACGGA GATTTCGAGT TAGCTAAGAA AATGGTAAAA GAGGCAAAAT 840

CTGCCGGGGT AGATGCAGTA AAATTCCAAA CTTTTAAAGC TGAGCAACTT ATCTCAAAAT 900

ATGCTCCTAA AGCAGAATAT CAAATAAAAG TTACCGGTAA TGAAGAGACT CAATTAGAGA 960

TGACTCGTAA GTTAGAGTTG CCATATGATG AGTTTATCAA GCTGGAAGAA TATGCAAAAG 1020

AACTTGGGTT GGATGTCTTC TCAACACCAT TTGATTTTGA TTCAATCGAT TTTCTTGCAT 1080

CCAGAAATCA AAAAGTTTGG AAGATTCCAT CAGGCGAATT ATTAAATTTA CCATATTTAG 1140

AGAAAATAGC CAAATTGCCA ATCGAAGATA AAAAAATTGT ACTATCAACT GGTATGGCAA 1200

CAGTTGATGA AATCAATTTG GCATTAAAGG TTTTTATTGA TAATGGCATT ACACATAGAA 1260

ATATAACAAT ATTACATTGT AATACAGAGT ATCCAACGCC ATTTGAAGAT GTAAATCTAA 1320

ATACAATTTC TGGATTCAAA AAAATATTTA GTCAATATAA TATTGGATTT TCTGATCATT 1380

CTCCAGGTTA TTTTGCTGGT ATTGCATCAG TGCCTTATGG GATCACTTTT ATTGAGAAAC 1440

ATTTTACACT GGATAAAAAT TTTGAAGGTC CAGATCATAA AGCATCAGTT ACACCTGAGG 1500

AACTTAAATT ACTATGCGAA GGAATACGTG CAGTAGAAAT TTCACTTGGT AGCTATGAAA 1560

AGTTAGTAAC AAATTCAGAA CGAAAGAATA AAGTAGTAGC TCGAAAATCA ATTGTTGCTA 1620

AATGTGCAAT AAAAAAAGGA GATATTTTTA CGACGGAAAA TATTACGACA AAGCGACCAG 1680

GAAACGGAAT TAGTCCAATG TACTGGTATG AGGTCTTAGG TAAAATAGCT GAAAAGGATT 1740

The termination orf3's of Orf2 is initial

TTGATGAGGA TCAACTAATT GAGCACTCTG ATTTTAGTAA ACAAGAGGTT TGAGTT GTGT1800

CATTAAAAAA AATTGCCATT ATACCTGCTC GGTCTGGATC TAAGGGCTTA CCAAATAAAA 1860

ATATACTAAT GTTATTGGAT CGCCCATTAA TTGCTTATAC TATCGAAGCA GCGATTAGCT 1920

CTAATATATT TGATAAAATT ATTGTTTCAA CAGATTCATT AGAATATAAA TATATTGCAG 1980

AGAAATATGG CGCGGAAGTA ATATTACGCA CAAAAGAATT ATCGTTGGAT TCGGCAACAT 2040

CTTTTATGGT AGTTCAAGAT GTGCTTGAAA AATGTCCAGG ATATGATTAT TTTGTGCTTC 2100

TACAACCAAC ATCACCATTT CGTAATTATA AGCATATTAA GAATGCTGTT GAGCAATTTG 2160

AAAATAATCA CGAAGCAAAA TTTTTGGTGT CTGTGGTAGA AAGTGATAAA AGTTCGGCGT 2220

TAATTAAACC AATTGATAAT TCACTGTCAT TAAGGAACTT TGACTGTGAT TTTAGTACAT 2280

ATCGTAGACA AAATAAAAAA GAATACTGTC CAAATGGTGC GATTTTTATT GGTTATGTTT 2340

CTAATTATTT AAGACAAAAA CATTTCTTTG GTGCTGACAG TATAGCCTAT ATTATGAATA 2400

AAGAAGATTC TATTGATATC GATGATCAGT TAGATTTTGA ATTGGCAATT CTAATCCAAA 2460

CAAAAAAAAA TAAAAAAAAT TTATTGGATA ATGCTATTAT CAAACGAATT GTCGATAAAA 2520

AAGATTTATT TAACAAAGTT GAGCAAATTA CACTGATTGG ACATTCAATT TTTGATTATT 2580

GGGATCTCAG CAACATATGT GGAATTAAGG TTAACAATTT AGGCATTGCT GGCATTGATA 2640

GCGAAAAATA TTACAAATAC ATCATTGAAA AAAACATGCT CACCAATATA GGTAAGTATG 2700

TGCTATTAAT TTCAGGCACA AACGACATTG TGAACGATGG TTGGACTATT GAATACACTA 2760

TAAAATGGAC AAAAAATCTG ATCAATAGAG TAAAAATTAT AAATCCTGAT GTTACTATCA 2820

TTCTGCTAGC AGTGCCGCCT GTTCGAGGAA GAGTTGATAG GGATAATAAT ACTATCAATA 2880

AATTAAATCT GGCAATGAAA CAATATTTTA GTAAATTAGA CAATGTTATT TGGATGCCGT 2940

TATCTCCATC TTTTTATGAT GAGTTCGGCA ATCTGAATGA AAATTATACT TATGATGGTT 3000

Orf4's is initial

TACATTTCAC GTTACAAGCT TACAAACAGT TAGAAAATGA TATTTCGAGT ATCTTAAA AT 3060

The termination of Orf3

GAAAAGAAAA TTGCTCTATG TCACCGGCTC AAGAGCTGAG TACGGTATTA TGAAACGACT 3120

ATTAAAAAGT CTAAAAGATG ATCCTGATAT TGATTTATCA ATTATTGCAA CAGGTATGCA 3180

TTGCGATTCT GAATATGGTT ATACATATAA AACAATCGAG AATGATGGTT TCTTAATTGA 3240

AAAATTAATT GATTTGCAGC TGAAAAATAA AACCAACGCA GATGTTTTGA AAACGATGTC 3300

GATATGCCAA CAAGCATTTG GTGAGCATTT CCAAGAGAAA AAATATGATG CTGTCATTAT 3360

ATTAGGTGAT CGATACGAAA TATTTTCAGT GGCAGTAGCA GCTTCTATGC ATAACCTACC 3420

TATTATTCAT CTTCATGGTG GAGAGAAAAC GTTAGGGAAT TATGATGAAT TTATACGACA 3480

TTCAATAACG AAAATGAGTC GTCTACATCT AGTATCAACT GATGAATATC GGAAAAGGGT 3540

TATCCAACTT GGTGAAGATC CTAATTGTGT ATTTAATGTA GGTGCGCTAG GGGCTGAAAA 3600

CAGTTTGCAA CTAGAATTAC CCTCTAAAAA GGAGCTTGAA AATAAGTACG GTGGGTTAAA 3660

TAGAAAATAT TTCGTAGTCG TATTTCATCC TGAAACGTTA TCAACTTTAC CTGTTAGAGA 3720

GCAGTATTTA GAATTACTAG AAGCATTAGC TAGTTTTAGC GAAGAATACG ATTATATCTT 3780

TATTGGCTCA AATGCTGATA CTGGTTCAGA ACAAATAAGA GAAATGACTC TCGATTTTTG 3840

TAGAGTATAT GGCTGTCGAT ATATGATTTC TGTGAGACCT GAAGAATATC TAGCTTTGAT 3900

AAAATATTCA TATGGCTTAA TAGGCAATTC GTCATCTGGG TTAATTGAGG TTCCAAGCTT 3960

AAAGGTTCCG ACGATTAATA TAGGCGATCG CCAAAAAGGA AGGGTTAGAG GTGATTCGGT 4020

AATAGATACC ATATGTAAAA AAGAAAGTAT AGAATGTGCC ATTGCTTATT CACAACAGAA 4080

AAGTTTCATT GAACGTGTAA AAAATGCTCA TAATCCTTAC TATAATAATG ATGTAATGAA 4140

TAGTATTGTT AAAATCATTA AAGATTTTTT AACTTATGAA AAGTTGGGTT ATAAAGACTT 4200

The termination of Orf4

CTATGATGTT GCATATGACT ATAGTCATTT AAAA TAAACA ACATGGGTAT TGTTTTTAAT 4260

AAGTTATTTG TATGATATTT TAAATTTAAA AACCTCATAT TAATATTTAA ATTATTGCAA 4320

Orf5's is initial

ATTTAATAAT CACAGATTAA AAACTTCCGA TTTTAACATT GGCCTTGG AT GTTTAATACT 4380

ATGCTTAAGT ATTACTCAAG TGTTGGATTA AGAGGGATTA CTCTACTTAC TAAATTTATT 4440

TTCATTGTTT TGCTTGCTCG ACTTTTACCA TCAACAGATT TAGGAGTGTA TGGATTAATT 4500

AATGCAGCTG TAGGATATGG TATTTTCGTT GTAGGTTTTG AGTTTTATAC GTATTCAACG 4560

AGAGAAATAA TTAACTCGCA AAAAAATAGG CTTTTTTTTA TACTAAAAAA TCAAGCTCTA 4620

TTTACTGTTA TATCTTATAT ACTATGTATA CCGGCATTTA TTTTTTTATT ATATTTAGAA 4680

ATATTACCAT CTGGAAGTGA ATACTGGTTT ATCCTACTTT TATTTTTTGA GCACTTATCA 4740

CAAGAGATTA ATAGAGTTCT AATAACAATA GAAAGTCAAT CGATTGCAAG TTTTATTCTT 4800

TTTGTAAGAC AAGGTGTATG GTGTTGGTTA GCTATAGCTG TGATGCTAGT GTATCCGAAC 4860

TTAAGAAATA TAACAGTTGT ATTTATTTTT TGGTTTGGTG GTACTGTGTC CGCGAGTGTG 4920

CTTGGAGTGG CTTATATTTT AAATAAAAAA AAACAAAGCG ATATTACAAA CTGGGATTGG 4980

ACGTGGATAA AAAAAGGTAT AAAGCTGTCT GTACCAATGC TAATTGCAGC CCTTGCACTA 5040

CGAGGCTTTT TCACGTTTGA TAGATTCGCG GTAGAAAAAA TATCGGGCCT AGAAGTTTTG 5100

GGAGGATATA CATTATTTGT TAGTATGACT TCAGCTATTC AATCATTTTT GGATACTATT 5160

TTGATATCTT TTTCATTTCC AAAGCTTGCC TTGTTATATT CAGGGAAAAA ATATATAAAA 5220

TTTAAATCTG AGTTAAGAAA ATTCACTTAT AAATTAATTT TACTACTATC TTTCTTGAGC 5280

ATCTGTTGCT TTTTTACTGG GATTATATTG GTTAAGTGGT TGGATAAACG AGATTACATA 5340

CAATTATTTC CTGTATTTAT ATTATTAATA GCAGCGACTT ATATCTATTG TATAAGTCTT 5400

ATTCCACATA TTGCTTTATA CGCGATGAGA GAAGATCGTT ACATATTAGT AAGTCAACTG 5460

ATATCATTTT TATCTTTTTT ACTATTTGTT TTTTTTAGCG TATATCAAAG TGATATCTAT 5520

TACTTGCTAA TTGGTATGAT AGCTAGTTTT GTATTACTTT TGATCTTAAA AATGATCCCG 5580

The termination orf6's of Orf5 is initial

TTATATAAAA TTCTAAAAAA GGTT TAAAAT AAAC GTGAAT ATAAAGAAAG ATAAGTTTAT5640

AAATGGAGTG ATTTTTTTTT GGTTAATTAT TTCTTCGTTA TATTACTTAA ATGCTATTTT 5700

TTCTGGTGTT GACACATTAA AATATAATGA AGATTTAACG CAAAAAATTA TAAAATATAT 5760

AGTTTGCTTA GTTATAAGTC TAAGTATCTT ATTTATTTAC AAGAAATTTA ATTATTTTTT 5820

TGTATTGTTT TTTTTCTTGT TCCTGTCTGT TGCTTCAGCC CTTTTCAGTG GTGCGGTAAC 5880

AATTTACGCA ACAACAATGT TGATTATTGC AACTATGATC AGCTTTTGCC TGATTATTCC 5940

TCTATTTTCT TATAATATGG TGAAAGTTAA TAGAGTTCTT TTATGGACAG GAGTTATTGT 6000

AGGCACGATT TCTGTATTAG AATTAACGGT ATTTTATAAT TATATGGTTT CATATTGGGC 6060

TGCCACTGAT GGGATTAGGT CAATATCTTC TCTTCTGAAT CCTACGAATA GTGGTGCTTA 6120

TTCAGCGATT ATTATTTTAA TCGCCTTGGT GACAAATATA AAAAGTCTTT TTAAAAGAGC 6180

TTTATTTCTT ATAATGCCGA TGATAACGTT AATTAGCAGT GGTTCGCGCA CAGCATGGTT 6240

ATCACTTGGT ATGACACTTT TATTAACAGT AGTATTGAGA GACAGTGCCA GCATTCGCTT 6300

GCGAAAAAAA ATATTTACTC TTGCAAGCAT TGGCACTGTT TGCGGTGCAT TGTACGCCAT 6360

ATTTTATATG GGCAGTATCT CTGGTATTGA ATCACAATAT CGAGGTCTTA ATACGTATAC 6420

TGCATCAATT CGAGTTGAAA ACTTTCTGAC ATATTTAAAT TTAGTTGATC TGAATATGTT 6480

GCTACCTGAT TTTTTAGATA AAAATATAAA TCTCATTTCA GATAACTTTT ATCTCGTAAT 6540

GTTTAATTAT GCCGGTCTAA TCGGCTTTTT TATTGTTTTA TTAATTTTAT TGCTGCTTAT 6600

CTTCTGGAAC ATACAATTTA AAATATTTAA TGAGTTAATG GCTGAAGATA TAGCCATTTG 6660

GAGAGTTGTT TTTATTTATT TCCTAATATC CGGGCTTTCA AATTCATTTA TAAATTCTTT 6720

TCCTGTAAAT CAATTGTTCT TTATCTCATG CGGATATTAT ATATATAAAT ATAAATTAGT 6780

The termination orf7's of Orf6 is initial

TAAAAGCTCT ATAGGAAGA T AAAT ATGCAA TTATCAATGT CGCAAATAAT AGAATGTTAT6840

TTACACGCAA CACAAAATGA AATAGATATC GATAAGTCTT TATCAAAAAG CATTACAAAA 6900

AAAATGCCAC CACTTCCCAG GACAAGTAAA TGGGCTGGGG AAAGGTGGAG TTCATCGAGT 6960

TTGCAAATTA TTTATGCAGT GATTATGTTT GCCTTTATTT TTGGGGGGTT TTTATTATAT 7020

GTTATATTAT TTTTTGCTAA ATATATATTG GCTAAATTTA AAAATTCCAT TTCAGAAATA 7080

AAGGTTGCTA ATAATAAGGA AGGTAAATTT TACTATTTTT CGTTTTCAGA TTTAGAAATG 7140

AGGCAAACTT GCTATTTTTT TAAAGATAAT GATAGTTACA AATTATCTCG AGATGAACTT 7200

ATTATAATAA AATTACCATG GGTAAATTAT ACACCAACGA GTTGTGACTT TAATGCTATT 7260

AATTTATATG AATTAACATC GTTTCTAGAT GTGGCAAGAG CATTCTTGTT ATCTATTTTT 7320

AGTTATATCT ATTATTTAAA ACCATCACGT ATAAAATGGC TTCTTCATAT TTATACAGCA 7380

CCTTCTTGGT TTTTGGTTGC TATGGGAATG AATAATATTA AAGGCAATCT CGCAAGTAGT 7440

GAACATTATG ATCGTTGGGC TGTTTTAACT GATTTTATAT GCAGAATAAA AAGGAAAAGA 7500

TATATATTAA TTCAACATGG CTCGTTACTG GCGTTAAAGA CTAAGGGTTA TGAATTTTTC 7560

TCTCTCTCAT ATAAACTGAA AGCTGTATCA GAGCTCGCTA TCTTTAACGA AATTGAGCTT 7620

GAATTATTTT TAGAGCATAT AATCTCACAG GCTAATGATT ACAATATAAA AATTCATTTC 7680

TATCAACAGC CATTTTATGT TTCATCAATT AATAATAAAG GATTATCAAT TCTAATTATT 7740

GGGCATTCAT TGTGTGAACG AGGACAACTA AGTCTTGGGT CACAACTTTC AACGTTATCA 7800

GATAATATTG TTCTTTATTA TAAAGAACAT CCAAAAGCTA GAGCATCTGA AAAAGCCAAA 7860

AAGACAAAAT GGAATTTCAT TACAGATGAT GATTATTTCC CTGATGTAGA TATTGTCATT 7920

TCGTATCCTT CCACTTTGGC ATATCAATAC AAAGAGTTGA ATAAAATAGT TATTCTTCAT 7980

GAATTAGATA ATATTGATCA AAATAAAATT GACGAGATAC TTATGACCAT TAGAAAAAAT 8040

The termination of the initial Orf7 of Orf8

AAGGGCGTGT ATGGAAAA TA AAAATTATCA AATTTGTAAT CATTGTATTA TGGATACATC8100

TGATCCTCAT ATTACTTTTG ATGATAGCGG AGTATGCAAT TACTGCGCCA ATTATAAAAA 8160

CAATATATTG CCGACATGGA GAAAAGGTTT GTTTTCAGAT GTTTCTTTAT CTGAAATGGC 8220

AAGTAAAATA AAGGCGGACG CAAAGAATAG TGATTTTGAT TGTATTATTG GACTATCAGG 8280

GGGGCTTGAT AGTTCATATG CTGCATATAT TGCAAAAGAA AAGATGGGGC TACGCCCATT 8340

ATTATTCCAT GTTGATGCCG GCTGGAATAC AGATCAGGCT GTTGGTAATA TTGAAAAACT 8400

TATTGAAGGC CTTGGTTTAG AACTTTATAC AGAGGTAGTA AACTGGGAGG AAATGAAAGA 8460

TCTTCAACTG TCTTTCTTAA AATCAGGAAT TCCAGATCAA GATCTAGTTC AGGACGCTTC 8520

CTTTTTTTCA TCATTATATA AGTTTGCTCG ACAACATAGA ATTAAACATG TCATTACGGG 8580

GTCCAATTTT TCTACTGAGT GTTGTCGTGA ACCAGAAGAA TGGGGAGGAT ATTTAGGCAT 8640

TGATACTCTG TTATTTAATG ATATCCATAC GAAATTTGGA GAAAAACCGT TAAAAACTTT 8700

TCCTTTAGTG GATATATTGG TTTATAAAAT TTACTATCAG AAAATTCTTG GTATGAAAGT 8760

GCATCATCCA CTAAATTTAG TTCCTTTTAA TAAGAAGGAT GCAGAAAATG AACTTAATAA 8820

AAAATTTGGC TGGCAACCCT TCCAGCATAA ACATCATGAG TCCCGTTTCA CCCGGTTTTA 8880

TGAAGATTAT TGGCTGCCAC GCAGATTTGG TTATGAGAAA CGAAGAGCTC ACTTTTCGAG 8940

TTTAATTATG ACGGGACAAA TGTCTCGTGA ACAAGCTCTT GAGAGGATTT CAAAACCAGA 9000

AATGGATGAG CACTTTCTTA AACAAGAGTT TGAGTATGTA GCGCACAAAC TTGGTATTAG 9060

TGTTGAAAAT TTGCAAGAGC TATTTTTTAT GCCCAAAAAA ACATATAAGG ATTATAAGAA 9120

TAAGCGTTGG TTGATTGGTT TAGGTGCGAA TGTCTTACGT GCACTTGGAT TAGAAAAAAG 9180

The termination of the initial Orf8 of Orf9

GTATTTCAG A TGATTACAAT CATTGATTAT GGTATTGGAA ATATACAAGC ATTTTTGAAT 9240

GTATATAAGC GACTAGGCAT TAATGCGGGT GTTGCCCGTT CTGTTGATGA TATTTTTAGT 9300

GCTACACATT TAATACCGCC TGGAGTTGGG GCTTTTGATC AAGCTATGAC TCTTTTTAAT 9360

AATTCAGGTT TACGAGATAG TATTGAAAAA CGTGTATATG AAGAAAAGAT TCCAATTATT 9420

GGTATCTGTG TGGGTATGCA AATGCTTGCG ACCTCTAGTG AGGAGGGGAG CATGCCAGGT 9480

TTAGGATGGA TTCCAGGTAC GGTTCGAGCA TTTAGTAGCA ATATCTCTTC ACAAAACCTC 9540

CCGATGCCCC ACATGGGATG GAATAATTTA ATTAAAAGAA CAAGTACACC ATTGCTGAAA 9600

GATTTCGCAG TAGAACCTTC ATTTTATTTT TTACACTCAT ACTATTACGA ATGTGATGAC 9660

ACAGGTGATG TATTGGCAAC TGCTAATTAT GGTCATAATT TTCATTGTAT AATTAGCAGG 9720

AATAACATAT ATGGTATTCA ATGTCATCCA GAGAAAAGCC ATTCATCGGG ATCCCAACTA 9780

The termination orf10's of Orf9 is initial

CTAAAAAATT TTGCAGAGAT C TAATAGC AT GCTTAGACCA AGAATCATAC CCTGTTTACT9840

TATCCACGAT TCTGGCTTAG TCAAAACAGT TAATTTTAAA TCCCCTAAGT ATGTTGGTGA 9900

TCCAATTAAT GCCGTCAAAA TTTTCAATGA GAAAGAAGCT GATGAATTGA TGGTCCTAGA 9960

TATTGATGCG ACATCCAGGG GACTCGAACC TAATTATGAT TTAATAAAAA AACTCGCAGC 10020

GGAATGCCGT ATGCCTTTAT GCTATGGAGG AGGCGTAACC AGCGTAGCAC AGGCTACAAA 10080

AATTATCTCT CTTGGTGTTG AAAAGGTATC AATAAGTTCA GCAGCTGTTG AAAATCCTAA 10140

TCTTGTTAGA GAATTGGCAG AAGCGGTTGG AAAACAAAGT GTTGTTGTTG TGCTGGATAT 10200

TATCAAACGA AAAGGTCTAT TCTCAAAGGG CTATGAACTT TCGACAAGGA ATAACACTCG 10260

CAAGCATAAA ATCGATCCCG TCTCGTTTGC AAAGGAAATG GCCGATCTGG GAGCTGGAGA 10320

AATTGTCATC AATTTTGTAG ATAATGATGG TGTCATGGGA GGATATGACG TTGCATATTG 10380

CTCAACTATC AAATCGCAAA TAAATATTCC TGTTACGTTT CTCGGGGGCG CTGGTAGTTA 10440

TGACCATCTT TCTGCACTGA TTGACCAATG TGGCATCGTT GGTGCGGCAG CTGGTAGTCT 10500

TTTTGTATTT AAAGGGAAGT ATAGGGCAGT ATTGATAAGC TATCCAACAC CTGAACAGAA 10560

The termination orf11's of orf10 is initial

AGATATTATT TGTAATGGTG TAATGAGTAA TCGTTTA TAA GGGGTT ATGA TGTTCAAAGA10620

TAAAATACTG TTAATCACTG GTGGTACTGG TTCATTTGGG AATGCTGTAT TAAATCGCTT 10680

TCTTTCCACA GATGTAAAAG AAATACGCAT ATTTAGTCGT GATGAAAAAA AGCAAGATGA 10740

TATGCGTAAA AATTATAACA ATAAAAAATT GAAATTCTAT ATTGGTGATG TTAGAGATTA 10800

TCGTAGTATT TTGAATGCGA CTCGCGGAGT TGATTTTATA TATCACGCAG CGGCACTTAA 10860

GCAAGTTCCA TCATGTGAGT TTCATCCTGT GGAGGCTGTT AAAACTAATG TTCTTGGTAC 10920

AGAAAATGTG CTTGAAGCTG CTATAGCGAA TGAAGTTAAA AGGGTTGTGT GTCTTAGTAC 10980

TGATAAAGCT GTATATCCGA TCAACGCTAT GGGAATTTCA AAAGCTATGA TGGAAAAAGT 11040

CATGGTTGCG AAATCGCGTA ATGTTGACTG CAACAAAACA GTAATATGTG GTACCCGTTA 11100

TGGGAATGTC ATGGCCTCTC GTGGATCTGT TATTCCATTA TTTGTTGATC TTATCAAATC 11160

TGGCAAGCCA CTAACAATTA CTGACCCTAA CATGACCCGT TTTATGATGA CTCTTGAGGA 11220

TGCTGTAGAT TTAGTACTTT ATGCATTCGA ACATGGTAAT AACGGAGATA TTTTTGTACA 11280

AAAAGCTCCA GCTGCAACTA TCGAAACATT AGCAACTGCT TTAAAGGATT TACTAAATGC 11340

TGCTCATCAC CCAGTAAATA TTATTGGTAC GCGTCATGGT GAGAAGTTAT TTGAAGCTTT 11400

GCTCAGTCGT GAGGAAATGC TCTCTGCTGA AGATATGGGG GATTATTATC GTATACCACC 11460

AGATCTTCGT GACCTTAATT ATGGAAAATA TGTTGAACAA GGTGATAGTC GTATATCCGA 11520

GGTTGAAGAT TACAACTCTC ATAATACACA ACGGTTGAAC GTTGAAGGCA TGAAAACGCT 11580

The termination orf12's of Orf11 is initial

TTTGCTCAAA TTGGATTTTA TTCGTGCACT TCATGCAGGT TAACAAC ATG GCTAAGACTC11640

ATGATATAAA AATATTGATT ACTGGTGCAA ATGGTTTTAT TGGCCGTAAT TTAAGTTTGA 11700

GGCTTGAGGA ACTAGGTTAC AAGAATCTTA TTAGAATTGA TCGGGATTCA ACAGAACAAG 11760

ATCTTGAACA AGGCTTACAG GACGCTGATT TCATTTATCA TTTGGCTGGA GTCAATAGAC 11820

CAAAGACTGA TGATGAATTT AAATCCGGAA ACAGTGATTT AACTAAATAC ATAGTTGAGT 11880

ATCTTATTTC TATAGGAAAG AAGATACCAA TTATGCTAAG TTCTTCTATA CAAGCAGAAC 11940

TTGACAATGC TTATGGGGTA AGCAAAGCTT TAGCTGAACG TTATATTGAA AAATATGCTA 12000

CTACTAGTGG TTCTCCATAT TATATTTTCA GATATCCAAA CGTTTTTGGC AAATGGTGCA 12060

AACCAAATTA TAATTCTTTT GTTGCGACTT TTTGCTATAA TATTGCGAAT GATCTTGCCA 12120

TAACTATCAA TGATGCATCT GCGCCAGTTA ATTTGGTTTA TATTGATGAT GTTTGTGCTA 12180

AAGCGATAGA GCTTCTCTCG GGGGCAACTG AAAGCGGATA TAAAGCTGTT GCACCAATTT 12240

ATTCAACTAC TGTTGGTGAA GTTGCAGAAT TAATTTATAG ATTCAAGAAT AGCCGTTCCA 12300

CCCTGATCAC AGAGGCTGTC GGGACGGGAT TTACCCGTGC ATTGTATTCT ACATGGCTGA 12360

CTTATTTACC AGCAGAGAAG TTTGCGTACA AGGTACCTTT TTATGGGGAT GCCCGCGGAG 12420

TCTTTTGTGA GATGTTGAAA ACGCCTTCAG CGGGGCAGTT TTCATTTTTT ACTGCTCACC 12480

CTGGTATTAC GCGTGGCGGA CATTACCATC ACAGTAAAAA TGAGAAGTTT TTGGTCATTC 12540

GAGGTCAGGC ATGCTTTAAA TTTGAACATG TGATTACCGG TGAGCGATAT GAACTGAATG 12600

TTTCATCGGA TGAGTTCAAG ATTGTTGAAA CAGTTCCTGG TTGGACACAT GACATTACAA 12660

ATATTGGAAC TGAGGAATTA ATAGTCATGC TCTGGGCAAA TGAAATTTTC AACCGTGATG 12720

The termination orf13's of Orf12 is initial

AGCCCGATAC TATTGCGAGA CCTCTA TAAT GAAAAAATTA AAAGTTATGT CTGTTGTTGG 12780

AACCCGTCCT GAGATTATCC GTTTGTCGAG GGTTCTTGCT AAGTTTGATG AATACTGCGA 12840

GCATATTATT GTCCATACTG GTCAAAATTA TGATTACGAA TTAAATGAAG TATTCTTCAA 12900

TGACTTGGGT GTTCGAAAAC CTGATTATTT TTTAAATGCA GCGGGAAAAA ATGCGGCGGA 12960

AACCATTGGC CAAGTTATTA TTAAGGTAGA TGAAGTATTA GAAATCGAAA AACCTGAAGC 13020

AATACTGGTA TTGGGCGATA CGAATTCATG TATTTCTGCC ATTCCGGCCA AACGCCATAA 13080

AGTTCCTATA TTTCATATGG AAGCAGGTAA CCGTTGTTTC GATCAACGCG TGCCTGAAGA 13140

AACCAACAGA CGTATTGTTG ACCATACGGC TGATATCAAT ATGACCTACA GTGATATTGC 13200

TCGTGAATAT CTCTTGGCTG AAGGTATCCC AGCTGATCGG ATCATAAAAA CTGGTAGCCC 13260

TATGTTTGAG GTTCTTTCAT ATTATATGCC CCAAATTGAT GGTTCAGATG TGCTATCGCG 13320

TTTGAATCTA CAGTCTGGTG AGTTTTTTGT AGTAAGTGCG CATCGTGAAG AGAATGTTGA 13380

TTCTCCAAAA CAGCTCGTAA AGCTTGCGAA CATTCTAAAT ACTGTTGCTG AAAAATATAA 13440

TCTTCCAGTT ATTGTCTCCA CACACCCAAG GACACGTAAC CGAATCCGTG AGCAAGGAAT 13500

TGAATTTCAT TCAAATATAA ATCTACTGAA ACCATTGGGT TTCCATGATT ATAACCACTT 13560

GCAGAAGAAC TCACGAGCTG TGCTTTCAGA TAGCGGTACT ATCACTGAAG AGTCATCCAT 13620

CATGAATTTC CCAGCGGTAA ACATCCGGGA AGCGCATGAG CGTCCGGAAG GCTTTGAGGA 13680

AGCATCCGTC ATGATGGTGG GGTTAGAGTG TGAACGCGTA TTACAAGCGC TGGATATTCT 13740

GGCAACACAA CCGCGAGGTG AAGTCCGTCT TTTACGTCAG GTTAGTGATT ACAGCATGCC 13800

AAATGTGTCG GATAAAGTTG TCAGAATTGT TCACTCTTAC ACAGATTATG TTAAGAGAGT 13860

The termination orf14's of Orf13 is initial

TGTCTGGAAA GAATAT TGAT GAAACTTGCT TTAATCATAG ATGATTACCT GCCCAACAGT 13920

ACTCGTGTTG GTGCAAAAAT GTTTCATGAA CTTGCTCAAG AATTTATCCA GCGTGGGCAC 13980

GATGTTACGG TAATTACTCC TGGTACGGGC ATGCAAGAAG AGATTTCTTT TGATACCTTT 14040

CAGGGGGTAA AAACATGGCG TTTTAAAAGC GGGCCGCTCA AGGATGTAAG TAAAATTCAG 14100

CGAGTGGTCA ATGAAACGCT TTTGTCCTAT CGGGCGTGGA AAGCCATCAA AAAATGGGTA 14160

AAAAAAGAGA CCTTTGAGGG TGTGATTTAT TATTCACCTT CCATATTCTG GGGGTATTTA 14220

GTTAAAAAAA TTAAAGCTCG TTGCCAATGT CCTGCTTATC TTATTTTAAG AGATATGTTT 14280

CCACAATGGG TAATTGATGC AGGAATCCTT AATGCTGGTT CCCCAATAGA ACGCTACTTT 14340

CGTCTTTTTG AAAAAATATC TTGTCGTCAG GCAAATCGTA TTGGACTTAT GTCTGATAAG 14400

AATCTTGATG TTTTTCGGAA AGATAATAAA GGCTATCCGT GCGAAGTTTT GCGTAATTGG 14460

GCATCCCTAA CACCAACGAT CATACCCAAG GATTACATAC CACTACGTAA GCGACTTGGC 14520

CTAGAGGATA AAACCATTTT CTTCTATGGT GGAAACATAG GTCATGCACA GGACATGACA 14580

AACTTGATGC GACTTGTGAG AAACATGGCA GCATATCCTC AAGCTCATTT CCTATTTATT 14640

GGCCAGGGTG ATGAAGTTGA ATTAATTAAT TCATTAGCCT CTGAGTGGGC ATTGACGAAT 14700

TTCACCTATT TGCCCTCGGT TAATCAGGAT GAATTTAAGT TCATTTTGTC GGAAATGGAT 14760

ATCGGCTTGT TTTCTCTTTC CGCTAGACAC TCTTCCCATA ATTTTCCTGG TAAGTTATTA 14820

GGCTATATGG TTCAGTCGCT ACCTATTTTA GGTAGCGTAA ATGCCGGAAA TGATTTGCTC 14880

GACATTGTCA ATCAAAATAA TGCCGGATTA ATCCATGTCA ATGGTGAGGA CGATAAATTA 14940

TGTCAATCTG CGCTATTAAT GTTGCATGAT ATTGATGTGC GCCGGCAACT TGGTTCGGGG 15000

GCGAATATAT TGTTGAAAGA ACAATTCTCC GTTGAGTCTG CGGCACAGAC GATAGAAATG 15060

The termination of the initial Orf14 of Orf15

AGGTTGGAGG CATGCA ATGC GATTAAT TGA TAATGACCAA CTCGACAAAT TATATGATCA15120

AGCCGAGCAA TCGGAACGTT TACGTTCCCA CCTTATTATG CACGGCTCGC ATCAAGAAAA 15180

GGTACAGCGT TTACTTATTG CATTAGTAAA GGGCAGCTAT GTTGAACCGC ATTATCACAA 15240

ACTTCCTCAT CAGTGGGAAA TGTTCATTGT TATGGAGGGG CAACTTCAGG TTTGTTTGTA 15300

TGGTAGAAAT GGTGAGGTTA TAAAGCAATT TATAGCAGGA GATAATATTG GAATAAGCAT 15360

TGTGGAGTTT TCTCCGGGCG ATATACACAG TGTCGAATGC CTATCTCCGC GTGCTCTTAT 15420

The termination of Orf15

GGTGGAAGTT AAAGAGGGGC CATTTGACCC TTCTTTTGCA AAATCGTTCG TG TGATGCTT 15480

GTCTAAAGTA CATCTTCTGC TATCTACTCA AGCTAAACCT GAGTTAACAT CCATACCATA 15540

TTTCAAGCTG CGCATATCTT GCGCGGTGAC CACCCCCTGA CAGGAGTATG TAATGTCCAA 15600

GCAACAGATT GGCGTAGTCG GTATGGCAGT GATGGGGCGC AATCTTGCGC TTAACATCGA 15660

AAGCCGTGGT TATACCGTCT CTATTTTCAA CCGTTCCCGT GAAAAGACGG AAGAAGTGAT 15720

TGCCGAAAAT CCAGGCAAAA AACTGGTTCC TTACTATACG GTGAAAGAGT TCGTTGAATC 15780

TCTGGAAACG CCTCGTCGCA TCCTGTTAAT GGTTAAAGCA GGTGCAGGCA CGGATGCTGC 15840

TATTGATTCC CTCAAGCCAT ACCTCGATAA AGGTGACATC ATTATTGACG GTGGTAATAC 15900

CTTCTTCCAG GACACTATTC GTCGTAATCG TGAGCTTTCT GCAGAAGGCT TTAATTTCAT 15960

TGGTACCGGT GTTTCCGGTG GGGAAGAAGG TGCACTGAAA GGACCTTCCA TTATGCCTGG 16020

TGGCCAGAAA GAAGCCTATG AACTGGTTGC ACCGATCCTG ACCAAAATCG CCGCAGTGGC 16080

TGAAGACGGT GAGCCATGCG TTACCTATAT TGGTGCCGAT GGCGCGGGTC ACTATGTGAA 16140

GATGGTTCAC AACGGTATTG AATACGGTGA TATGCAGCTG ATTGCTGAAG CTTATTCTCT 16200

GCTTAAAGGT GGCCTGAACC TCACCAACGA AGAACTGGCA CAGACCTTTA CCGAGTGGAA 16260

TAACGGTGAA CTGAGCAGCT ACCTGATCGA CATCACCAAA GATATCTTCA CTAAAAAAGA 16320

TGAAGACGGT AACTACCTGG TTGATGTGAT TCTGGATGAA GCGGCTAACA AAGGTACCGG 16380

TAAATGGACC AGCCAGAGCG CGCTGGATCT CGGCGAACCG CTGTCGCTGA TTACCGAGTC 16440

TGTGTTTGCA CGTTATATCT CTTCTCTGAA AGATCAGCGT GTTGCCGCGT CTAAAGTTCT 16500

CTCTGGCCCG CAAGCGCAGC CAGCTGGCTA CAAAGCTGAG TTCATTGAAA AAGTTCGTCG 16560

TGCGCTGTAT CTGGGCAAAA TCGTTTCTTA CGCTCAGGGC TTCTCTCAGC TGCGTGCTGC 16620

GTCTGAAGAA TACAACTGGG ATCTGAACTA CGGCGAAATC GCGAAGATTT TCCGTGCTGG 16680

CTGCATCATC CGTGCGCAGT TCCTGCAGAA AATCACCGAT GCCTATGCCG AAAATCCGCA 16740

GATCGCTAAC CTGCTGCTGG CTCCGTACTT CAAGCAAATT GCCGATGACT ATCAGCAGGC 16800

GCTGCGCGAT GTCGTTGCTT ATGCAGTACA GAACGGTATC CCGGTTCCGA CCTTCGCCGC 16860

TGCGGTTGCC TATTATGACA GCTACCGTTC CGCTGTTCTG CCTGCGAACC TAATCCAGGC 16920

GCAGCGCGAC TA 16932

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 O145 type, it is characterized in that: its wzx sequence is that one or more Nucleotide are inserted, lack or replaced to the Nucleotide of 4369 to 5607 bases among the SEQ ID NO:1 or Nucleotide that the wzy sequence is 5615 to 6802 bases among the SEQ IDNO:1 or above-mentioned sequence, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O145 type.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O145 type, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 4468 to 4488 bases among the SEQ ID NO:1 and the Nucleotide of 5059 to 5077 bases, the Nucleotide of 5054 to 5074 bases among the SEQ ID NO:1 and the Nucleotide of 5532 to 5552 bases; Or the oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6061 to 6078 bases among the SEQID NO:1 and the Nucleotide of 6687 to 6706 bases; The Nucleotide of 5849 to 5866 bases among the SEQID NO:1 and the Nucleotide of 6280 to 6297 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 O145 type of 2.

4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O145 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.