CN1285727C - O-antigen specific nucleotide of E.coli 0139 type - Google Patents

Abstract

The present invention provides a nucleotide specific to an O-antigen of Escherichia coli O139, which is a complete nucleotide sequence of a gene cluster for controlling the synthesis of the O-antigen in Escherichia coli O139, such as the separated nucleotide disclosed in SEQ ID NO: 1 with the full length of 12654 basic groups, or the nucleotide of SEQ ID NO: 1 comprising one or a plurality of inserted, deleted or substituted basic groups and maintaining the functions of the separated nucleotide simultaneously. The present invention also comprises an oligonucleotide of a glycosyltransferase gene and an oligosaccharide unit processing gene derived from an O-antigen gene cluster of Escherichia coli O139. In the present invention, PCR indicates that the oligonucleotide has high specificity on the O-antigen of Escherichia coli O139. The present invention also discloses a method for detecting and identifying Escherichia coli O139 in human bodies and in environments by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O139 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O139 type (Escherichia coli O139), particularly relate in the intestinal bacteria O139 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O139 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. (1935) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O139 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.Nineteen thirty-five, Paton, the A.W et.al serotype [" Molecular microbiologicalinvestigation 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 ofthe Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152 and and Shigella O antigens " J.clinMicrobiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O139 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O139 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 O139 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O139 type: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; The rhamnosyl synthase gene comprises orf1, orf2, orf3, orf4 or with orf1, orf2, orf3, orf4 the gene of identity function is arranged; Glycosyltransferase gene comprises orf6, orf8, orf9, orf10 gene.Wherein said gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come 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 O139 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O139 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O139 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 O139 type of these methods detections and identification of escherichia coli O139 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 O139 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

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

The present invention is to the Nucleotide of the O-antigen-specific of intestinal bacteria O139 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,12654 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type, it is by 10 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type, described gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; The rhamnosyl synthase gene comprises orf1, orf2, orf3, orf4 or with orf1, orf2, orf3, orf4 the gene of identity function is arranged; Glycosyltransferase gene comprises orf6, orf8, orf9, orf10 gene.Wherein said gene.Wherein said gene: wzx is the Nucleotide of 7016 to 8236 bases among the SEQID NO:1; Wzy is the Nucleotide of 4922 to 6160 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1140 to 2225 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 2225 to 3124 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 3182 to 4063 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4065 to 4595 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6157 to 7029 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8226 to 9284 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9277 to 10434 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 10424 to 11080 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type, wherein it is to come from described wzx gene, wzy gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 7219 to 7236 bases among the SEQ ID NO:1 and the Nucleotide of 7708 to 7725 bases; The Nucleotide of 7712 to 7729 bases among the SEQ ID NO:1 and the Nucleotide of 8100 to 8117 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 5150 to 5167 bases among the SEQ ID NO:1 and the Nucleotide of 5393 to 5410 bases; The Nucleotide of 5344 to 5362 bases among the SEQ ID NO:1 and the Nucleotide of 6046 to 6063 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type, and can provide the O-antigen of expressing intestinal bacteria O139 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O139 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O139 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 liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor 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 O139 type bunch: with the genome of intestinal bacteria O139 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O139 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 O139 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O139 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 O139 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 10 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 O139 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O139 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 the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O139 type all is high special.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O139 type, its complete sequence shown in SEQ ID NO:1,12654 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 O139 type by method of the present invention, as shown in table 3, it is altogether by 10 genomic constitutions, all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O139 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); Rhamnosyl synthase gene (orf1, orf2, orf3, orf4 or the gene of identity function arranged with orf1, orf2, orf3, orf4); Glycosyltransferase gene (orf6, orf8, orf9, orf10 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 O139 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O139 type is provided or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function, the oligonucleotide of gene are arranged with wzx, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, any product or product is arranged does not all increase in other groups, but size is different with the product in the 13rd group, so the O-antigen of wzx, wzy gene pairs intestinal bacteria O139 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O139 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O139 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene.

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

As used herein, " oligonucleotide " mainly refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 7016 to 8236 bases from SEQ ID NO:1); It is high special to intestinal bacteria O139 type that wzy gene (nucleotide position is the Nucleotide of 4922 to 6160 bases from SEQ ID NO:1) comes from above intragenic oligonucleotide.

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 O139 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 O139 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 O139 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 O139 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 O139 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 O139 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 O139 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 liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol, supernatant liquor 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 O139 type bunch:

With the genome of intestinal bacteria O139 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGATTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 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 competent cell bacillus coli DH 5 mouth.Get a ring bacillus coli DH 5 mouth 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 liquor, 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 liquor, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O139 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 O139 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O139 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 O139 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 10 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 O139 type at last, as shown in table 3.

By retrieving and comparing, find orf1,2, the albumen of rmlBDAC genes encoding has very high consensus amino acid sequence in 3,4 encoded protein and the Shigella flexneri O-antigen gene bunch, by the search to Pfam protein-based order sequenced data storehouse, find orf1, the homology desired value of 2,3,4 encoded protein and known consensus sequence is very high.The rmlBDAC gene is responsible for rhamnosyl synthetic gene, and we infer orf1, and 2,3,4 also is to be responsible for rhamnosyl synthetic gene, and contains rhamnosyl in the O-antigen of O139.

Orf5 and orf7 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O139 kind.The O-antigen transferring enzyme of orf7 encoded protein and Yersinia enterocolitica has 23% sequence identity, and 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 orf7 is wzx.The O-antigen polysaccharase of orf5 encoded protein and Streptococcus pneumoniae has 21% consistence, 45% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf5 is wzy.

Orf6, the albumen of 8,9,10 4 genes encodings and other known glycosyltransferases have the sequence identity of 34-47% and the sequence similarity of 45-67%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these four genes encodings and known glycosyltransferase family 1 and 2 is 1 * e ^-12To 5 * e ^-17, so we infer this four genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O139 may be made up of four monose.Because the definite function of these four genes can't be determined, so we are with the temporary called after orf6 of these four genes, 8,9,10.

Embodiment 6: the screening of specific gene:

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O139 type bunch, the position of these genes in nucleotide sequence sees Table 1.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O139 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 O139 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 8-10 bacterium, and 13 groups altogether, all list in the table in their source.

The genomic dna that contains intestinal bacteria O139 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 O139 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O139 type, screen gene by PCR: wzx, wzy and five glycosyltransferase genes to the O-antigen high special of intestinal bacteria O139 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O139 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 O139 type.These all oligonucleotide all can be used for the intestinal bacteria O139 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 O139 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O139 type, altogether by 12 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O139 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O139 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQUENCE LISTING

＜110〉Nankai University

＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O139 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O139 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>12654

<212>DNA

<213>Escherichia coli

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atcattgtgg ctgcagggat caaagaaatc ctcctggtaa ctcatgcgtc caagaacgca 60

gtcgaaaacc acttcgacac ctcttatgaa ttagaatctc tccttgaaca gcgcgtgaag 120

cgtcaactgc tggcggaagt acagtccatt tgcccgccgg gcgtgacaat tatgaacgtg 180

cgtcagggcg aacctttagg tttgggccac tccattttat gtgcacgacc tgccattggt 240

gacaatccat ttgtcgtggt gctgccagac gttgtgatcg acgacgccag cgccgacccg 300

ctgcgctaca accttgctgc catgattgcg cgcttcaacg aaacgggccg cagccaggtg 360

ctggcaaaac gtatgccggg tgacctctct gaatactccg tcattcagac caaagagccg 420

ctggaccgtg aaggcaaagt cagccgcatt gttgaattca tcgaaaaacc ggatcaaccg 480

cagacgctgg actcagacat catggccgtt ggtcgctatg tgctttctgc cgatatttgg 540

cctgaactgg agcgtactca acctggagca tggggacgta ttcagttgac tgatgccatt 600

gctgagttgg caaaaaaaca agcagttgac gcaatgctga tgactggggg acagttacga 660

ctgcggaaag aaaatgggtt atatgcaagc gtttgtgaag tatgggctgc gtaacctgaa 720

agaaggggcg aagttccgta aagggattga gaagctgtta agcgaataat gaaatctgac 780

cggatgtaac ggttgataag aaaattataa cggcagtgaa gattcgtggc gaaagtaatt 840

tgttgcgaat attcctgccg ttgttttata taaacaatca gaataacaac gagttagcaa 900

taggatttta gtcaaagttt tccaggattt tccttgtttc cagagcggat tggtaagaca 960

attagcgttt gagtttttcg agttaagcgc gagtgggtaa cgctcgtcac atcgtaggca 1020

tgcatgcagt gctctggtag ctgtaaagcc aggggcggta gcgtgcatta atacctctat 1080

taatcaacct aagagccgct tatttcacag catgctctga agtaatatgg aataataaag 1140

tgaagatact tgttactggt ggtgcaggat ttattggttc tgctgtagtt cgtcacatta 1200

taaataatac gcaagatagt gttgttaatg tcgataaatt aacgtacgcc ggaaacctgg 1260

aatcacttgc tgatgtttct gattctgaac gctatgtttt tgaacatgcg gatatttgcg 1320

atgcagctgc aatggcacgg atttttgctc agcatcagcc agatgcagta atgcacctgg 1380

ctgctgaaag ccatgttgac cgttcaatta caggtcctgc ggcatttatt gaaaccaata 1440

ttgttggtac atatgtcctt ttggaagccg ctcgcaatta ctggtctgct cttgatagcg 1500

acaagaaaac tagcttccgt tttcatcata tttctactga cgaagtctat ggtgatttgc 1560

ctcatccaga tgaagtaaat aataacgaag aattaccctt atttactgag acgacagctt 1620

acgcgccaag cagtccttat tccgcatcaa aagcatccag cgatcattta gttcgcgcgt 1680

ggaaacgtac ctatggttta ccgaccattg tgactaactg ttcgaataac tacggtcctt 1740

atcactttcc ggaaaaattg attccactag taattcttaa tgctctggaa ggtaaggcat 1800

tacctattta tggtaaaggg gatcaaattc gtgactggct gtatgttgaa gatcatgcgc 1860

gtgcgttata taccgtcgta accgaaggta aagcgggtga aacttataac attggtggac 1920

acaacgaaaa gaaaaacatc gatgtagtgc tcactatttg tgatttgttg gatgagattg 1980

tcccgaaaga gaaatcttac cgcgagcaaa ttacttatgt taccgatcgt ccgggacacg 2040

atcgccgtta tgcgattgat gctgagaaga ttggtcgcga attgggttgg aaaccacagg 2100

aaacgtttga gagcgggatt cggaagacag tggaatggta cctgtccaat acaaaatggg 2160

ttgataatgt gaaaagtggt gcctatcaat cgtggattga acagaactat gagggccgcc 2220

agtaatgaat atcctccttt tcggcaaaac agggcaggta ggttgggaac tacagcgtgc 2280

gctggcacct ttgggtaatt tgattgctct tgatgttcac tctactgatt attgtggtga 2340

ttttagtaac ccagaaggtg tggctgaaac cgtcaaaaaa attcgtcctg acgttattgt 2400

taatgctgct gctcataccg cggtagataa ggctgagtca gaacccgaat ttgcacaatt 2460

actcaatgcg acaagtgttg aagcgattgc aaaagcggct aatgaagttg gggcttgggt 2520

aattcattac tcaactgact acgtattccc tggaaatggc gacacgccat ggctggagac 2580

ggatgcaacc gcaccgctaa atgtttacgg tgaaaccaag ttagccggag aaaaagcgtt 2640

acaggaacat tgcgcgaagc atcttatttt ccggaccagc tgggtatatg caggtaaagg 2700

aaacaatttt gccaagacaa tgttacgtct ggcaaaagag cgcgaagaat tagctgtcat 2760

taacgatcag tttggtgcac caacaggtgc tgaactgctg gctgattgta cggcacatgc 2820

aattcgtatg gcactgaata aaccagaagt cgcaggcttg taccatctgg tagccactgg 2880

taccacaacc tggcacgatt atgctgcgct ggtttttgaa gaggcacgaa aagcaggtat 2940

tccccttgca ctcaacaagc tcaacgcagt accaacaaca gcctatccaa caccagctcg 3000

tcgtccacat aactctcgcc ttaatacaga aaaatttcag caaaattttg cgcttgtttt 3060

gcctgactgg caggttggcg tgaaacgaat gctcaacgaa ttatttacga ctacagcaat 3120

ttaatagttt ttgcatcttg ttcgtgatga tggagcaaga tgaattaaaa ggaatgatga 3180

aatgaaaacg cgtaaaggta ttattttagc gggtggttct ggtactcgtc tttatcctgt 3240

gactatggca gtcagtaaac agctgttacc tatttatgat aaaccgatga tctattaccc 3300

gctttctaca ctgatgttag cgggtattcg cgatattctg attatcagta caccacagga 3360

tactcctcgt tttcaacaac tgctgggtga cgggagccag tgggggctaa atcttcagta 3420

caaagtgcaa ccgactccag atggacttgc gcaggcgttt attatcggtg aagagtttat 3480

tggtggtgat gattgcgctt tggttcttgg tgataatatc ttctacggcc acgatctgcc 3540

gaagttaatg gacacagcag ttaataaaga aagtggcgca acggtatttg cctatcacgt 3600

taatgatcct gaacgctacg gtgtcgttga gtttgataaa aacggtacgg caataagcct 3660

ggaagaaaaa ccgctacaac caaaaagtaa ttatgcggta accgggcttt atttctatga 3720

taacgacgtt gtcgaaatgg cgaaaaacct taagccttct gcccgtggtg aactggaaat 3780

taccgatatt aaccgtattt atatggaaca ggggcgttta tccgttgcca tgatggggcg 3840

tggttatgcg tggctggaca cggggacaca tcaaagcctg attgaggcaa gcaacttcat 3900

tgctactatt gaagaacgtc aggggctgaa agtctcctgc ccggaagaaa ttgcttaccg 3960

taaagggttt gttgatgctg agcaggtgaa agtattagct gaacctctga aaaaaaatgc 4020

ttatggtcag tatctgctga aaatgattaa aggttattca taaaatgaac gtaattaaaa 4080

cagaaattcc tgatgtactg atttttgaac cgaaagtttt tggtgatgag cgtggtttct 4140

tttttgagag ctttaaccaa aagatttttg aggaagctgt aggccgcaaa gttgaatttg 4200

tccaggataa ccattcgaag tccacgaaag gtgttttacg cggactgcat tatcagttag 4260

aaccttatgc gcaagggaaa ttggtgcgct gcgttgttgg tgaggtttat gatgttgcgg 4320

ttgatattcg taaatcgtca cctacttttg gcaagtgggt tggggtgaat ttatctgctg 4380

agaataagcg ccagttgtgg atccctgagg gatttgcaca tggttttttg gtgctgagtg 4440

agacggcgga atttgtttat aagacgacga attattatca tcctgagagt gaaagaggaa 4500

ttatttggga tgataaaaat ataaatatcc attggcctat agttgatatt cctatattgt 4560

caaagaaaga tcataataac acttcattat tatgattagt aaatttggga catgattttt 4620

taaatgccgc cttttaaaag gcggtaaaaa actactgacc ggatgcttta atagaaaata 4680

taaaaaaata aatttcattt tacaaaattt gaatgtcaag ttagagaaat aaaactgtta 4740

tatgttagaa atactggtca cagcatgtaa attttcttga gtcagaatga attatatgtg 4800

tactatttct taaaaactat tgacattttt tagttggaaa taaaggttaa aaataaaaaa 4860

ctattcatat tgagagttaa atattacgta tgctaaaaat tttcctgtca gaggatacat 4920

tgtgagtttt attcatatct tattgctaat tattttattt tatttaccgt ttattttttt 4980

aaaatttggt ttacgaaaga aaataacgca tccgccaact ttattttgtg caatattttc 5040

atttgtacta tcaatggctt atatagctca cataactttg gggttttatg aaatatcata 5100

tgaaagctat gctatttatg gattagggaa tttatcattt attgctgggg ggatgattac 5160

tgatgcttta aatagacgaa acataaaaaa cagcactcaa cttgttatta acaagcaaga 5220

aataacccta ctaaagattc ttatttttac gctattgtta tattttccaa ttgcatatag 5280

cgagtttaaa catgcgacac ctgatatgcc tctcgcgtta aaaatattac gcattcgtga 5340

acgaggatta gaagagcagg tatatagtac cataacaaac aacatgataa tattgtcgtc 5400

gtgtttagtt atgtatatga catttatttt ctcaatcaaa aagataaatg tttattatta 5460

ttctctgaca tttgtcctct tctgtgctta taatttcatg actggcacga gggcagcaat 5520

tattcttatt tcaatcgcgt gtatttttat ttacttacta aataccaata aaaataataa 5580

aaccagtaag ctttttcttt tatcggtcgg gatgactgcc ataattctag gtgctctagt 5640

tgcgattttt atgggcaaag atggaatgga acgtgatgaa agtttattag ctaatttaaa 5700

taaggttgtt gataattatt tttcatatac tgttcaaggg gtgattttat ttgataatta 5760

tgtaattgga agagaaaaga ttactcctaa ttgggatata ttatcaggct cagctgaagt 5820

aattaataaa ataacaagct caaacatatt caaaactgac tcgaaattca gtgaattcag 5880

tcattttgca aaagataaag atggtaatgt atgtacaatt tattttgcta tttatccatt 5940

atatggttta gttggagtaa tattgttttt tttgttatat ggaaccgtat gtacaatact 6000

ttataatcat gggcctggca ttgtttccgt attagtcgga tatattaacg ccactctatg 6060

tttaaatata tttaatgaac aagtgttcat caatattata ttcacaatta aatttatttg 6120

ttttctgcta ttaatacgct gtttcgagag gataaaatga acgttggtgt tgtgattgtt 6180

tggtataacc ctacaaaggc tcagatagaa aatgtttata gtctcgcaaa tactagcgag 6240

ttaaaaatat gtatagtgga taactcaaat gatgagaatt tatttgaaaa aaaaacgttg 6300

agcagtaaaa acatatcata tattcataat agtaataaag gtggcatcgc tggagcattt 6360

aatagaggaa taaataaatt gttcgctgag agaactatga aacatatttt tacaatggat 6420

caggactcag agttaagcga agactatttc ttaagtatgt ggaagtttgt tagagataat 6480

aattgcgaaa tagcttgccc aaatttttat gaccggaatt caaaaacata tggtacgttt 6540

attaatttaa ctccgttttt ctataaggct gtaaataatg gtacaacaac attttgtatt 6600

tcatctggta tgattttttc ccgaaaagca tgggagatac tcggggattt tgacgaaaga 6660

cttataattg atcatgtcga tacagattat tgtttaaaag ctaataatca taaagtaaca 6720

atattggtta attatgagca gtgcttaaat catgctattg gggaacgaga gagtcatcga 6780

attttagggg ttactcttaa accaaaccat cataattata tcaggaaata ttatattgta 6840

agaaatggta catatttggc attaaaatat tttaaatttt cgaagggata ctttaacctc 6900

aatattctgc gcgtaattca tgaaacagta tgtgtgattc tatatgaaaa tgataaaata 6960

agaaaattat cgtatatgtt acgtggatta cgggatggta ttaaggggaa gttaggtgcc 7020

attaagtaaa agtatttatg caatcttaag tatttcatgt ttgctgttag gattatatat 7080

tcaatttctc tttgcggaat tttatcaagg agatttttta aatttattct atttgggggt 7140

tagtttttgt agtgttgtca taagtatgtc ttctggggga ttaggcggct ttttaatttc 7200

aagattttct aatctttcag aaacattacg ccttgatatt gcagtgtttt catttactat 7260

tgtcttttcg ataataggtg tgggtgtttt ctgtgctctt tcatatactt cattatttgt 7320

gcttaaaatg gcactgcatc agtcattaat tattgctttt ttattttttt tatatctttt 7380

atctcttgct ttaaatctaa tttttcaatc atactcctac tcgaaaaaag ggatttcagg 7440

gccaatattt tatgagtttt gtagccttat atcttatatt ctggttgcat ttcttcttgt 7500

gattaataaa gaagattata ttatttgctg catattgttt tgtgtgcgtg gttattttca 7560

actgattttg tactatattt tttgcaagga taaaatagct tgcaggctaa ttaacggcga 7620

cgaagttaaa tcttttttta atgaaattaa atacttaatg agtggtagtg catattataa 7680

atcagagcca ttaattgata ggttttttct tgttgccagt tctcataatt tagcagcata 7740

tcacttaatt agccaaattt ataatgcagc ccttggtctc tggtttaaaa tttctgtatc 7800

acctttagtt aataaactga ctcaaaatac aacaaatacc attgtattta tgaaattatt 7860

ccgtcgcggt ttattaagtc aatgcataat ctctatagta tctgctataa tccttttttt 7920

tacaccaaca cttcactatt tagaatcgat aaatatattt cgccccatcg ctcaaaatgc 7980

tgatgttgtc tatttacttt tcattttttt tgttgcgtct cttttggggc aagtagtttc 8040

taatgcatac tatagtattg gacaccataa aacaccggtt atagtttctt gtataacata 8100

taccctgttt ctaccagcaa aatattatct tgtcaaaaga tacggggtct ctggactgtg 8160

tattctagtt tctttatatc acacggttaa ttttattatt ctgtggttaa tgtttagagt 8220

taaagatgaa agttaataat atcgtaagaa ttatggcggt tacctccgga attaaaacgc 8280

catcaagcag atttagaatt cgtcaacata ttgaacaatt aaggaagtat aatttatatg 8340

tagatgaaat aatccctcct attgataaaa ataagtctgt gccatttcta tcaaaaatta 8400

gccctaaata ttatatccct ttatatatac cttggcagat aataaaaatt ctacagcgtc 8460

tccctgttct tcttaagcaa acgaggtata aatatatatg gttaaataga gagctgctta 8520

ctggaatacc aacattagaa gtatttttct gcaagaaaat tatacttgat gttgatgatg 8580

ctatctggaa aaatccccct atgggagaga taacagcgag atttttagca aagaaggcat 8640

ataaaataat atgtgggaat aattaccttg ccgaatggtt tagaagatat aataataatg 8700

tttatgtgat ccctacagca gtggatgtgc ataaatttat acctagatgt aataagagga 8760

aagatatagt tttgggatgg attggaacac atggtaattt aaaatacctt gaaaagattt 8820

atccaacttt gttaaaaatt ctgaatagca atcgtaatgt atatttgaat attgtttcag 8880

acaaaagacc cgagttttgt attgataact cacgtgcaaa ttacattgag tggtcagcgg 8940

aagatgaagt acaaaacttc caatcaatag atattggact tatgcctctt gatgataatg 9000

aatggacgag aggtaaatgt agctataaat tgttacagca tatggcctgt ggttcattag 9060

ttgttggatc gccggttggt atgaataagg aaattctgtt agaggataat ggtgcgtatc 9120

cggcaaatac tgaaaacgag tggatggata cgttatcgta cttaatatcg aattttgaag 9180

aaatttattg cattcagtct aaaaaagcaa gacgtttcat tgagtgtgaa tattccgcag 9240

atacaattag ggaaaaattg gcaagggtaa tgattgatga ctaaggttat aatggtgtta 9300

ccaaggtatt tacccatcat tggtggggct gaagttcaat gtagccgcct catccgcgaa 9360

tttaataaaa ctggtaaagt taacatagtg agcattgtta caagaagagt aagcaatacc 9420

ttggaaaaaa aagaatacat tgataatata cctgtaacga gaatcccacc aagcggaact 9480

ggaatgctcg ttgaatacat tttttgctta ttattgatat tctatctttt gcttaatcat 9540

aagaagtatg atgtaattca ttgccacgct tcctctattt ttggtctttc ttgttcctta 9600

gttggatttc tactcaaaaa gaaagtgatt attaagatct ctactaatgg tgaaattggc 9660

tcaatgcaga gttcgtctat aaaaagatgt atttcacggt ttctatcaaa atattgtacc 9720

tatatagcgt taaattctga gggctattct gaagtaaatt gttatttatc taaagctcat 9780

aaatgtatta ttcccaatgg aattgatttc aatatcaacc atgaaaaaaa tttggaattg 9840

gccactgtga ttagaaaaaa attacataat atgtatgggg aagatatata catatgtgtt 9900

tttgtcggtc gttttgttac aagaaaaggg ataagggaaa tatatgaatg tggtgcagac 9960

ctgactgcta atggtaaaaa aatcatattc gtgttaattg gtgattcgga attacaacgt 10020

gatgctatca atttcgaata tgatcatctt cataatttct atatcgctgg taggaaaaaa 10080

aatgtgttcc cctatttgat tgctggagat ttattcattt caccatcaca caaagaaggt 10140

ttgccaaaca ctgtccttga agctctttcc gttggcaagg tttgtattct ttcagatata 10200

cttccacatc aagagattgc ttatgaacat cctaaaaacg tcatactttt taaaacagga 10260

gacgctcagg agttgaagca aactataata aattatctgg aattaaatcc tcataaatca 10320

agtttgcata gttatgtgca gattgaggac aatgtcttat tagacaaata ttctatcagt 10380

tgtatagcgg aacaatattg tctactttac aaggacaagt ctaatgcctc gtgatgatta 10440

taaaatatat atatctatag tttcccataa tcacggtgag ttaataaaga atttagattg 10500

tattaagaat ttatctcaag agttcatagt ggttattaag aataactgtt atgatgaagt 10560

attattggac tatttaaaag atagcaatgc ctatctcatc aatgaagaat ataataaagg 10620

atttggctat aataataatg tcgtttttag ttattgtcgt ttgcaactaa atatgcaacc 10680

ttccgattat tttatagtac ttaatccaga tttggtcatc aaaaactcgt ctatcaaaga 10740

gttggttaac aaaatgcatt gcagcaatgt caaagctgca actattaacc tttacctaga 10800

tgataaactt catgactatg attattctgt gaggcatttt ccccgcttat atgactttat 10860

tttatcttta gtattaggta agaataaaac taaaatagac aaatcaataa ttaccaatcc 10920

aacagaagta gattggtgtg caggttcttt tatggcttta agtagtcatg tttatcaatc 10980

cattcaagga tttgatacag ggttatttta tgtattgcga ggatatcgat ctttgttttc 11040

gtttaaagaa aaaaaatatt ggaataattt attatcctga gattacaggc attcacaaag 11100

ctcagcataa aaatagaagt ttttttcgaa acattttatt tggcatgtta aaagtgtatt 11160

cagatttatg tttgctaaaa acgggttaat taggaataag tccaatattt gtcactaaag 11220

attgatttgt gcttagaaag aaaaattaaa ccagagtctt ttagatatct tttaaagcag 11280

tatatgctct atccccccct gacaggagta aacaatgtca aagcaacaga tcggcgtcgt 11340

cggtatggca gtgatggggc gcaaccttgc gctcaatatc gaaagccgtg gttataccgt 11400

ctctattttc aaccgttccc gtgagaaaac ggaagaagtg attgccgaaa atccgggcaa 11460

gaaactggtt ccttactata cggtgaaaga atttgttgaa tctctggaaa cgcctcgtcg 11520

catcctgtta atggtgaaag caggtgcagg cacggatgct gctattgatt ctctcaagcc 11580

atacctcgat aaaggtgaca tcatcattga tggcggtaac accttcttcc aggatactat 11640

ccgtcgtaac cgtgagcttt ctgccgaagg ctttaacttc attggtaccg gtgtctccgg 11700

tggtgaagaa ggcgcgctga aaggtccttc cattatgcct ggtgggcaga aagaagccta 11760

tgaactggtt gcaccgatcc tgaccaaaat cgccgcagta gctgaagacg gtgagccatg 11820

cgttacctat attggtgccg atggcgcagg tcactatgtg aagatggttc acaacggtat 11880

tgaatacggc gatatgcagc tgattgctga agcctattct ctgcttaaag gtggcctgaa 11940

tctttccaac gaagaactgg cgcagacgtt taccgagtgg aataacggtg aactgagcag 12000

ctacctgatc gacatcacca aagatatctt caccaaaaaa gatgaagacg gtaactacct 12060

ggttgatgtg attctggatg aagcagcaaa caaaggtacg ggcaaatgga ccagccagag 12120

cgcgctggat ctcggagaac cgctgtcgct gattaccgag tctgtgtttg cacgttatat 12180

ctcttctctg aaagatcagc gcgttgccgc atctaaagtt ctctctggtc cgcaagctca 12240

gtcagcaggc gacaaggctg agttcatcga aaaagttcgc cgtgcgctgt atctgggcaa 12300

aatcgtttct tacgctcagg gcttctctca gctgcgtgct gcgtctgaag aatacaactg 12360

ggatctgaac tacggcgaaa tcgcgaagat tttccgtgct ggctgcatta tccgtgcgca 12420

gttcctgcag aaaatcaccg atgcatatgc cgaaaatccg cagatcgcta acctgctgct 12480

ggctccgtac ttcaagcaaa ttgccgatga ctatcagcag gcgctgcgcg atgtcgtcgc 12540

ttacgcagta cagaacggta tcccggttcc gaccttcgcc gctgcggttg cctattatga 12600

cagctaccgt gccgctgttc tgcctgcgaa cctgatccag gcccagcgtg acta 12654

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O139 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	O-antigen transhipment enzyme	7016-8236	7219-7236	7708-7725	507bp	0 *	55
			7712-7729	8100-8117	406bp	0 *	55
								Wzy	O-antigen polysaccharase	4922-6160	5150-5167	5393-5410	261bp	0 *	55
			5344-5362	6046-6063	720bp	0 *	55

^*Only in intestinal bacteria O139 type, obtain a correct band

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

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli	O1，O2，O5，O7，O12，O13，O14，O15，O16，O17，O19ab，O20， O21，O22，O23，O24，O59	IMVS a
2, wild-type e. coli	O25，O26，O27，O28，O29，O30，O32，O33，O35，O36，O37， O38，O40，O41，O42，O43	IMVS a
			3, wild-type e. coli	O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62	IMVS a
4, wild-type e. coli	O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78，	IMVS a

	O79，O80，O81，O82，O83
			5, wild-type e. coli	O84，O85，O86，O87，O88，O89，O91，O92，O98，O99，O101， O102，O103，O104，O106	IMVS a
6, wild-type e. coli	O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128	IMVS a
			7, wild-type e. coli	O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O141，O142，O143，O144，O145	IMVS a
8, wild-type e. coli	O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166	IMVS a b
			9, wild-type e. coli and Shigellae	O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12	c d
10, wild-type Shigellae	B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18	d
			11, wild-type Shigellae	F1a，F1b，F2a，F2b，F3，F4b，F5(v:4)，F5(v:7)，F6，F X becomes，F Y becomes，DS，DR，	d
12, wild-type e. coli	O3，O11，O39，O59，O64，O73，O96，O95，O100，O114，O151， O155，O124	IMVS a
			13, the 7th group of bacterial strain adds the intestinal bacteria reference culture	O139	IMVS

^*For the convenience that detects, we are divided into one group with every 13-17 bacterium, 13 groups altogether

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 O139 type O antigen gene structure iron

Table 4 intestinal bacteria O139 type O antigen gene cluster gene position

ATCATTGTGG CTGCAGGGAT CAAAGAAATC CTCCTGGTAA CTCATGCGTC CAAGAACGCA 60

GTCGAAAACC ACTTCGACAC CTCTTATGAA TTAGAATCTC TCCTTGAACA GCGCGTGAAG 120

CGTCAACTGC TGGCGGAAGT ACAGTCCATT TGCCCGCCGG GCGTGACAAT TATGAACGTG 180

CGTCAGGGCG AACCTTTAGG TTTGGGCCAC TCCATTTTAT GTGCACGACC TGCCATTGGT 240

GACAATCCAT TTGTCGTGGT GCTGCCAGAC GTTGTGATCG ACGACGCCAG CGCCGACCCG 300

CTGCGCTACA ACCTTGCTGC CATGATTGCG CGCTTCAACG AAACGGGCCG CAGCCAGGTG 360

CTGGCAAAAC GTATGCCGGG TGACCTCTCT GAATACTCCG TCATTCAGAC CAAAGAGCCG 420

CTGGACCGTG AAGGCAAAGT CAGCCGCATT GTTGAATTCA TCGAAAAACC GGATCAACCG 480

CAGACGCTGG ACTCAGACAT CATGGCCGTT GGTCGCTATG TGCTTTCTGC CGATATTTGG 540

CCTGAACTGG AGCGTACTCA ACCTGGAGCA TGGGGACGTA TTCAGTTGAC TGATGCCATT 600

GCTGAGTTGG CAAAAAAACA AGCAGTTGAC GCAATGCTGA TGACTGGGGG ACAGTTACGA 660

CTGCGGAAAG AAAATGGGTT ATATGCAAGC GTTTGTGAAG TATGGGCTGC GTAACCTGAA 720

AGAAGGGGCG AAGTTCCGTA AAGGGATTGA GAAGCTGTTA AGCGAATAAT GAAATCTGAC 780

CGGATGTAAC GGTTGATAAG AAAATTATAA CGGCAGTGAA GATTCGTGGC GAAAGTAATT 840

TGTTGCGAAT ATTCCTGCCG TTGTTTTATA TAAACAATCA GAATAACAAC GAGTTAGCAA 900

TAGGATTTTA GTCAAAGTTT TCCAGGATTT TCCTTGTTTC CAGAGCGGAT TGGTAAGACA 960

ATTAGCGTTT GAGTTTTTCG AGTTAAGCGC GAGTGGGTAA CGCTCGTCAC ATCGTAGGCA 1020

TGCATGCAGT GCTCTGGTAG CTGTAAAGCC AGGGGCGGTA GCGTGCATTA ATACCTCTAT 1080

Orf1 is initial

TAATCAACCT AAGAGCCGCT TAAATCACAG CATGCTCTGA AGTAATATGG AATAATAAA G 1140

TGAAGATACT TGTTACTGGT GGTGCAGGAT TTATTGGTTC TGCTGTAGTT CGTCACATTA 1200

TAAATAATAC GCAAGATAGT GTTGTTAATG TCGATAAATT AACGTACGCC GGAAACCTGG 1260

AATCACTTGC TGATGTTTCT GATTCTGAAC GCTATGTTTT TGAACATGCG GATATTTGCG 1320

ATGCAGCTGC AATGGCACGG ATTTTTGCTC AGCATCAGCC AGATGCAGTA ATGCACCTGG 1380

CTGCTGAAAG CCATGTTGAC CGTTCAAAAA CAGGTCCTGC GGCATTTATT GAAACCAATA 1440

TTGTTGGTAC ATATGTCCTT TTGGAAGCCG CTCGCAATTA CTGGTCTGCT CTTGATAGCG 1500

ACAAGAAAAC TAGCTTCCGT TTTCATCATA TTTCTACTGA CGAAGTCTAT GGTGATTTGC 1560

CTCATCCAGA TGAAGTAAAT AATAACGAAG AATTACCCTT ATTTACTGAG ACGACAGCTT 1620

ACGCGCCAAG CAGTCCTTAT TCCGCATCAA AAGCATCCAG CGATCATTTA GTTCGCGCGT 1680

GGAAACGTAC CTATGGTTTA CCGACCATTG TGACTAACTG TTCGAATAAC TACGGTCCTT 1740

ATCACTTTCC GGAAAAATTG ATTCCACTAG TAATTCTTAA TGCTCTGGAA GGTAAGGCAT 1800

TACCTATTTA TGGTAAAGGG GATCAAATTC GTGACTGGCT GTATGTTGAA GATCATGCGC 1860

GTGCGTTATA TACCGTCGTA ACCGAAGGTA AAGCGGGTGA AACTTATAAC ATTGGTGGAC 1920

ACAACGAAAA GAAAAACATC GATGTAGTGC TCACTATTTG TGATTTGTTG GATGAGATTG 1980

TCCCGAAAGA GAAATCTTAC CGCGAGCAAA TTACTTATGT TACCGATCGT CCGGGACACG 2040

ATCGCCGTTA TGCGATTGAT GCTGAGAAGA TTGGTCGCGA ATTGGGTTGG AAACCACAGG 2100

AAACGTTTGA GAGCGGGATT CGGAAGACAG TGGAATGGTA CCTGTCCAAT ACAAAATGGG 2160

TTGATAATGT GAAAAGTGGT GCCTATCAAT CGTGGATTGA ACAGAACTAT GAGGGCCGCC 2220

It is initial that orf1 stops orf2

AG TAATGAAT ATCCTCCTTT TCGGCAAAAC AGGGCAGGTA GGTTGGGAAC TACAGCGTGC 2280

GCTGGCACCT TTGGGTAATT TGATTGCTCT TGATGTTCAC TCTACTGATT ATTGTGGTGA 2340

TTTTAGTAAC CCAGAAGGTG TGGCTGAAAC CGTCAAAAAA ATTCGTCCTG ACGTTATTGT 2400

TAATGCTGCT GCTCATACCG CGGTAGATAA GGCTGAGTCA GAACCCGAAT TTGCACAATT 2460

ACTCAATGCG ACAAGTGTTG AAGCGATTGC AAAAGCGGCT AATGAAGTTG GGGCTTGGGT 2520

AATTCATTAC TCAACTGACT ACGTATTCCC TGGAAATGGC GACACGCCAT GGCTGGAGAC 2580

GGATGCAACC GCACCGCTAA ATGTTTACGG TGAAACCAAG TTAGCCGGAG AAAAAGCGTT 2640

ACAGGAACAT TGCGCGAAGC ATCTTATTTT CCGGACCAGC TGGGTATATG CAGGTAAAGG 2700

AAACAATTTT GCCAAGACAA TGTTACGTCT GGCAAAAGAG CGCGAAGAAT TAGCTGTCAT 2760

TAACGATCAG TTTGGTGCAC CAACAGGTGC TGAACTGCTG GCTGATTGTA CGGCACATGC 2820

AATTCGTATG GCACTGAATA AACCAGAAGT CGCAGGCTTG TACCATCTGG TAGCCACTGG 2880

TACCACAACC TGGCACGATT ATGCTGCGCT GGTTTTTGAA GAGGCACGAA AAGCAGGTAT 2940

TCCCCTTGCA CTCAACAAGC TCAACGCAGT ACCAACAACA GCCTATCCAA CACCAGCTCG 3000

TCGTCCACAT AACTCTCGCC TTAATACAGA AAAATTTCAG CAAAATTTTG CGCTTGTTTT 3060

GCCTGACTGG CAGGTTGGCG TGAAACGAAT GCTCAACGAA TTATTTACGA CTACAGCAAT 3120

Orf2 stops

T TAATAGTTT TTGCATCTTG TTCGTGATGA TGGAGCAAGA TGAATTAAAA GGAATGATGA 3180

Orf3 is initial

A ATGAAAACG CGTAAAGGTA TTATTTTAGC GGGTGGTTCT GGTACTCGTC TTTATCCTGT 3240

GACTATGGCA GTCAGTAAAC AGCTGTTACC TATTTATGAT AAACCGATGA TCTATTACCC 3300

GCTTTCTACA CTGATGTTAG CGGGTATTCG CGATATTCTG ATTATCAGTA CACCACAGGA 3360

TACTCCTCGT TTTCAACAAC TGCTGGGTGA CGGGAGCCAG TGGGGGCTAA ATCTTCAGTA 3420

CAAAGTGCAA CCGACTCCAG ATGGACTTGC GCAGGCGTTT ATTATCGGTG AAGAGTTTAT 3480

TGGTGGTGAT GATTGCGCTT TGGTTCTTGG TGATAATATC TTCTACGGCC ACGATCTGCC 3540

GAAGTTAATG GACACAGCAG TTAATAAAGA AAGTGGCGCA ACGGTATTTG CCTATCACGT 3600

TAATGATCCT GAACGCTACG GTGTCGTTGA GTTTGATAAA AACGGTACGG CAATAAGCCT 3660

GGAAGAAAAA CCGCTACAAC CAAAAAGTAA TTATGCGGTA ACCGGGCTTT ATTTCTATGA 3720

TAACGACGTT GTCGAAATGG CGAAAAACCT TAAGCCTTCT GCCCGTGGTG AACTGGAAAT 3780

TACCGATATT AACCGTATTT ATATGGAACA GGGGCGTTTA TCCGTTGCCA TGATGGGGCG 3840

TGGTTATGCG TGGCTGGACA CGGGGACACA TCAAAGCCTG ATTGAGGCAA GCAACTTCAT 3900

TGCTACTATT GAAGAACGTC AGGGGCTGAA AGTCTCCTGC CCGGAAGAAA TTGCTTACCG 3960

TAAAGGGTTT GTTGATGCTG AGCAGGTGAA AGTATTAGCT GAACCTCTGA AAAAAAATGC 4020

It is initial that orf3 stops orf4

TTATGGTCAG TATCTGCTGA AAATGATTAA AGGTTATTCA TAAA ATGAAC GTAATTAAAA 4080

CAGAAATTCC TGATGTACTG ATTTTTGAAC CGAAAGTTTT TGGTGATGAG CGTGGTTTCT 4140

TTTTTGAGAG CTTTAACCAA AAGATTTTTG AGGAAGCTGT AGGCCGCAAA GTTGAATTTG 4200

TCCAGGATAA CCATTCGAAG TCCACGAAAG GTGTTTTACG CGGACTGCAT TATCAGTTAG 4260

AACCTTATGC GCAAGGGAAA TTGGTGCGCT GCGTTGTTGG TGAGGTTTAT GATGTTGCGG 4320

TTGATATTCG TAAATCGTCA CCTACTTTTG GCAAGTGGGT TGGGGTGAAT TTATCTGCTG 4380

AGAATAAGCG CCAGTTGTGG ATCCCTGAGG GATTTGCACA TGGTTTTTTG GTGCTGAGTG 4440

AGACGGCGGA ATTTGTTTAT AAGACGACGA ATTATTATCA TCCTGAGAGT GAAAGAGGAA 4500

TTATTTGGGA TGATAAAAAT ATAAATATCC ATTGGCCTAT AGTTGATATT CCTATATTGT 4560

Orf4 stops

CAAAGAAAGA TCATAATAAC ACTTCATTAT TA TGATTAGT AAATTTGGGA CATGATTTTT 4620

TAAATGCCGC CTTTTAAAAG GCGGTAAAAA ACTACTGACC GGATGCTTTA ATAGAAAATA 4680

TAAAAAAATA AATTTCATTT TACAAAATTT GAATGTCAAG TTAGAGAAAT AAAACTGTTA 4740

TATGTTAGAA ATACTGGTCA CAGCATGTAA ATTTTCTTGA GTCAGAATGA ATTATATGTG 4800

TACTATTTCT TAAAAACTAT TGACATTTTT TAGTTGGAAA TAAAGGTTAA AAATAAAAAA 4860

CTATTCATAT TGAGAGTTAA ATATTACGTA TGCTAAAAAT TTTCCTGTCA GAGGATACAT 4920

Orf5 is initial

T GTGAGTTTT ATTCATATCT TATTGCTAAT TATTTTATTT TATTTACCGT TTATTTTTTT 4980

AAAATTTGGT TTACGAAAGA AAATAACGCA TCCGCCAACT TTATTTTGTG CAATATTTTC 5040

ATTTGTACTA TCAATGGCTT ATATAGCTCA CATAACTTTG GGGTTTTATG AAATATCATA 5100

TGAAAGCTAT GCTATTTATG GATTAGGGAA TTTATCATTT ATTGCTGGGG GGATGATTAC 5160

TGATGCTTTA AATAGACGAA ACATAAAAAA CAGCACTCAA CTTGTTATTA ACAAGCAAGA 5220

AATAACCCTA CTAAAGATTC TTATTTTTAC GCTATTGTTA TATTTTCCAA TTGCATATAG 5280

CGAGTTTAAA CATGCGACAC CTGATATGCC TCTCGCGTTA AAAATATTAC GCATTCGTGA 5340

ACGAGGATTA GAAGAGCAGG TATATAGTAC CATAACAAAC AACATGATAA TATTGTCGTC 5400

GTGTTTAGTT ATGTATATGA CATTTATTTT CTCAATCAAA AAGATAAATG TTTATTATTA 5460

TTCTCTGACA TTTGTCCTCT TCTGTGCTTA TAATTTCATG ACTGGCACGA GGGCAGCAAT 5520

TATTCTTATT TCAATCGCGT GTATTTTTAT TTACTTACTA AATACCAATA AAAATAATAA 5580

AACCAGTAAG CTTTTTCTTT TATCGGTCGG GATGACTGCC ATAATTCTAG GTGCTCTAGT 5640

TGCGATTTTT ATGGGCAAAG ATGGAATGGA ACGTGATGAA AGTTTATTAG CTAATTTAAA 5700

TAAGGTTGTT GATAATTATT TTTCATATAC TGTTCAAGGG GTGATTTTAT TTGATAATTA 5760

TGTAATTGGA AGAGAAAAGA TTACTCCTAA TTGGGATATA TTATCAGGCT CAGCTGAAGT 5820

AATTAATAAA ATAACAAGCT CAAACATATT CAAAACTGAC TCGAAATTCA GTGAATTCAG 5880

TCATTTTGCA AAAGATAAAG ATGGTAATGT ATGTACAATT TATTTTGCTA TTTATCCATT 5940

ATATGGTTTA GTTGGAGTAA TATTGTTTTT TTTGTTATAT GGAACCGTAT GTACAATACT 6000

TTATAATCAT GGGCCTGGCA TTGTTTCCGT ATTAGTCGGA TATATTAACG CCACTCTATG 6060

TTTAAATATA TTTAATGAAC AAGTGTTCAT CAATATTATA TTCACAATTA AATTTATTTG 6120

The initial orf5 of orf6 stops

TTTTCTGCTA TTAATACGCT GTTTCGAGAG GATAAA ATGA ACGTTGGTGT TGTGATTGTT 6180

TGGTATAACC CTACAAAGGC TCAGATAGAA AATGTTTATA GTCTCGCAAA TACTAGCGAG 6240

TTAAAAATAT GTATAGTGGA TAACTCAAAT GATGAGAATT TATTTGAAAA AAAAACGTTG 6300

AGCAGTAAAA ACATATCATA TATTCATAAT AGTAATAAAG GTGGCATCGC TGGAGCATTT 6360

AATAGAGGAA TAAATAAATT GTTCGCTGAG AGAACTATGA AACATATTTT TACAATGGAT 6420

CAGGACTCAG AGTTAAGCGA AGACTATTTC TTAAGTATGT GGAAGTTTGT TAGAGATAAT 6480

AATTGCGAAA TAGCTTGCCC AAATTTTTAT GACCGGAATT CAAAAACATA TGGTACGTTT 6540

ATTAATTTAA CTCCGTTTTT CTATAAGGCT GTAAATAATG GTACAACAAC ATTTTGTATT 6600

TCATCTGGTA TGATTTTTTC CCGAAAAGCA TGGGAGATAC TCGGGGATTT TGACGAAAGA 6660

CTTATAATTG ATCATGTCGA TACAGATTAT TGTTTAAAAG CTAATAATCA TAAAGTAACA 6720

ATATTGGTTA ATTATGAGCA GTGCTTAAAT CATGCTATTG GGGAACGAGA GAGTCATCGA 6780

ATTTTAGGGG TTACTCTTAA ACCAAACCAT CATAATTATA TCAGGAAATA TTATATTGTA 6840

AGAAATGGTA CATATTTGGC ATTAAAATAT TTTAAATTTT CGAAGGGATA CTTTAACCTC 6900

AATATTCTGC GCGTAATTCA TGAAACAGTA TGTGTGATTC TATATGAAAA TGATAAAATA 6960

Orf7 is initial

AGAAAATTAT CGTATATGTT ACGTGGATTA CGGGATGGTA TTAAGGGGAA GTTAG GTGCC 7020

Orf6 stops

ATTAAG TAAA AGTATTTATG CAATCTTAAG TATTTCATGT TTGCTGTTAG GATTATATAT 7080

TCAATTTCTC TTTGCGGAAT TTTATCAAGG AGATTTTTTA AATTTATTCT ATTTGGGGGT 7140

TAGTTTTTGT AGTGTTGTCA TAAGTATGTC TTCTGGGGGA TTAGGCGGCT TTTTAATTTC 7200

AAGATTTTCT AATCTTTCAG AAACATTACG CCTTGATATT GCAGTGTTTT CATTTACTAT 7260

TGTCTTTTCG ATAATAGGTG TGGGTGTTTT CTGTGCTCTT TCATATACTT CATTATTTGT 7320

GCTTAAAATG GCACTGCATC AGTCATTAAT TATTGCTTTT TTATTTTTTT TATATCTTTT 7380

ATCTCTTGCT TTAAATCTAA TTTTTCAATC ATACTCCTAC TCGAAAAAAG GGATTTCAGG 7440

GCCAATAAAT TATGAGTTTT GTAGCCTTAT ATCTTATATT CTGGTTGCAT TTCTTCTTGT 7500

GATTAATAAA GAAGAAAATA TTATTTGCTG CATATTGTTT TGTGTGCGTG GTTATTTTCA 7560

ACTGATTTTG TACTATATTT TTTGCAAGGA TAAAATAGCT TGCAGGCTAA TTAACGGCGA 7620

CGAAGTTAAA TCTTTTTTTA ATGAAATTAA ATACTTAATG AGTGGTAGTG CATATTATAA 7680

ATCAGAGCCA TTAATTGATA GGTTTTTTCT TGTTGCCAGT TCTCATAATT TAGCAGCATA 7740

TCACTTAATT AGCCAAATTT ATAATGCAGC CCTTGGTCTC TGGTTTAAAA TTTCTGTATC 7800

ACCTTTAGTT AATAAACTGA CTCAAAATAC AACAAATACC ATTGTATTTA TGAAATTATT 7860

CCGTCGCGGT TTATTAAGTC AATGCATAAT CTCTATAGTA TCTGCTATAA TCCTTTTTTT 7920

TACACCAACA CTTCACTATT TAGAATCGAT AAATATATTT CGCCCCATCG CTCAAAATGC 7980

TGATGTTGTC TATTTACTTT TCATTTTTTT TGTTGCGTCT CTTTTGGGGC AAGTAGTTTC 8040

TAATGCATAC TATAGTATTG GACACCATAA AACACCGGTT ATAGTTTCTT GTATAACATA 8100

TACCCTGTTT CTACCAGCAA AATATTATCT TGTCAAAAGA TACGGGGTCT CTGGACTGTG 8160

TATTCTAGTT TCTTTATATC ACACGGTTAA TTTTATTATT CTGTGGTTAA TGTTTAGAGT 8220

The initial orf7 of orf8 stops

TAAAG ATGAA AGT TAATAAT ATCGTAAGAA TTATGGCGGT TACCTCCGGA ATTAAAACGC 8280

CATCAAGCAG ATTTAGAATT CGTCAACATA TTGAACAATT AAGGAAGTAT AATTTATATG 8340

TAGATGAAAT AATCCCTCCT ATTGATAAAA ATAAGTCTGT GCCATTTCTA TCAAAAATTA 8400

GCCCTAAATA TTATATCCCT TTATATATAC CTTGGCAGAT AATAAAAATT CTACAGCGTC 8460

TCCCTGTTCT TCTTAAGCAA ACGAGGTATA AATATATATG GTTAAATAGA GAGCTGCTTA 8520

CTGGAATACC AACATTAGAA GTATTTTTCT GCAAGAAAAT TATACTTGAT GTTGATGATG 8580

CTATCTGGAA AAATCCCCCT ATGGGAGAGA TAACAGCGAG ATTTTTAGCA AAGAAGGCAT 8640

ATAAAATAAT ATGTGGGAAT AATTACCTTG CCGAATGGTT TAGAAGATAT AATAATAATG 8700

TTTATGTGAT CCCTACAGCA GTGGATGTGC ATAAATTTAT ACCTAGATGT AATAAGAGGA 8760

AAGATATAGT TTTGGGATGG ATTGGAACAC ATGGTAATTT AAAATACCTT GAAAAGATTT 8820

ATCCAACTTT GTTAAAAATT CTGAATAGCA ATCGTAATGT ATATTTGAAT ATTGTTTCAG 8880

ACAAAAGACC CGAGTTTTGT ATTGATAACT CACGTGCAAA TTACATTGAG TGGTCAGCGG 8940

AAGATGAAGT ACAAAACTTC CAATCAATAG ATATTGGACT TATGCCTCTT GATGATAATG 9000

AATGGACGAG AGGTAAATGT AGCTATAAAT TGTTACAGCA TATGGCCTGT GGTTCATTAG 9060

TTGTTGGATC GCCGGTTGGT ATGAATAAGG AAATTCTGTT AGAGGATAAT GGTGCGTATC 9120

CGGCAAATAC TGAAAACGAG TGGATGGATA CGTTATCGTA CTTAATATCG AATTTTGAAG 9180

AAATTTATTG CATTCAGTCT AAAAAAGCAA GACGTTTCAT TGAGTGTGAA TATTCCGCAG 9240

The initial orf8 of orf9 stops

ATACAATTAG GGAAAAATTG GCAAGGGTAA TGATTG ATGA C TAAGGTTAT AATGGTGTTA 9300

CCAAGGTATT TACCCATCAT TGGTGGGGCT GAAGTTCAAT GTAGCCGCCT CATCCGCGAA 9360

TTTAATAAAA CTGGTAAAGT TAACATAGTG AGCATTGTTA CAAGAAGAGT AAGCAATACC 9420

TTGGAAAAAA AAGAATACAT TGATAATATA CCTGTAACGA GAATCCCACC AAGCGGAACT 9480

GGAATGCTCG TTGAATACAT TTTTTGCTTA TTATTGATAT TCTATCTTTT GCTTAATCAT 9540

AAGAAGTATG ATGTAATTCA TTGCCACGCT TCCTCTATTT TTGGTCTTTC TTGTTCCTTA 9600

GTTGGATTTC TACTCAAAAA GAAAGTGATT ATTAAGATCT CTACTAATGG TGAAATTGGC 9660

TCAATGCAGA GTTCGTCTAT AAAAAGATGT ATTTCACGGT TTCTATCAAA ATATTGTACC 9720

TATATAGCGT TAAATTCTGA GGGCTATTCT GAAGTAAATT GTTATTTATC TAAAGCTCAT 9780

AAATGTATTA TTCCCAATGG AATTGATTTC AATATCAACC ATGAAAAAAA TTTGGAATTG 9840

GCCACTGTGA TTAGAAAAAA ATTACATAAT ATGTATGGGG AAGATATATA CATATGTGTT 9900

TTTGTCGGTC GTTTTGTTAC AAGAAAAGGG ATAAGGGAAA TATATGAATG TGGTGCAGAC 9960

CTGACTGCTA ATGGTAAAAA AATCATATTC GTGTTAATTG GTGATTCGGA ATTACAACGT 10020

GATGCTATCA ATTTCGAATA TGATCATCTT CATAATTTCT ATATCGCTGG TAGGAAAAAA 10080

AATGTGTTCC CCTATTTGAT TGCTGGAGAT TTATTCATTT CACCATCACA CAAAGAAGGT 10140

TTGCCAAACA CTGTCCTTGA AGCTCTTTCC GTTGGCAAGG TTTGTATTCT TTCAGATATA 10200

CTTCCACATC AAGAGATTGC TTATGAACAT CCTAAAAACG TCATACTTTT TAAAACAGGA 10260

GACGCTCAGG AGTTGAAGCA AACTATAATA AATTATCTGG AATTAAATCC TCATAAATCA 10320

AGTTTGCATA GTTATGTGCA GATTGAGGAC AATGTCTTAT TAGACAAATA TTCTATCAGT 10380

The initial orf9 of orf1 stops

TGTATAGCGG AACAATATTG TCTACTTTAC AAGGACAAGT CTA ATGCCTC G TGATGATTA 10440

TAAAATATAT ATATCTATAG TTTCCCATAA TCACGGTGAG TTAATAAAGA ATTTAGATTG 10500

TATTAAGAAT TTATCTCAAG AGTTCATAGT GGTTATTAAG AATAACTGTT ATGATGAAGT 10560

ATTATTGGAC TATTTAAAAG ATAGCAATGC CTATCTCATC AATGAAGAAT ATAATAAAGG 10620

ATTTGGCTAT AATAATAATG TCGTTTTTAG TTATTGTCGT TTGCAACTAA ATATGCAACC 10680

TTCCGATTAT TTTATAGTAC TTAATCCAGA TTTGGTCATC AAAAACTCGT CTATCAAAGA 10740

GTTGGTTAAC AAAATGCATT GCAGCAATGT CAAAGCTGCA ACTATTAACC TTTACCTAGA 10800

TGATAAACTT CATGACTATG ATTATTCTGT GAGGCATTTT CCCCGCTTAT ATGACTTTAT 10860

TTTATCTTTA GTATTAGGTA AGAATAAAAC TAAAATAGAC AAATCAATAA TTACCAATCC 10920

AACAGAAGTA GATTGGTGTG CAGGTTCTTT TATGGCTTTA AGTAGTCATG TTTATCAATC 10980

CATTCAAGGA TTTGATACAG GGTTATTTTA TGTATTGCGA GGATATCGAT CTTTGTTTTC 11040

Orf10 stops

GTTTAAAGAA AAAAAATATT GGAATAATTT ATTATCC TGAGATTACAGGC ATTCACAAAG 11100

CTCAGCATAA AAATAGAAGT TTTTTTCGAA ACATTTTATT TGGCATGTTA AAAGTGTATT 11160

CAGATTTATG TTTGCTAAAA ACGGGTTAAT TAGGAATAAG TCCAATATTT GTCACTAAAG 11220

ATTGATTTGT GCTTAGAAAG AAAAATTAAA CCAGAGTCTT TTAGATATCT TTTAAAGCAG 11280

TATATGCTCT ATCCCCCCCT GACAGGAGTA AACAATGTCA AAGCAACAGA TCGGCGTCGT 11340

CGGTATGGCA GTGATGGGGC GCAACCTTGC GCTCAATATC GAAAGCCGTG GTTATACCGT 11400

CTCTATTTTC AACCGTTCCC GTGAGAAAAC GGAAGAAGTG ATTGCCGAAA ATCCGGGCAA 11460

GAAACTGGTT CCTTACTATA CGGTGAAAGA ATTTGTTGAA TCTCTGGAAA CGCCTCGTCG 11520

CATCCTGTTA ATGGTGAAAG CAGGTGCAGG CACGGATGCT GCTATTGATT CTCTCAAGCC 11580

ATACCTCGAT AAAGGTGACA TCATCATTGA TGGCGGTAAC ACCTTCTTCC AGGATACTAT 11640

CCGTCGTAAC CGTGAGCTTT CTGCCGAAGG CTTTAACTTC ATTGGTACCG GTGTCTCCGG 11700

TGGTGAAGAA GGCGCGCTGA AAGGTCCTTC CATTATGCCT GGTGGGCAGA AAGAAGCCTA 11760

TGAACTGGTT GCACCGATCC TGACCAAAAT CGCCGCAGTA GCTGAAGACG GTGAGCCATG 11820

CGTTACCTAT ATTGGTGCCG ATGGCGCAGG TCACTATGTG AAGATGGTTC ACAACGGTAT 11880

TGAATACGGC GATATGCAGC TGATTGCTGA AGCCTATTCT CTGCTTAAAG GTGGCCTGAA 11940

TCTTTCCAAC GAAGAACTGG CGCAGACGTT TACCGAGTGG AATAACGGTG AACTGAGCAG 12000

CTACCTGATC GACATCACCA AAGATATCTT CACCAAAAAA GATGAAGACG GTAACTACCT 12060

GGTTGATGTG ATTCTGGATG AAGCAGCAAA CAAAGGTACG GGCAAATGGA CCAGCCAGAG 12120

CGCGCTGGAT CTCGGAGAAC CGCTGTCGCT GATTACCGAG TCTGTGTTTG CACGTTATAT 12180

CTCTTCTCTG AAAGATCAGC GCGTTGCCGC ATCTAAAGTT CTCTCTGGTC CGCAAGCTCA 12240

GTCAGCAGGC GACAAGGCTG AGTTCATCGA AAAAGTTCGC CGTGCGCTGT ATCTGGGCAA 12300

AATCGTTTCT TACGCTCAGG GCTTCTCTCA GCTGCGTGCT GCGTCTGAAG AATACAACTG 12360

GGATCTGAAC TACGGCGAAA TCGCGAAGAT TTTCCGTGCT GGCTGCATTA TCCGTGCGCA 12420

GTTCCTGCAG AAAATCACCG ATGCATATGC CGAAAATCCG CAGATCGCTA ACCTGCTGCT 12480

GGCTCCGTAC TTCAAGCAAA TTGCCGATGA CTATCAGCAG GCGCTGCGCG ATGTCGTCGC 12540

TTACGCAGTA CAGAACGGTA TCCCGGTTCC GACCTTCGCC GCTGCGGTTG CCTATTATGA 12600

CAGCTACCGT GCCGCTGTTC TGCCTGCGAA CCTGATCCAG GCCCAGCGTG ACTA 12654

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 O139 type, it is characterized in that: its wzx sequence is that the Nucleotide of 7016 to 8236 bases among the SEQ ID NO:1 or wzy sequence are the Nucleotide of 4922 to 6160 bases among the SEQ IDNO:1 or have one or more Nucleotide are inserted, lack or replaced to above-mentioned sequence, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O139 type.

2, a kind of oligonucleotide of the O-antigen high special to intestinal bacteria O139 type, it is characterized in that, the oligonucleotide that comes from the wzx gene of doing is right: the Nucleotide of 7219 to 7236 bases among the SEQ ID NO:1 and the Nucleotide of 7708 to 7725 bases, the Nucleotide of 7712 to 7729 bases among the SEQ ID NO:1 and the Nucleotide of 8100 to 8117 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 5150 to 5167 bases among the SEQID NO:1 and the Nucleotide of 5393 to 5410 bases, the Nucleotide of 5344 to 5362 bases among the SEQID NO:1 and the Nucleotide of 6046 to 6063 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 O139 type of 2.

4, the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O139 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.