CN1256433C - Nucleotide peculiar to 0-antige of 036 type bacillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O36. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O36 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 15248 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the oligonucleotides of oligosaccharide unit processing genes such as wzx genes or genes with the similar functions of wzx and wzy genes or genes with the similar functions of wzy stemmed from the O-antigen gene clusters of Escherichia coli O36, and glycosyl transferase gene orf8. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O36 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Escherichia coli O36 in human bodies and environment.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O36

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster among the intestinal bacteria O36 (Escherichia coli O36), particularly relate among the intestinal bacteria O36 oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O36 in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Intestinal bacteria O36 is a kind of pathogenic bacterium, infect birds, cause the acute septicemia of intestines [Cheville NF, Arp LH.1978, " Comparative pathologic findings ofEscherichia coli infection in birds " J Am Vet Med Assoc.1 of birds; 173 (5 Pt2): 584-7], therefore need the method that can fast, accurately detect intestinal bacteria 036.

The lipopolysaccharides that is positioned at the intestinal bacteria surface is the morbific inducements of intestinal bacteria, and O-antigen is lipopolysaccharides outermost layer structure, is the target of immune system recognition and the site of phage absorption.The antigenic disappearance of O-can cause the serum sensitivity of many pathogenic agent, perhaps seriously undermines the virulence [Frank et al (1987) " The function of antibody and complement in the lysis of bacteria " .RevInfect Dis 177:1750-1753.Pluschke G et al " Role of the capsule andthe O-antigen in resistance of O18:K1Escherichia coli tocomplement-mediated king " .J Bacteriol 42:907-913] of pathogenic agent.Intestinal bacteria are kinds, and the bacterial strain in planting is generally identified by O-antigen and H-antigen (sometimes by K-antigen).Wherein O-antigen has the height diversity, intestinal bacteria have 166 kinds of different O-antigens, the antigenic variation of O-may be colibacillary origin and keep its multifarious major cause [Reeves, P.R (1992) " Variation in antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516].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 o-antigen transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In intestinal bacteria, Shigellae and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichiacoli O111 and Salmonella enterica O35 gene clusters:gene clusters encodingthe same colitose-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 o-antigen 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 ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of S.enterica majors erogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiologicalinvestigation of an outbreak of Hemolytic-Uremic Syndrome caused bydry fermented sausage contaminated with Shiga-like toxin producingEscherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.and Reeves of a supposition, P.R. (1995) " Sequence and analysis of 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.

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O36.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O36, is the special Nucleotide that comes from o-antigen transhipment enzyme gene, pol gene and glycosyltransferase gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O36.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O36: transhipment enzyme gene 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 orf8, orf10, orf11, orf12 gene; Sugar synthesis path gene comprises rmlB, rmlD, rmlA, rmlC; The gene of two Unknown Function comprises orf5, orf7.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.

Another purpose of the present invention has provided oligonucleotide, and they come from the gene of coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O36 respectively, comprises the wzx gene or with wzx the gene of identity function is arranged; Come from the gene of coding polysaccharase, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from the gene of encoding glycosyl transferring enzyme, comprise orf8, orf10, orf11, off12 gene.They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O36; Especially the gene and the gene of polysaccharase and the oligonucleotide of orf8 gene that come from coding transhipment enzyme listed in the table 1, they are high specials to the O-antigen of intestinal bacteria O36, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O36.The above-mentioned oligonucleotide that another object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect O-antigen and detection and identification of escherichia coli O36 with identification of escherichia coli O36 by these methods.

A further object of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O36.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 to the Nucleotide of the O-antigen-specific of intestinal bacteria O36 it is the isolating Nucleotide shown in SEQ IDNO:1,15248 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 O36 is characterized in that it comprises called after rmlB, rmlD, rmlA, rmlC, orf5, wzx, orf7, orf8, wzy, orf10, orf11,12 genomic constitutions of orf12 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O36 is characterized in that the gene that has high degree of specificity in the described gene comprises: transhipment enzyme gene 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 orf8, orf10, orf11, orf12 gene.Wherein said transhipment enzyme gene is the Nucleotide of 5982 to 7496 bases among the SEQ ID NO:1; Described pol gene is the Nucleotide of 9583 to 10659 bases among the SEQ ID NO:1; Described orf8 gene is the Nucleotide of 8631 to 9515 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 10679 to 11587 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 11590 to 12738 bases among the SEQ ID NO:1; Orf12 is the Nucleotide of 12758 to 13783 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O36 is characterized in that it also comprises the oligonucleotide that comes from described wzx gene or wzy gene or glycosyltransferase gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to intestinal bacteria O36, it is characterized in that, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 6084 to 6101 bases among the SEQ ID NO:1 and the Nucleotide of 7138 to 7155 bases, the Nucleotide of 6259 to 6277 bases among the SEQ ID NO:1 and the Nucleotide of 6967 to 6984 bases; The oligonucleotide of the described wzy of coming from gene is to being: the Nucleotide of 9697 to 9714 bases among the SEQ ID NO:1 and the Nucleotide of 10570 to 10587 bases, the Nucleotide of 9814 to 9832 bases among the SEQ ID NO:1 and the Nucleotide of 10428 to 10445 bases; The oligonucleotide of the described orf8 of coming from gene is to being: the Nucleotide of 8656 to 8673 bases among the SEQ ID NO:1 and the Nucleotide of 9330 to 9347 bases, the Nucleotide of 8758 to 8775 bases among the SEQ ID NO:1 and the Nucleotide of 9475 to 9492 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O36 is in the application that detects 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 O36 is providing the O-antigen of expressing intestinal bacteria O36 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 O36 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 O36 is characterized in that, comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O36 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 O36 type bunch: with the genome of intestinal bacteria O36 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

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

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

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O36 type;

(6) screening of specific gene: at wzx, wzy and the orf8 gene design primer in the O-antigen gene of intestinal bacteria O36 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy and orf8 gene pairs intestinal bacteria O36 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O36, 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 O36.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O36 is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O36 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 3ul10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, 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 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene among the pcr amplification intestinal bacteria O36 bunch: with the genome of intestinal bacteria O36 is that template is passed through its O-antigen gene of Long pcr amplification bunch; At first according to the galF gene design upstream primer (5 '-ATT GTG GCT GCA GGG ATC AAAGAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCGCGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the ExpandLong Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of sex change were 10 seconds then, 61 ℃ of annealing 30 seconds, 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product; Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(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; Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction; Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water; In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ were reacted 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 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and this mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged; Use the dehydrated alcohol precipitation of the 3M NaAc (pH 5.2) 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-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, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG, 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, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O36;

(4) to the cloning and sequencing in the library: from the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage, carry out backward sequencing by the sequence that will interrelate again and survey logically obtaining remaining 20% sequence, thereby obtain all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical ResearchCouncil) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O36, the quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O36 is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base; Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O36 bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 12 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 O36 at last;

(6) screening of specific gene: at wzx, wzy, the orf8 gene design primer in the O-antigen gene of intestinal bacteria O36 bunch; Respectively designed two pairs of primers in each gene, the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity; Is that template is carried out PCR with these primers with the intestinal bacteria of 166 kinds of serotypes and the genome of 43 strain Shigellaes, all primers all obtain positive findings in intestinal bacteria O36, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, so wzx, wzy, orf8 gene pairs intestinal bacteria O36 and O-antigen thereof all are high specials.

(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 O36 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 6 pairs of oligonucleotide, the Nucleotide of 6084 to 6101 bases among the SEQ ID NO:1 and the Nucleotide of 7138 to 7155 bases, the Nucleotide of 6259 to 6277 bases among the SEQ ID NO:1 and the Nucleotide of 6967 to 6984 bases, the Nucleotide of 9697 to 9714 bases among the SEQ ID NO:1 and the Nucleotide of 10570 to 10587 bases, the Nucleotide of 9814 to 9832 bases among the SEQ ID NO:1 and the Nucleotide of 10428 to 10445 bases, the Nucleotide of 8656 to 8673 bases among the SEQ ID NO:1 and the Nucleotide of 9330 to 9347 bases, the Nucleotide of 8758 to 8775 bases among the SEQ ID NO:1 and the Nucleotide of 9475 to 9492 bases, carry out the PCR reaction, 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 6 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 6 pairs of primers reaction.Illustrate that these 6 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O36 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 O36, its complete sequence shown in SEQ ID NO:1,15248 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 O36 by method of the present invention, as described in Table 3, it it comprise called after rmlB, rmlD, rmlA, rmlC, orf5, wzx, orf7, orf8, wzy, orf10, orf11,11 genome Chengdu of orf12 are between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O36, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf8, orf10, orf11, orf12 gene.Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises rmlB, rmlD, rmlA, rmlC gene; The gene of two Unknown Function comprises orf5, orf7.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 o-antigen 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 special, and the o-antigen that the present invention relates to transhipment enzyme gene, pol gene and glycosyltransferase gene are special to the O-antigen of intestinal bacteria O36.

The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O36 is provided or the gene of identity function and wzx gene is arranged or with wzx the oligonucleotide of gene of identity function and the oligonucleotide that glycosyltransferase gene comprises orf8, orf10, orf11, orf12 gene are arranged with wzy, they are any one section oligonucleotide in these genes.But, be to list in the wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O36 in the table 1 or the gene, wzx gene of identity function arranged or the oligonucleotide of gene of identity function is arranged to right preferentially with the oligonucleotide that comes from the orf8 gene with wzx with wzy by usefulness.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O36 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to intestinal bacteria O36 and their O-antigen.

The separation and the authentication method of the Nucleotide of described O-antigen-specific to intestinal bacteria O36 comprise the steps: 1) genomic extraction; 2) the O-antigen gene among the pcr amplification intestinal bacteria O36 bunch; 3) structure in O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis finally obtain the structure of O-antigen gene bunch; 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 described herein, " oligonucleotide " mainly is meant gene, the gene of coding polysaccharase and one section nucleic acid molecule in the encoding glycosyl transferase gene of the coding transhipment enzyme that derives from the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes.Especially come from wzx gene (nucleotide position is 5982 to 7496 bases from SEQ ID NO:1), wzy gene (nucleotide position is 9583 to 10659 bases from SEQ ID NO:1) and the oligonucleotide that comes from the orf8 gene (nucleotide position is 8631 to 9515 bases from SEQ ID NO:1) all are high specials to intestinal bacteria O36.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function are arranged; Come from pol gene, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from glycosyltransferase gene, comprise orf8, orf10, orf11, orf12 gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from transhipment enzyme gene, come from pol gene and come from the combination of the oligonucleotide in the glycosyltransferase gene.

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) coding transhipment enzyme gene, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged, comprise the wzy gene or the gene of identity function is arranged with wzy with wzx.(iii) the encoding glycosyl transferase gene comprises orf8,0rf10, orf11, orf12 gene.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O36.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.

The present inventor considers following situation: when single 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 and comes from that coding transhipment enzyme gene comprises the wzx gene or have the gene of gene, the coding polysaccharase of identity function to comprise the wzy gene with wzx or with wzy the oligonucleotide of gene of identity function and the oligonucleotide that the encoding glycosyl transferase gene comprises orf8, orf10, orf11, orf12 gene are arranged.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O36.

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 coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf8, orf10, orf11, orf12 gene.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 O36.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 and be used to detect the many to oligonucleotide of the inventive method, many here is that the gene that comes from coding transhipment enzyme comprises the wzx gene or with wzx the gene of identity function arranged to oligonucleotide; The gene that comes from the coding polysaccharase comprises the wzy gene or with wzy the gene of identity function is arranged; The gene that comes from the encoding glycosyl transferring enzyme comprises orf8, orf10, orf11, orf12 gene.This cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprise orf8, orf10, orf11, orf12 gene.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O36.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from the wzx gene or the gene of identity function and wzy gene are arranged or have the gene of identity function and glycosyltransferase gene to comprise on the sequence of orf8, orf10, orf11, orf12 gene with wzy with wzx 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 oligonucleotide mixture to detect mixed base because of so that detection specificity 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-.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 O36 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 O36 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 O36 in the LB of 5mL substratum, centrifugal collecting cell.With 500ul50mM 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, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution 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, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene among the pcr amplification intestinal bacteria O36 bunch

With the genome of intestinal bacteria O36 is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF gene design upstream primer (5 '-ATT GTGGCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of BoehringerMannheim 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, 61 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

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

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares 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 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 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 O36 with the EcoRI enzyme.

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

From the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is surveyed logical obtaining by backward sequencing and with some sequence again, obtains all sequences of O-antigen gene bunch at last.

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 O36 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O36 is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O36 bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 12 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 O36 at last, as shown in table 3.

By retrieving and comparing, find orf1, orf2, orf3 and orf4 have very high homogeny (identity) with four synthase genes of the dTDP-rhamnosyl of the synthetic rhamnosyl precursors of many bacterium, wherein: the RmlB of orf1 and Shigella bogdii has 98% homogeny in 353 amino acid, 99% similarity; The RmlD of Orf2 and Shigella bogdii has 97% homogeny in 299 amino acid, 98% similarity; The RmlA of orf3 and Shigella bogdii has 97% homogeny in 291 amino acid, 98% similarity, the RmlC of Orf4 and Shigella bogdii has 63% homogeny in 171 amino acid, 75% similarity, illustrate that they all have high homology, can determine that orf1, orf2, orf3, Orf4 are respectively rmlB gene, rmlD gene, rmlA gene, the rmlC gene of synthetic dTDP-rhamnosyl, therefore difference called after rmlB, rmlD, rmlA, rmlC.O-antigenic structure the unknown of intestinal bacteria O36 is a kind of monose in the O-antigen of intestinal bacteria O36 but above result shows rhamnosyl.In genbank, seek conservative functional domain, find that orf5 and Coenzyme F420-reducinghydrogenase have 24% homogeny, 45% similarity; Because do not know the antigenic structure of the O-of intestinal bacteria O36, so the Unknown Function of orf5, called after orf5 temporarily.The O-antigen transhipment enzyme of Orf6 and ShigelLa flexneri2a str.301 has 21% homogeny, 42% similarity in 418 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf6 has 14 potential transmembrane domains, it and many Wzx protein similars, and about 40 amino acid whose conservative motifs are arranged at the proteic aminoterminal of Wzx, so can determine orf6 is the wzx gene, called after wzx.The conservative hypothetical protein of Orf7 and Bacteroides thetaiotaomicron VPI-5482 has 32% homogeny respectively, 51% similarity in 386 amino acid; Therefore can not determine the function of orf7, temporarily called after orf7.Seek conservative functional domain in genbank, find that orf8 is similar to the glycosyltransferase family of pfam00535, the E value is 2 * e ^-07Relatively find by carrying out blast, the glycosyltransferase of orf8 and Clostridium acetobutylicum has 32% homogeny respectively in 449 amino acid, 53% similarity, illustrate higher homology is arranged between them, therefore infer that orf8 also is a glycosyltransferase gene, temporarily called after orf8.Blast comparison shows that orf9 and many Wzy protein similars, for example and Shigella boydii in 350 amino acid, 22% homogeny is arranged, 41% similarity.Learn that by the Eisenberg algorithm orf9 has 9 potential transmembrane domains in addition, similar secondary structure is arranged, have the feature of typical O-antigen polysaccharase, so determine that orf9 is the wzy gene, called after wzy to other O-antigen polysaccharase.Relatively find by carrying out blast, the glycosyltransferase of the rhamnosyl of orf10 and Shigella dysenteriae has 42% homogeny, 65% similarity, and illustrating has higher homology between them, therefore infer that orf10 also is the glycosyltransferase gene of rhamnosyl, temporarily called after orf10.The WbnE albumen of the albumen of Orf11 genes encoding and Escherichia coli has kinship, and both have 42% homogeny and 65% similarity in 392 amino acid.WbnE albumen is a glycosyltransferase, therefore infers that orf11 also is the gene of a glycosyltransferase, temporarily called after orf11.Blast relatively finds; the acetyltransferase of orf12 and Lactobacillus plantarumWCFS1 has 21% homogeny and 32% similarity in 354 amino acid; therefore infer that orf12 also is an acetyltransferase gene, temporarily called after orf12.

Embodiment 6: the screening of specific gene

At wzx, wzy in the O-antigen gene of intestinal bacteria O36 bunch and the orf8 gene design primer of a glycosyltransferase, the position of these genes in nucleotide sequence sees Table 1.

Transhipment enzyme gene, pol gene and orf8 gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O36 in table 1, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer (5 '-TTC ATC CTA AACTCC TTA TT-3 ') and (5 '-TAA TCG CAG GGG AAA GCA GG-3 '), extract genome then from the intestinal bacteria of 166 kinds of serotypes, method as previously mentioned.With this to primer from the colibacillary genome of 166 kinds of serotypes PCR with identification of escherichia coli and detect its genomic quality.

Table 2 is intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen 166 kinds of serotypes of specific gene, and for the convenience that detects, we are divided into one group, 13 groups altogether with their every 12-19 bacterium.All list in the table in their source.

In the 2nd group, contain the genomic dna of intestinal bacteria O36 as positive control.In the 13rd group is the genomic dna that does not contain intestinal bacteria O36, as negative 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 2 minutes, 95 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get l0ul PCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy, orf8 gene, each gene all has two pairs of primers detected, and every pair of primer has obtained except be PCR in the 2nd group after the correct band of expection size, and the correct band of any size does not all increase in other groups.So wzx, wzy, orf8 gene pairs intestinal bacteria O36 and O-antigen thereof all are high specials.

At last, from intestinal bacteria O36, screen gene by PCR: wzx, wzy, orf8 gene to the O-antigen high special of intestinal bacteria O36.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O36, 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 O36.These all oligonucleotide all can be used for the intestinal bacteria O36 in the human body and environment rapidly and accurately, and can identify their O-antigen.

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 O36 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 6 pairs of oligonucleotide, the Nucleotide of 6084 to 6101 bases among the SEQ ID NO:1 and the Nucleotide of 7138 to 7155 bases, the Nucleotide of 6259 to 6277 bases among the SEQ ID NO:1 and the Nucleotide of 6967 to 6984 bases, the Nucleotide of 9697 to 9714 bases among the SEQ ID NO:1 and the Nucleotide of 10570 to 10587 bases, the Nucleotide of 9814 to 9832 bases among the SEQ ID NO:1 and the Nucleotide of 10428 to 10445 bases, the Nucleotide of 8656 to 8673 bases among the SEQ ID NO:1 and the Nucleotide of 9330 to 9347 bases, the Nucleotide of 8758 to 8775 bases among the SEQ ID NO:1 and the Nucleotide of 9475 to 9492 bases, carry out the PCR reaction, 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 6 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 6 pairs of primers reaction.Illustrate that these 6 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O36 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 Microbiology1993,7:239-252).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 O36 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ ID NO:1) according to the O-antigen-specific to intestinal bacteria O36 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 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O36, has listed the structure of the O-antigen gene bunch of intestinal bacteria O36 in table, altogether by 12 genomic constitutions, and each gene box indicating, and in square frame, write the title of gene.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 location tables of the gene in the O-antigen gene bunch of intestinal bacteria O36, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O36, 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 bacterium: 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 O36

<160>1

<170>PatentIn version 3.1

<210>1

<211>15248

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaactgcg cgtgaagcgt 120

caactactgg cggaagttca gtccatctgc ccgccgggcg tgaccattat gaacgtacgt 180

cagggcgaac ctttaggttt aggtcactcc attttgtgtg cacgacccgc cattggtgac 240

aacccatttg tcgtggtgct gccagacgct gtgatcgacg acgccagtgc cgatccgctg 300

cgttacaacc ttgctgccat gattgcgcgt ttcaatgaaa cgggccgtag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtta ttcagaccaa agaaccactg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgcaga tatttggccg 540

gaacttgaac gcactcagcc aggtgcatgg ggacgtatcc aactgacaga tgccattgcc 600

gaactggcga aaaaacagtc tgttgatgca atgctgatga ctggtgacag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaggcattt gtgaagtatg gactacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg tattgagaaa ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa atcataacgg cagtgaagat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag ggcggattgg taagacaatt 960

agcgtttgaa tttttcgggt taagcgcgag tgggtaacgc tcgctacatc gtaggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

tcaaactaag agccgctaat ttcacagcat gctctgaagt aatatggaat aaaaaagtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc agtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgat tctgaacgct atgtttttga acatgcggat atttgcgatg 1320

ctgctgcaat ggcgcggatt tttgctcagc atcagccgga tgcagtgatg cacctggctg 1380

ctgaaaagcc atgtggatca attacaggcc ctgcggcatt tattgaaacc aatattgttg 1440

gtacttatgt ccttttggaa gcggctcgca attactggtc tgctcttgat ggcgacaaga 1500

aaaatagctt ccgttttcat catatttcta ctgacgaagt ctatggcgat ctgcctcatc 1560

ctgacgaagt aaataataaa gaacaattac ccctctttac tgagacgaca gcttacgcgc 1620

ctagtagtcc ttattccgca tcaaaagcat ccagcgatca tttagtccgt gcgtggaaac 1680

gtacctatgg tttaccgacc attgtgacta actgttcgaa taactacggt ccttatcact 1740

ttccggaaaa attgattcca ctagtaattc ttaatgctct ggaaggtaag gcattaccta 1800

tttatggcaa aggggatcaa attcgtgact ggctgtatgt tgaagatcat gcgcgtgcgt 1860

tatatatcgt cgtaaccgaa ggtaaagcgg gtgaaactta taacattggt ggacacaacg 1920

aaaagaaaaa catcgatgta gtgctcacta tttgtgattt gttggatgag attgtaccga 1980

aagagaaatc ttaccgtgag caaattactt atgttgccga tcgcccggga cacgatcgcc 2040

gttatgcgat tgatgcagag aagattagcc gcgaattggg ctggaaaccg caggaaacgt 2100

ttgagagcgg gattcgtaaa acggtggaat ggtacctgtc caatacaaaa tgggttgata 2160

atgtgaaaag tggtgcttat caatcgtgga ttgaacagaa ctatgagggc cgccagtaat 2220

gaatattctc cttttcggca aaacagggca ggtcggttgg gaactacagc gtgctctggc 2280

acctttgggt aatttgattg ctcttgatgt tcactccact gattattgtg gtgattttag 2340

taatcctgaa ggtgtagctg aaaccgtcaa aaaaattcgc cctgatgtta ttgttaatgc 2400

tgcggctcat accgcagtag ataaggctga gtcagaaccc gaatttgcac aattactcaa 2460

tgcgaccagc gttgaatcaa ttgcaaaagc ggctaatgaa gttggggcct gggtaattca 2520

ttactcaact gactacgtat ttccggggac cggtgaaata ccatggcagg aggcggatgc 2580

aaccgcaccg ctaaatgttt acggtgaaac caagttagct ggagaaaaag cattacaaga 2640

gcattgtgcg aagcacctaa ttttccgtac cagctgggtc tatgcaggta aaggaaataa 2700

cttcgccaaa acgatgttgc gtctggcaaa agagcgtgaa gaattagccg ttattaatga 2760

tcagtttggt gcgccaacag gtgctgaact gctggctgat tgtacggcac atgcaattcg 2820

tgtggcactg aataaaccag aagtcgcagg cttgtaccat ctggtagcca gtggtaccac 2880

aacctggcac gattatgctg cgctggtttt tgaagaggcg cgcaaagcag gcattcccct 2940

tgcactcaac aagctcaacg cagtatcaac aacagcctat cctacaccag ctcgtcgtcc 3000

gcataactct cgccttaata cagaaaaatt tcagcagaac tttgcgcttg ttttgcctga 3060

ctggcaggtt ggtgtgaaac gcatgctcaa cgaattattt acgactacag caatttaata 3120

gtttttgtat cttgttcgtg atggtggaac aagataaatt aaaaggaatg atggaatgaa 3180

aacgcgtaaa ggtattattt tagcgggtgg ttctggtaca cgtctttatc ctgtgactat 3240

ggctgtcagt aaacagctat tacctattta tgataaaccg atgatctatt atccgctctc 3300

tacactgatg ttggcgggta ttcgcgatat tctaattata agtacgccac aggatactcc 3360

tcgttttcaa caactgttgg gtgacgggag ccagtggggc ctgaatcttc agtacaaagt 3420

gcaaccaagt ccagatggtc ttgcgcaagc atttatcatc ggtgaagaat ttatcggtgg 3480

tgatgattgt gctttggttc ttggtgataa tattttttac ggtcacgatc tgccgaagtt 3540

aatggatgcc gctgttaaca aagaaagtgg tgcaacggtg tttgcctatc acgttaatga 3600

tcctgaacgc tatggtgtcg ttgagtttga taaaaacggt acggcaatta gcctggaaga 3660

aaaaccgcta cagccaaaaa gtaattatgc ggtaactggg ctttatttct atgataacga 3720

cgttgtagaa atggcgaaaa atcttaagcc ttctgcccgc ggtgaactgg aaattaccga 3780

tattaaccgt atctacatgg aacaagggcg tttatctgtt gccatgatgg gacgtggata 3840

tgcatggtta gacacgggga cacatcaaag cctgattgag gcaagcaact tcattgctac 3900

aattgaagag cgtcaagggt tgaaagtttc ctgtccagaa gaaattgctt accgtaaagg 3960

gtttatcaat gctgagcagg tgaaaatatt agctgaaccg ttgaagaaaa atgcttatgg 4020

tcagtacctg ctaaaaatga ttaaaggtta ttaataaaat gaacgtaatt aaaacagaga 4080

ttccagacgt attaattttc gaaccgaaag tttttggtga cgagcgcggt ttctttttcg 4140

agagctttaa ccagaaggtt tttgaggaag ctgtaggccg caaagttgaa tttgtccagg 4200

ataaccattc gaagtcttgt aaaggtgttt tacgcggact gcattatcag ttagaacctt 4260

atgcacaagg aaagttggtg cgctgcgttg ttggtgaagt ttttgacgta gctgttgata 4320

ttcgtaaatc gtcgcctacc tttggtaaat gggttggggt gaatttatct gctgagaata 4380

agcggcaatt gtggatccct gagggatttg cgcatgggtt tttggtgctg agcgacgagg 4440

cagaatttgt ttataagaca aacaattatt attatcaaaa gcatgaaaga agcattcttt 4500

ggaacgataa aattctcaac gtagaatggc catttacttc taacttaatt ctttcttcaa 4560

aagatatgag cgcgtcatta ttcactgttg cagaaaaatt ttaattggtt ttggaaataa 4620

aatgtctata caaaatgtaa tagataatgg ttactgcgtc ggatgtggtg cttgtaaatt 4680

ggctcttgga gagtcagtgg aaataaaaat gtatgaagat ggcttttata atgcttcgat 4740

aaaacctaat gcaaatattg aaattgccga gaaaatttgt ccattttctg atagttcaac 4800

aaatgaaact atcttaggcg catcacttta tggagataaa aaatttgaca agcgtgtagg 4860

atattttgac gacatttata ttgggtatgt agttgataat caggaccgac taacttccag 4920

ttcaggtgga cttactacct ggtttgctaa aaagttatta ataaataaag aggttgatgc 4980

ggtaattcat gtagggcatg ggacgcatat gttggactac attattactg agaatatttt 5040

agatcttgat ttacagaata ataaaaaatc cagatattat cctgtaagtt tttctgaatt 5100

gataaataaa ataaaccaat cagataaaaa ataccttttc attggcattc catgttttgt 5160

taaatcaata agattagcgc agaaagaagg aatggttcag aatattaaat ttgtgatttc 5220

attactctgc ggacatatga agtccaaaga tttcgctacg agtcttgcgt ggcagattgg 5280

cgttaaacct gatgatctgg gtagtgtaga ctttcgtgtc aaggaggata ataaaaaagc 5340

aaatgattat aatatagaag ttgaagatac ttgtaataag aaatatatgc atcaaagctc 5400

ttcgttgttt ggcactaatt gggggctagg ctttttcaaa catcatgcat gtgatttttg 5460

tgatgacatt gcaggtgaat tggcggatgc aactttaggt gatgcttggt taccgaagta 5520

tataaatgat tcacaaggta ctaatatact tattgtaaga aacgatgttt taaataaatt 5580

acttgaacaa tatcaagaag aaatagtgct tgaaactgcg aatgtcgagg atttttatga 5640

aagtcaagct gggaattttc gtaatcgacg tgaagggtta cttgttcgtg tccaaaatga 5700

aaaaaaatgg actcctagaa aaaggttgga aattattaac tccgatattc cattacaacg 5760

tcgtaaattg tatttagttc gtcagaagtt aagtgaacaa agtataaaga aatttcaaat 5820

agctaaaaag ataggttcaa tatattcatt caaattgtta atggttcctg atatttttag 5880

atataaaatg attgagaaga atttaatatc ggcgttaaaa gaaactataa gaatagtggc 5940

gccaagaaaa cttgtaaaaa tattcaaaaa acggtagctt tatgcatagg caagtttttc 6000

gaaacgtatt tctaaatgtg acgacatttt tatttaatgt agttactgga ttatatttaa 6060

ctccattttt gattaaatat ttagggttgg aagtgtatgg tatacttcct ttagcattgt 6120

ttttaacctt ttatattggc gtgataactc aggcccttac ggcatcagta aatcgatttt 6180

ttattgcaaa tttacaaact aataacatta aagaggctaa tgttgttttt aacactgctt 6240

tttgtatcat agttacatat tcggtattgc agtttgctct cctatactat cctatacaaa 6300

atatagacca gtatttttct ataccgacgc atagcagaga tgatgcccgg agattgtttg 6360

aatcagtttt actcagcttc tctcttgcgc ttttaacatc catttttagt gtttcgcttt 6420

ttgcattaaa caggttggat ttgattcaat gcgtccagtt aatcaaaatt attataaagt 6480

tcattaccat aatattattc tttgaacatc attgcatagg tatccagttt gtaggaatag 6540

caatgattct gtcagagtta atagctttaa ttttgacgat ttgtttatgg agaaaattga 6600

caccggaaat aaatttaaaa ctattttact tcagtaaaaa gaaagtagga gagctttcaa 6660

agtttgcaat gtggttgatt attgatcagg ttggatatgt tctttttatt aaaatggatt 6720

tattattggt aaacaaattc tttggggcga aggaatctgg gcgctattca attgcaacgc 6780

aatttagtga tttactaaga tcatttgcag ggcttatggc gggagttctt gcacctgtaa 6840

taatgatact acacgccaag aatgaattag aaagaattgt aaccgttacc aaaacgtttg 6900

tgatgattct atcgctgaca atagcgatac caatttctat aatctgtgta ttttctcaag 6960

agctgattca tttctggcta ggacaagata taaatatcca aactttaatt tggattgtga 7020

cttttccatt aataattaat ttgggcgtct taccattatt ttctataaat gtagcgttta 7080

aaaaagttaa actacctagt gttctaaata tcgtattggc actagttggc gtatttgtct 7140

caattatact tattcgaaat actgaactgg gtttacttgc agtagcggct gggtttggtt 7200

tttccctgac cctcaaaaat gcggtattta tacctattta cgcggcacta aatttaagaa 7260

tagataaatt aacatttcta actgtacatg taagaactat tttatttaca tttttttatg 7320

tatgttttct gattttagtt aagtcaaaac tgtctagtaa tctttacggg tttttagctg 7380

aattaacagt gcttattata tttggcttta taattagtat ctttttttat tctcgggatg 7440

aaaggaagta tattactaat acactttcag ataaattaaa agcgaaggta aaatgaaaaa 7500

taaaattggt attctaaatt ttcaatattc ggatcataat tatggtgcag tcttacaagc 7560

tgttgcacta gaaaatgttt taaaacagct agggaaaaat gctgaacata ttaattttat 7620

tccaaagaag aaaggtaaca aaaatatcaa acaatttata attgaatttt tagcttctat 7680

tggtttaaaa cctttcattt atcgggtatt taacaaaaaa gttattataa aaaataaagt 7740

tgaaggatct gagatttttg aggatttcag gaataaatat ttgaacagaa caaaagcgtt 7800

tcattccctc aatgacctta tggaattaga ggatagttac agttctgtta tcgttggaag 7860

tgatcaagtc tggcgcccca aaatgtactc agaccttaca gaaattgaaa tttatttcct 7920

ttcttttatt tcacagaaaa caaaaaaaat ttcttatgca gcaagttttg gtgttgagcg 7980

atgggaatat gacaaaaatg ataatgtgac acataaagca caaaaatatc tgaacaaatt 8040

taattctatt tctgttagag aaaaatcagg agtagacatt tgtaatagtg tatttgatat 8100

tagagctgag catgttctgg atccaacact attgatcgga cgtgattttt tcgacaatct 8160

tattaaagac aaacaaccat tagttcaaaa gacaataagt tattataagt tagatctttg 8220

tgagtctttt ttagcgggaa tagagcatct tgctaatcta accggctatg agactaataa 8280

tatttactac aaacaactga ataatgggag ttatgaatat tatccagttt tagattggct 8340

aagcatgatt aattcatctt cgataataat aacagactct tttcattgtg tatgcttctc 8400

aattttattt aataaaagct tctattgttg tcttaataaa gaaagaggag catcgaggct 8460

tgaaagttta ttgaatgaac tggggttgca agacagatta atctctgaag aagaactatc 8520

gagaatttat acaatagttg atatcaatta ttctcctgta gaagatattt tgaataagca 8580

caggaaacag tcaatagaat ttttagtttc agcaattgat ggataattaa atgagtcata 8640

taattccagt agttattatg acacggaatg aaggtgctta ccttttgaaa tgtgtccaat 8700

caatcattaa tactgttact ataccctatc atatatatat tattgataac aattcttcaa 8760

cattggagca agaagaagtt ctaaaaatta ttgaatataa gtttaatgat aaagtaaata 8820

ttattagaaa tcgtactaac aggtgggttc tgggtttaaa taagacactt attgaattga 8880

gtaaaaataa agaacaaccc tatttctttt taactgatgg tgatattgac ttttcaagat 8940

gcaccgctaa gccatgttgg ttatcatatt tggttaccca tatgaattct aatgtatctc 9000

tggggaaaat tggcttatct ctcaattggg attacatttc aactacaaaa gaattggcgg 9060

ctgtttatga acaggagaga agtctatata atgaaagtaa aaaaataaat gatttatatg 9120

tgtctcctgt tgatacaaca gcggcattat ttcgatggga ttggtctata gaaggaagac 9180

caagcttata tccagaccat attagatacc ttagaccaga actttattca tgtcgaacac 9240

cattagatat taatgtagag catatgggat ggtataaata ttgtgcagaa tctggagata 9300

caaaaaattt agaggaaaaa atagtctgct tcactatggt tggtggtgat gttaaaaacg 9360

aaattttaga taaggttcgt tttttctata aggttatata taaatcattg tctggtccag 9420

taaaaaaagc atggtatttc aggagatatt attatctttt gaaatatttt ctttttaagg 9480

gcgttcgcag attcgatggt caaaactgtc tttgaaatgt aatgtaatta ttggagtgta 9540

actttgtata tatttcgaag tgagtttgta ctagcgatat ttatttttca tttgttattc 9600

ataatattgg ccttatatgc acgaaaactt gataataagg tattagatta tatatttctt 9660

atattggcaa ctacattagc cttaattatt tcactcttta gacctggctt tggagttgat 9720

gaaccttctt atcttgaggc atttattaat ttcaaaaata atgctgacac atttccattc 9780

gattattcat ttaaagtatt atatgaaata ttatctaata cgggcataca aattgagtat 9840

ttcaataaca cagtcagctc tttatatatt attttactta gtataacggc gatagtaaca 9900

tcgagtaaaa ataataagtg tttttatttc ttgatgttat tgttcagttt tacttcatta 9960

gatctaatgt ttaatgctta caggcaagca tttgcattta tatttattta taactcttta 10020

tttctttttt tgagagaaaa gaaaatcaag ggggctttat tagctgtaat ctcttttggg 10080

tttcattggt cagcagttat cctgatctta ttttattttt tatcgaggtt tcttaagaat 10140

aaatattctt accttatttt acttttatta tttccatttg tattaatctc gatgttttat 10200

ccattaggtt tgatggggat aggatataat gtgttgttat ttcttccttt gaatgaatta 10260

ttcaaagctc atgtgttagc gtatttagaa gttgataaca tttctagcgc gtcgttttat 10320

atacttaatc tctttggtcg actaccattg gctatatcgg tattaacttt ttatgctttt 10380

atactgattt tttacaaaaa aataagaatg aagcgtataa tagcaatgat tgtcttgatg 10440

gctgtttatt gtctagtgtt tatggagatg gcatatagtt ttagaaatta ttattgggtt 10500

ctaccatttt tccctattat tgctttagat tacgcagaat ctaatcaagc gaaaattcat 10560

tcaagaatga taggcattgc tgctcttcat atcttgatag cattgcctac tttttacagt 10620

tcaggtataa actctatgat tttcaatact ggcaattagt attgaatatg gaaaaaaaat 10680

gtgttcatta aaagttagcg ctttactcgt tagttacaat ccagatatta atagattaag 10740

tgaggttctt aatagtattc attctcaagt agatcatcta gttttagttg ataatggttc 10800

aaaaaataaa aatgatatac ttgctctcat tgccgattat gagggggttt atacactgct 10860

taatgataat aatataggtt tagcctctgc tcaaaatgtt ggtattgatt atattattga 10920

caataacctt tccgatttta tcgtgttctt cgaccaagat agtgtattgc agtcaggttt 10980

tattagtgcg ctactcgaaa cgtatactaa tttagtaact cagaatatta aattagcagc 11040

agtaggacca tcatttattg atcctgtaaa taataaacaa tatcctgcga ctgtttatgc 11100

aggaccattt attaaacgag tagatcttga aagaaaacct gtagaagcta ccttcataat 11160

tgcttctgga tgctttgttg ccattgaggc attgaaaaaa attgggaaaa tgaaggatga 11220

attgtttata gactatatcg atgttgaatg gtgtctcaga gctaggagtt taggatacaa 11280

aatatatatt tcacctaaag ctgtaatgaa acatactatt ggagataatc gagtatctat 11340

ctttgggcga acaatatctg ttcattcacc attacgtcga tattatcttg taaggaatag 11400

cttttatatg attagattgg attatatacc aattggatat aaaattagag aaatattttt 11460

taatgtagtt cgtatttgta tcagcttagc tctatctgat gataaaaaaa agatacttca 11520

ttatacaata actgcaatta aagatggtgt tagtaaaaaa ttcggacctt gtaaaaaaat 11580

actctaatta tgatcaaaat agctcatatt caattattgc caatgcttag tggtgttcag 11640

agagtgtgtt tggatgagct ctcaagattg gacaaaaaaa aatatcaaag atacttaatt 11700

tgtaaagaac caggtaaact cagtgatgag gctactgcta ttggcgttca atgtatcttt 11760

cttcccgagc ttcagcgcaa tatcagcttt aagatggact taatagcgtt gtacaaatta 11820

tatcggataa taaagatata taaattcgat attgttcata cacactcttc taagccaggt 11880

gttttagggc gtattgcagc caggctgaat ggtgttaaac tgatacttca cactgtgcac 11940

ggtttttctt ttcctgctgc acaaaatagg ttccaatatt atatattttg gatcatggaa 12000

aaaattggct caatatgtgg agataaatta atatgtctgc ataaagatga tgcagtgatt 12060

gctcaggaaa aattatttat tcataagaat aaaatttcca taattgaaaa tggcgtcgac 12120

actaacaaat tcaagaagag aaataataat gaaatagata aattatctac atattttaac 12180

atagatagaa attcggatgt tgtggtcggg atgataggta gactgtggcc acaaaaaaac 12240

cccatgcttt tattatatgc agcaaaaaat ataatcaaca ataataaaag agtaaaattc 12300

ttgtttgtcg gcgatggtga gttaaaagac tcaatgaacg attatattat tcaaaataaa 12360

cttacccaga atattaccat ttgtggctgg tgtaatgatg ttagttctta tttgaatata 12420

tttgatattt ttgtattacc atctttatgg gagggtatgc ctttggcaat tttagaagcc 12480

gaaagtagtt cgttgccctg tattgtttca aatatcccgg gaaacaggag tataatcagg 12540

catacagttg atggctatct cttttcatta gataacccat ctgaacttga atcttacatt 12600

aatatattaa ttgaggataa agaaagacgt attatcttgg gtaataacgc cagagtcaaa 12660

attcttgaaa gctataaaat agaaaatagg attttgaaat tagatgaact gtatagttca 12720

atttctttaa aagaatagcg ttgtatgttg aggggatatg cggataaaaa tactcgacca 12780

tatgaaaggt gctgctattt tagctgtggt atttattcat tcttcaggtt atcttgctgt 12840

ttctaatata ggaacatttg aatggtacat cggtataata tcgcggcaat ttgttaattt 12900

tgcagtgcct atttttttat ttatatctgg ttttttatct ttttctcgag atatacatga 12960

tacaccatac tatctcaaga aaaaattatt aagaattatt ataccttatc tctcatggtc 13020

ttgtttttac ttctttcttg tattcatatt taataaccaa agtttcgctg tcaaaaatat 13080

tatagctcag ttgattcttg gtacgggaat aggtgtggga tattttgtga tcgttttagt 13140

gcaatttatt atgctgacac cgttaattaa tgcgattaca tctataaaaa tacatattat 13200

gattatatgt ggttttacta tcataggtgt ggggattaat tatatgtctg tgaagattga 13260

ttgcttgaaa ctgttttcac agtttcctta ttccgccatt cctttttttg tatggtatcc 13320

tttttatcat ttgggctttg ttttcaataa gtataatata tcttgttcaa agtggtctaa 13380

tagttccatc ttattttgga ttttatcttt ttcacttaca ttagccattt ctgaagggat 13440

attttggggt tatactggga actattcatt tgctgtgtct caacttaaac tatcatcttt 13500

ggttctctct ttatgcgttt gtatggttgt ttatttttat ctaaaccatg atataaatta 13560

taaatacctt tctcaattag ggacaatgtc ctacgggatt tatcttactc atatgttgtt 13620

tgtttggttc tatcattatc tcttttcgta tacgagtgtg tttataaggc atttagttat 13680

ctcgtgtggc tttatcttta tattaagtac aatatcatca gttaccttag ttttttttat 13740

aagaaaaatt ttggggagat attccgtata tattgtaggg taggtgattg cttgatattt 13800

atactgtttt aagaagaagc ctaatttatc tcaatactgg gcatttcact agagctgtat 13860

tagtatattt gtcacattct catccgtatt tctaatgtat ttttcagagc aggagactta 13920

aatgtcaaag caacagatcg gcgtcgtcgg tatggcagtg atggggcgca accttgcgct 13980

caacatcgaa agccgtggtt ataccgtctc tattttcaac cgttcccgtg aaaagacgga 14040

agaagtgatt gccgaaaatc caggcaaaaa actggttcct tactatacgg tgaaagagtt 14100

tgttgaatct ctggaaacgc ctcgtcgcat cctgttaatg gtgaaagcag gtgcaggcac 14160

ggatgctgct attgattccc tcaaaccata tctcgataaa ggcgacatca tcattgatgg 14220

tggtaatacc ttcttccagg acaccattcg tcgtaatcgt gagctttctg cagaaggttt 14280

taacttcatt ggtaccggtg tttccggcgg tgaagaaggt gcgctgaaag gtccttccat 14340

tatgcctggt gggcagaaag aagcctatga acttgttgcg ccgatcctga ccaaaatcgc 14400

cgcagtggct gaagacgggg agccatgcgt tacctatatt ggtgccgatg gtgcaggtca 14460

ctatgtgaag atggttcaca acggtattga atacggtgat atgcagctga ttgctgaagc 14520

ctattctctc ctgaaaggcg gcctgaatct ctctaacgaa gaactggcac agacctttac 14580

cgagtggaat aacggtgaac tgagtagcta cctgatcgac atcaccaaag atatcttcac 14640

caaaaaagat gaagacggta actacctggt tgatgtgatt ctggatgaag ccgctaacaa 14700

aggtaccggt aaatggacca gccaaagtgc gctggatctc ggcgaaccgc tgtcgctgat 14760

taccgagtct gtgtttgcac gttatatatc ttctttgaaa gatcagcgtg ttgccgcctc 14820

taaagtactg tctggtccgc aaggacagcc agcaggcgac aaagctgagt tcattgaaaa 14880

agttcgccgt gcgctgtatt tgggcaaaat cgtttcttac gcccagggct tctctcagct 14940

gcgtgcggcg tctgaagagt acaactggga tctgaactac ggcgaaatcg cgaaaatttt 15000

ccgtgctggc tgtattatcc gtgcgcagtt cctgcagaaa atcaccgatg cttatgccga 15060

aaatccgcag atcgctaacc tgctgctggc tccgtacttc aagcaaattg ccgatgacta 15120

ccagcaggcg ctgcgtgatg tcgttgctta tgcagtacag aacggtatcc cggttccgac 15180

cttcgccgct gcggttgcct attatgacag ctaccgtgcc gctgttctgc ctgcgaacct 15240

gatccagg 15248

Wzx gene, wzy gene and Orf8 gene and wherein primer and PCR data in the O antigen gene of table 1 intestinal bacteria O36 bunch

Gene	Function	The base position of gene	The forward primer position	The reverse primer position	PCR product length	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzx wzy Orf8	O-antigen transhipment enzyme O-antigen polymerase glycosyl transferase	5982-7496 9538-10659 8631-9515	6084-6101 6259-6277 9697-9714 9814-9832 8656-8673 8758-8775	7138-7155 6967-6984 10570-10587 10428-10445 9330-9347 9475-9492	1089bp 730bp 891bp 632bp 700bp 635bp	0 0 0 0 0 0	53 53 53 55 55 53

The intestinal bacteria of 166 kinds of serotypes of table 2 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, wild-type e. coli	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, O145, 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, D1O, 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 removes the 2nd group of bacterium of intestinal bacteria O36	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d 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 is structural tables of the O-antigen gene bunch of intestinal bacteria O36

galF rmlB rmlD rmlA rmlC orf5 wzx orf7 orf8 wzy orf10 orf11 orf12 gnd

1kb

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O36

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTTC TTGAACTGCG CGTGAAGCGT 120

CAACTACTGG CGGAAGTTCA GTCCATCTGC CCGCCGGGCG TGACCATTAT GAACGTACGT 180

CAGGGCGAAC CTTTAGGTTT AGGTCACTCC ATTTTGTGTG CACGACCCGC CATTGGTGAC 240

AACCCATTTG TCGTGGTGCT GCCAGACGCT GTGATCGACG ACGCCAGTGC CGATCCGCTG 300

CGTTACAACC TTGCTGCCAT GATTGCGCGT TTCAATGAAA CGGGCCGTAG CCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTTA TTCAGACCAA AGAACCACTG 420

GATCGTGAAG GTAAAGTCAG CCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGCTGGACT CAGACATCAT GGCCGTTGGT CGCTATGTGC TTTCTGCAGA TATTTGGCCG 540

GAACTTGAAC GCACTCAGCC AGGTGCATGG GGACGTATCC AACTGACAGA TGCCATTGCC 600

GAACTGGCGA AAAAACAGTC TGTTGATGCA ATGCTGATGA CTGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGCTATAT GCAGGCATTT GTGAAGTATG GACTACGCAA CCTGAAAGAA 720

GGGGCGAAGT TCCGTAAAGG TATTGAGAAA CTGTTAAGCG AATAATGAAA ATCTGACCGG 780

ATGTAACGGT TGATAAGAAA ATCATAACGG CAGTGAAGAT TCGTGGCGAA AGTAATTTGT 840

TGCGAATATT CCTGCCGTTG TTTTATATAA ACAATCAGAA TAACAACGAG TTAGCAATAG 900

GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG GGCGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TAAGCGCGAG TGGGTAACGC TCGCTACATC GTAGGCATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGCATTAATA CCTCTATTAA 1080

TCAAACTAAG AGCCGCTAAT TTCACAGCAT GCTCTGAAGT AATATGGAAT AAAAAAGTGA 1140

AGATACTTGT TACTGGTGGC GCAGGATTTA TTGGTTCTGC AGTAGTTCGT CACATTATAA 1200

ATAATACGCA GGATAGTGTT GTTAATGTCG ATAAATTAAC GTACGCCGGA AACCTGGAAT 1260

CACTTGCTGA TGTTTCTGAT TCTGAACGCT ATGTTTTTGA ACATGCGGAT ATTTGCGATG 1320

Orf1's is initial

CTGCTGCA AT GGCGCGGATT TTTGCTCAGC ATCAGCCGGA TGCAGTGATG CACCTGGCTG 1380

CTGAAAAGCC ATGTGGATCA ATTACAGGCC CTGCGGCATT TATTGAAACC AATATTGTTG 1440

GTACTTATGT CCTTTTGGAA GCGGCTCGCA ATTACTGGTC TGCTCTTGAT GGCGACAAGA 1500

AAAATAGCTT CCGTTTTCAT CATATTTCTA CTGACGAAGT CTATGGCGAT CTGCCTCATC 1560

CTGACGAAGT AAATAATAAA GAACAATTAC CCCTCTTTAC TGAGACGACA GCTTACGCGC 1620

CTAGTAGTCC TTATTCCGCA TCAAAAGCAT CCAGCGATCA TTTAGTCCGT GCGTGGAAAC 1680

GTACCTATGG TTTACCGACC ATTGTGACTA ACTGTTCGAA TAACTACGGT CCTTATCACT 1740

TTCCGGAAAA ATTGATTCCA CTAGTAATTC TTAATGCTCT GGAAGGTAAG GCATTACCTA 1800

TTTATGGCAA AGGGGATCAA ATTCGTGACT GGCTGTATGT TGAAGATCAT GCGCGTGCGT 1860

TATATATCGT CGTAACCGAA GGTAAAGCGG GTGAAACTTA TAACATTGGT GGACACAACG 1920

AAAAGAAAAA CATCGATGTA GTGCTCACTA TTTGTGATTT GTTGGATGAG ATTGTACCGA 1980

AAGAGAAATC TTACCGTGAG CAAATTACTT ATGTTGCCGA TCGCCCGGGA CACGATCGCC 2040

GTTATGCGAT TGATGCAGAG AAGATTAGCC GCGAATTGGG CTGGAAACCG CAGGAAACGT 2100

TTGAGAGCGG GATTCGTAAA ACGGTGGAAT GGTACCTGTC CAATACAAAA TGGGTTGATA 2160

The termination of Orf1

ATGTGAAAAG TGGTGCTTAT CAATCGTGGA TTGAACAGAA CTATGAGGGC CGCCAG TAAT 2220

Orf2's is initial

GAATATTCTC CTTTTCGGCA AAACAGGGCA GGTCGGTTGG GAACTACAGC GTGCTCTGGC 2280

ACCTTTGGGT AATTTGATTG CTCTTGATGT TCACTCCACT GATTATTGTG GTGATTTTAG 2340

TAATCCTGAA GGTGTAGCTG AAACCGTCAA AAAAATTCGC CCTGATGTTA TTGTTAATGC 2400

TGCGGCTCAT ACCGCAGTAG ATAAGGCTGA GTCAGAACCC GAATTTGCAC AATTACTCAA 2460

TGCGACCAGC GTTGAATCAA TTGCAAAAGC GGCTAATGAA GTTGGGGCCT GGGTAATTCA 2520

TTACTCAACT GACTACGTAT TTCCGGGGAC CGGTGAAATA CCATGGCAGG AGGCGGATGC 2580

AACCGCACCG CTAAATGTTT ACGGTGAAAC CAAGTTAGCT GGAGAAAAAG CATTACAAGA 2640

GCATTGTGCG AAGCACCTAA TTTTCCGTAC CAGCTGGGTC TATGCAGGTA AAGGAAATAA 2700

CTTCGCCAAA ACGATGTTGC GTCTGGCAAA AGAGCGTGAA GAATTAGCCG TTATTAATGA 2760

TCAGTTTGGT GCGCCAACAG GTGCTGAACT GCTGGCTGAT TGTACGGCAC ATGCAATTCG 2820

TGTGGCACTG AATAAACCAG AAGTCGCAGG CTTGTACCAT CTGGTAGCCA GTGGTACCAC 2880

AACCTGGCAC GATTATGCTG CGCTGGTTTT TGAAGAGGCG CGCAAAGCAG GCATTCCCCT 2940

TGCACTCAAC AAGCTCAACG CAGTATCAAC AACAGCCTAT CCTACACCAG CTCGTCGTCC 3000

GCATAACTCT CGCCTTAATA CAGAAAAATT TCAGCAGAAC TTTGCGCTTG TTTTGCCTGA 3060

The termination of Orf2

CTGGCAGGTT GGTGTGAAAC GCATGCTCAA CGAATTATTT ACGACTACAG CAATT TAATA 3120

Orf3's is initial

GTTTTTGTAT CTTGTTCGTG ATGGTGGAAC AAGATAAATT AAAAGGAATG ATGGA ATGAA 3180

AACGCGTAAA GGTATTATTT TAGCGGGTGG TTCTGGTACA CGTCTTTATC CTGTGACTAT 3240

GGCTGTCAGT AAACAGCTAT TACCTATTTA TGATAAACCG ATGATCTATT ATCCGCTCTC 3300

TACACTGATG TTGGCGGGTA TTCGCGATAT TCTAATTATA AGTACGCCAC AGGATACTCC 3360

TCGTTTTCAA CAACTGTTGG GTGACGGGAG CCAGTGGGGC CTGAATCTTC AGTACAAAGT 3420

GCAACCAAGT CCAGATGGTC TTGCGCAAGC ATTTATCATC GGTGAAGAAT TTATCGGTGG 3480

TGATGATTGT GCTTTGGTTC TTGGTGATAA TATTTTTTAC GGTCACGATC TGCCGAAGTT 3540

AATGGATGCC GCTGTTAACA AAGAAAGTGG TGCAACGGTG TTTGCCTATC ACGTTAATGA 3600

TCCTGAACGC TATGGTGTCG TTGAGTTTGA TAAAAACGGT ACGGCAATTA GCCTGGAAGA 3660

AAAACCGCTA CAGCCAAAAA GTAATTATGC GGTAACTGGG CTTTATTTCT ATGATAACGA 3720

CGTTGTAGAA ATGGCGAAAA ATCTTAAGCC TTCTGCCCGC GGTGAACTGG AAATTACCGA 3780

TATTAACCGT ATCTACATGG AACAAGGGCG TTTATCTGTT GCCATGATGG GACGTGGATA 3840

TGCATGGTTA GACACGGGGA CACATCAAAG CCTGATTGAG GCAAGCAACT TCATTGCTAC 3900

AATTGAAGAG CGTCAAGGGT TGAAAGTTTC CTGTCCAGAA GAAATTGCTT ACCGTAAAGG 3960

GTTTATCAAT GCTGAGCAGG TGAAAATATT AGCTGAACCG TTGAAGAAAA ATGCTTATGG 4020

The termination Orf4's of Orf3 is initial

TCAGTACCTG CTAAAAATGA TTAAAGGTTA T TAATAAA AT GAACGTAATT AAAACAGAGA4080

TTCCAGACGT ATTAATTTTC GAACCGAAAG TTTTTGGTGA CGAGCGCGGT TTCTTTTTCG 4140

AGAGCTTTAA CCAGAAGGTT TTTGAGGAAG CTGTAGGCCG CAAAGTTGAA TTTGTCCAGG 4200

ATAACCATTC GAAGTCTTGT AAAGGTGTTT TACGCGGACT GCATTATCAG TTAGAACCTT 4260

ATGCACAAGG AAAGTTGGTG CGCTGCGTTG TTGGTGAAGT TTTTGACGTA GCTGTTGATA 4320

TTCGTAAATC GTCGCCTACC TTTGGTAAAT GGGTTGGGGT GAATTTATCT GCTGAGAATA 4380

AGCGGCAATT GTGGATCCCT GAGGGATTTG CGCATGGGTT TTTGGTGCTG AGCGACGAGG 4440

CAGAATTTGT TTATAAGACA AACAATTATT ATTATCAAAA GCATGAAAGA AGCATTCTTT 4500

GGAACGATAA AATTCTCAAC GTAGAATGGC CATTTACTTC TAACTTAATT CTTTCTTCAA 4560

The termination of Orf4

AAGATATGAG CGCGTCATTA TTCACTGTTG CAGAAAAATT T TAATTGGTT TTGGAAATAA 4620

Orf5's is initial

A ATGTCTATA CAAAATGTAA TAGATAATGG TTACTGCGTC GGATGTGGTG CTTGTAAATT 4680

GGCTCTTGGA GAGTCAGTGG AAATAAAAAT GTATGAAGAT GGCTTTTATA ATGCTTCGAT 4740

AAAACCTAAT GCAAATATTG AAATTGCCGA GAAAATTTGT CCATTTTCTG ATAGTTCAAC 4800

AAATGAAACT ATCTTAGGCG CATCACTTTA TGGAGATAAA AAATTTGACA AGCGTGTAGG 4860

ATATTTTGAC GACATTTATA TTGGGTATGT AGTTGATAAT CAGGACCGAC TAACTTCCAG 4920

TTCAGGTGGA CTTACTACCT GGTTTGCTAA AAAGTTATTA ATAAATAAAG AGGTTGATGC 4980

GGTAATTCAT GTAGGGCATG GGACGCATAT GTTGGACTAC ATTATTACTG AGAATATTTT 5040

AGATCTTGAT TTACAGAATA ATAAAAAATC CAGATATTAT CCTGTAAGTT TTTCTGAATT 5100

GATAAATAAA ATAAACCAAT CAGATAAAAA ATACCTTTTC ATTGGCATTC CATGTTTTGT 5160

TAAATCAATA AGATTAGCGC AGAAAGAAGG AATGGTTCAG AATATTAAAT TTGTGATTTC 5220

ATTACTCTGC GGACATATGA AGTCCAAAGA TTTCGCTACG AGTCTTGCGT GGCAGATTGG 5280

CGTTAAACCT GATGATCTGG GTAGTGTAGA CTTTCGTGTC AAGGAGGATA ATAAAAAAGC 5340

AAATGATTAT AATATAGAAG TTGAAGATAC TTGTAATAAG AAATATATGC ATCAAAGCTC 5400

TTCGTTGTTT GGCACTAATT GGGGGCTAGG CTTTTTCAAA CATCATGCAT GTGATTTTTG 5460

TGATGACATT GCAGGTGAAT TGGCGGATGC AACTTTAGGT GATGCTTGGT TACCGAAGTA 5520

TATAAATGAT TCACAAGGTA CTAATATACT TATTGTAAGA AACGATGTTT TAAATAAATT 5580

ACTTGAACAA TATCAAGAAG AAATAGTGCT TGAAACTGCG AATGTCGAGG ATTTTTATGA 5640

AAGTCAAGCT GGGAATTTTC GTAATCGACG TGAAGGGTTA CTTGTTCGTG TCCAAAATGA 5700

AAAAAAATGG ACTCCTAGAA AAAGGTTGGA AATTATTAAC TCCGATATTC CATTACAACG 5760

TCGTAAATTG TATTTAGTTC GTCAGAAGTT AAGTGAACAA AGTATAAAGA AATTTCAAAT 5820

AGCTAAAAAG ATAGGTTCAA TATATTCATT CAAATTGTTA ATGGTTCCTG ATATTTTTAG 5880

ATATAAAATG ATTGAGAAGA ATTTAATATC GGCGTTAAAA GAAACTATAA GAATAGTGGC 5940

The termination Orf6's of Orf5 is initial

GCCAAGAAAA CTTGTAAAAA TATTCAAAAA ACGG TAGCTT T ATGCATAGG CAAGTTTTTC6000

GAAACGTATT TCTAAATGTG ACGACATTTT TATTTAATGT AGTTACTGGA TTATATTTAA 6060

CTCCATTTTT GATTAAATAT TTAGGGTTGG AAGTGTATGG TATACTTCCT TTAGCATTGT 6120

TTTTAACCTT TTATATTGGC GTGATAACTC AGGCCCTTAC GGCATCAGTA AATCGATTTT 6180

TTATTGCAAA TTTACAAACT AATAACATTA AAGAGGCTAA TGTTGTTTTT AACACTGCTT 6240

TTTGTATCAT AGTTACATAT TCGGTATTGC AGTTTGCTCT CCTATACTAT CCTATACAAA 6300

ATATAGACCA GTATTTTTCT ATACCGACGC ATAGCAGAGA TGATGCCCGG AGATTGTTTG 6360

AATCAGTTTT ACTCAGCTTC TCTCTTGCGC TTTTAACATC CATTTTTAGT GTTTCGCTTT 6420

TTGCATTAAA CAGGTTGGAT TTGATTCAAT GCGTCCAGTT AATCAAAATT ATTATAAAGT 6480

TCATTACCAT AATATTATTC TTTGAACATC ATTGCATAGG TATCCAGTTT GTAGGAATAG 6540

CAATGATTCT GTCAGAGTTA ATAGCTTTAA TTTTGACGAT TTGTTTATGG AGAAAATTGA 6600

CACCGGAAAT AAATTTAAAA CTATTTTACT TCAGTAAAAA GAAAGTAGGA GAGCTTTCAA 6660

AGTTTGCAAT GTGGTTGATT ATTGATCAGG TTGGATATGT TCTTTTTATT AAAATGGATT 6720

TATTATTGGT AAACAAATTC TTTGGGGCGA AGGAATCTGG GCGCTATTCA ATTGCAACGC 6780

AATTTAGTGA TTTACTAAGA TCATTTGCAG GGCTTATGGC GGGAGTTCTT GCACCTGTAA 6840

TAATGATACT ACACGCCAAG AATGAATTAG AAAGAATTGT AACCGTTACC AAAACGTTTG 6900

TGATGATTCT ATCGCTGACA ATAGCGATAC CAATTTCTAT AATCTGTGTA TTTTCTCAAG 6960

AGCTGAT CA TTTCTGGCTA GGACAAGATA TAAATATCCA AACTTTAATT TGGATTGTGA 7020

CTTTTCCATT AATAATTAAT TTGGGCGTCT TACCATTATT TTCTATAAAT GTAGCGTTTA 7080

AAAAAGTTAA ACTACCTAGT GTTCTAAATA TCGTATTGGC ACTAGTTGGC GTATTTGTCT 7140

CAATTATACT TATTCGAAAT ACTGAACTGG GTTTACTTGC AGTAGCGGCT GGGTTTGGTT 7200

TTTCCCTGAC CCTCAAAAAT GCGGTATTTA TACCTATTTA CGCGGCACTA AATTTAAGAA 7260

TAGATAAATT AACATTTCTA ACTGTACATG TAAGAACTAT TTTATTTACA TTTTTTTATG 7320

TATGTTTTCT GATTTTAGTT AAGTCAAAAC TGTCTAGTAA TCTTTACGGG TTTTTAGCTG 7380

AATTAACAGT GCTTATTATA TTTGGCTTTA TAATTAGTAT CTTTTTTTAT TCTCGGGATG 7440

The termination of the initial Orf6 of Orf7

AAAGGAAGTA TATTACTAAT ACACTTTCAG ATAAATTAAA AGCGAAGGTA AA ATGAAAAA 7500

TAAAATTGGT ATTCTAAATT TTCAATATTC GGATCATAAT TATGGTGCAG TCTTACAAGC 7560

TGTTGCACTA GAAAATGTTT TAAAACAGCT AGGGAAAAAT GCTGAACATA TTAATTTTAT 7620

TCCAAAGAAG AAAGGTAACA AAAATATCAA ACAATTTATA ATTGAATTTT TAGCTTCTAT 7680

TGGTTTAAAA CCTTTCATTT ATCGGGTATT TAACAAAAAA GTTATTATAA AAAATAAAGT 7740

TGAAGGATCT GAGATTTTTG AGGATTTCAG GAATAAATAT TTGAACAGAA CAAAAGCGTT 7800

TCATTCCCTC AATGACCTTA TGGAATTAGA GGATAGTTAC AGTTCTGTTA TCGTTGGAAG 7860

TGATCAAGTC TGGCGCCCCA AAATGTACTC AGACCTTACA GAAATTGAAA TTTATTTCCT 7920

TTCTTTTATT TCACAGAAAA CAAAAAAAAT TTCTTATGCA GCAAGTTTTG GTGTTGAGCG 7980

ATGGGAATAT GACAAAAATG ATAATGTGAC ACATAAAGCA CAAAAATATC TGAACAAATT 8040

TAATTCTATT TCTGTTAGAG AAAAATCAGG AGTAGACATT TGTAATAGTG TATTTGATAT 8100

TAGAGCTGAG CATGTTCTGG ATCCAACACT ATTGATCGGA CGTGATTTTT TCGACAATCT 8160

TATTAAAGAC AAACAACCAT TAGTTCAAAA GACAATAAGT TATTATAAGT TAGATCTTTG 8220

TGAGTCTTTT TTAGCGGGAA TAGAGCATCT TGCTAATCTA ACCGGCTATG AGACTAATAA 8280

TATTTACTAC AAACAACTGA ATAATGGGAG TTATGAATAT TATCCAGTTT TAGATTGGCT 8340

AAGCATGATT AATTCATCTT CGATAATAAT AACAGACTCT TTTCATTGTG TATGCTTCTC 8400

AATTTTATTT AATAAAAGCT TCTATTGTTG TCTTAATAAA GAAAGAGGAG CATCGAGGCT 8460

TGAAAGTTTA TTGAATGAAC TGGGGTTGCA AGACAGATTA ATCTCTGAAG AAGAACTATC 8520

GAGAATTTAT ACAATAGTTG ATATCAATTA TTCTCCTGTA GAAGATATTT TGAATAAGCA 8580

The termination Orf8's of Orf7 is initial

CAGGAAACAG TCAATAGAAT TTTTAGTTTC AGCAAT TGAT GGATAATTAA ATGAGTCATA8640

TAATTCCAGT AGTTATTATG ACACGGAATG AAGGTGCTTA CCTTTTGAAA TGTGTCCAAT 8700

CAATCATTAA TACTGTTACT ATACCCTATC ATATATATAT TATTGATAAC AATTCTTCAA 8760

CATTGGAGCA AGAAGAAGTT CTAAAAATTA TTGAATATAA GTTTAATGAT AAAGTAAATA 8820

TTATTAGAAA TCGTACTAAC AGGTGGGTTC TGGGTTTAAA TAAGACACTT ATTGAATTGA 8880

GTAAAAATAA AGAACAACCC TATTTCTTTT TAACTGATGG TGATATTGAC TTTTCAAGAT 8940

GCACCGCTAA GCCATGTTGG TTATCATATT TGGTTACCCA TATGAATTCT AATGTATCTC 9000

TGGGGAAAAT TGGCTTATCT CTCAATTGGG ATTACATTTC AACTACAAAA GAATTGGCGG 9060

CTGTTTATGA ACAGGAGAGA AGTCTATATA ATGAAAGTAA AAAAATAAAT GATTTATATG 9120

TGTCTCCTGT TGATACAACA GCGGCATTAT TTCGATGGGA TTGGTCTATA GAAGGAAGAC 9180

CAAGCTTATA TCCAGACCAT ATTAGATACC TTAGACCAGA ACTTTATTCA TGTCGAACAC 9240

CATTAGATAT TAATGTAGAG CATATGGGAT GGTATAAATA TTGTGCAGAA TCTGGAGATA 9300

CAAAAAATTT AGAGGAAAAA ATAGTCTGCT TCACTATGGT TGGTGGTGAT GTTAAAAACG 9360

AAATTTTAGA TAAGGTTCGT TTTTTCTATA AGGTTATATA TAAATCATTG TCTGGTCCAG 9420

TAAAAAAAGC ATGGTATTTC AGGAGATATT ATTATCTTTT GAAATATTTT CTTTTTAAGG 9480

The termination Orf9's of Orf8 is initial

GCGTTCGCAG ATTCGATGGT CAAAACTGTC TT TGAAATGT AATGTAATTA TTGGA GTGTA9540

ACTTTGTATA TATTTCGAAG TGAGTTTGTA CTAGCGATAT TTATTTTTCA TTTGTTATTC 9600

ATAATATTGG CCTTATATGC ACGAAAACTT GATAATAAGG TATTAGATTA TATATTTCTT 9660

ATATTGGCAA CTACATTAGC CTTAATTATT TCACTCTTTA GACCTGGCTT TGGAGTTGAT 9720

GAACCTTCTT ATCTTGAGGC ATTTATTAAT TTCAAAAATA ATGCTGACAC ATTTCCATTC 9780

GATTATTCAT TTAAAGTATT ATATGAAATA TTATCTAATA CGGGCATACA AATTGAGTAT 9840

TTCAATAACA CAGTCAGCTC TTTATATATT ATTTTACTTA GTATAACGGC GATAGTAACA 9900

TCGAGTAAAA ATAATAAGTG TTTTTATTTC TTGATGTTAT TGTTCAGTTT TACTTCATTA 9960

GATCTAATGT TTAATGCTTA CAGGCAAGCA TTTGCATTTA TATTTATTTA TAACTCTTTA 10020

TTTCTTTTTT TGAGAGAAAA GAAAATCAAG GGGGCTTTAT TAGCTGTAAT CTCTTTTGGG 10080

TTTCATTGGT CAGCAGTTAT CCTGATCTTA TTTTATTTTT TATCGAGGTT TCTTAAGAAT 10140

AAATATTCTT ACCTTATTTT ACTTTTATTA TTTCCATTTG TATTAATCTC GATGTTTTAT 10200

CCATTAGGTT TGATGGGGAT AGGATATAAT GTGTTGTTAT TTCTTCCTTT GAATGAATTA 10260

TTCAAAGCTC ATGTGTTAGC GTATTTAGAA GTTGATAACA TTTCTAGCGC GTCGTTTTAT 10320

ATACTTAATC TCTTTGGTCG ACTACCATTG GCTATATCGG TATTAACTTT TTATGCTTTT 10380

ATACTGATTT TTTACAAAAA AATAAGAATG AAGCGTATAA TAGCAATGAT TGTCTTGATG 10440

GCTGTTTATT GTCTAGTGTT TATGGAGATG GCATATAGTT TTAGAAATTA TTATTGGGTT 10500

CTACCATTTT TCCCTATTAT TGCTTTAGAT TACGCAGAAT CTAATCAAGC GAAAATTCAT 10560

TCAAGAATGA TAGGCATTGC TGCTCTTCAT ATCTTGATAG CATTGCCTAC TTTTTACAGT 10620

The termination Orf10's of Orf9 is initial

TCAGGTATAA ACTCTATGAT TTTCAATACT GGCAAT TAGT ATTGAATATG GAAAAAAA AT10680

GTGTTCATTA AAAGTTAGCG CTTTACTCGT TAGTTACAAT CCAGATATTA ATAGATTAAG 10740

TGAGGTTCTT AATAGTATTC ATTCTCAAGT AGATCATCTA GTTTTAGTTG ATAATGGTTC 10800

AAAAAATAAA AATGATATAC TTGCTCTCAT TGCCGATTAT GAGGGGGTTT ATACACTGCT 10860

TAATGATAAT AATATAGGTT TAGCCTCTGC TCAAAATGTT GGTATTGATT ATATTATTGA 10920

CAATAACCTT TCCGATTTTA TCGTGTTCTT CGACCAAGAT AGTGTATTGC AGTCAGGTTT 10980

TATTAGTGCG CTACTCGAAA CGTATACTAA TTTAGTAACT CAGAATATTA AATTAGCAGC 11040

AGTAGGACCA TCATTTATTG ATCCTGTAAA TAATAAACAA TATCCTGCGA CTGTTTATGC 11100

AGGACCATTT ATTAAACGAG TAGATCTTGA AAGAAAACCT GTAGAAGCTA CCTTCATAAT 11160

TGCTTCTGGA TGCTTTGTTG CCATTGAGGC ATTGAAAAAA ATTGGGAAAA TGAAGGATGA 11220

ATTGTTTATA GACTATATCG ATGTTGAATG GTGTCTCAGA GCTAGGAGTT TAGGATACAA 11280

AATATATATT TCACCTAAAG CTGTAATGAA ACATACTATT GGAGATAATC GAGTATCTAT 11340

CTTTGGGCGA ACAATATCTG TTCATTCACC ATTACGTCGA TATTATCTTG TAAGGAATAG 11400

CTTTTATATG ATTAGATTGG ATTATATACC AATTGGATAT AAAATTAGAG AAATATTTTT 11460

TAATGTAGTT CGTATTTGTA TCAGCTTAGC TCTATCTGAT GATAAAAAAA AGATACTTCA 11520

TTATACAATA ACTGCAATTA AAGATGGTGT TAGTAAAAAA TTCGGACCTT GTAAAAAAAT 11580

The termination Orf11's of Orf10 is initial

ACTC TAATT A TGATCAAAAT AGCTCATATT CAATTATTGC CAATGCTTAG TGGTGTTCAG11640

AGAGTGTGTT TGGATGAGCT CTCAAGATTG GACAAAAAAA AATATCAAAG ATACTTAATT 11700

TGTAAAGAAC CAGGTAAACT CAGTGATGAG GCTACTGCTA TTGGCGTTCA ATGTATCTTT 11760

CTTCCCGAGC TTCAGCGCAA TATCAGCTTT AAGATGGACT TAATAGCGTT GTACAAATTA 11820

TATCGGATAA TAAAGATATA TAAATTCGAT ATTGTTCATA CACACTCTTC TAAGCCAGGT 11880

GTTTTAGGGC GTATTGCAGC CAGGCTGAAT GGTGTTAAAC TGATACTTCA CACTGTGCAC 11940

GGTTTTTCTT TTCCTGCTGC ACAAAATAGG TTCCAATATT ATATATTTTG GATCATGGAA 12000

AAAATTGGCT CAATATGTGG AGATAAATTA ATATGTCTGC ATAAAGATGA TGCAGTGATT 12060

GCTCAGGAAA AATTATTTAT TCATAAGAAT AAAATTTCCA TAATTGAAAA TGGCGTCGAC 12120

ACTAACAAAT TCAAGAAGAG AAATAATAAT GAAATAGATA AATTATCTAC ATATTTTAAC 12180

ATAGATAGAA ATTCGGATGT TGTGGTCGGG ATGATAGGTA GACTGTGGCC ACAAAAAAAC 12240

CCCATGCTTT TATTATATGC AGCAAAAAAT ATAATCAACA ATAATAAAAG AGTAAAATTC 12300

TTGTTTGTCG GCGATGGTGA GTTAAAAGAC TCAATGAACG ATTATATTAT TCAAAATAAA 12360

CTTACCCAGA ATATTACCAT TTGTGGCTGG TGTAATGATG TTAGTTCTTA TTTGAATATA 12420

TTTGATATTT TTGTATTACC ATCTTTATGG GAGGGTATGC CTTTGGCAAT TTTAGAAGCC 12480

GAAAGTAGTT CGTTGCCCTG TATTGTTTCA AATATCCCGG GAAACAGGAG TATAATCAGG 12540

CATACAGTTG ATGGCTATCT CTTTTCATTA GATAACCCAT CTGAACTTGA ATCTTACATT 12600

AATATATTAA TTGAGGATAA AGAAAGACGT ATTATCTTGG GTAATAACGC CAGAGTCAAA 12660

ATTCTTGAAA GCTATAAAAT AGAAAATAGG ATTTTGAAAT TAGATGAACT GTATAGTTCA 12720

The termination Orf12's of Orf11 is initial

ATTTCTTTAA AAGAA TAGCG TTGTATGTTG AGGGGAT ATG CGGATAAAAA TACTCGACCA12780

TATGAAAGGT GCTGCTATTT TAGCTGTGGT ATTTATTCAT TCTTCAGGTT ATCTTGCTGT 12840

TTCTAATATA GGAACATTTG AATGGTACAT CGGTATAATA TCGCGGCAAT TTGTTAATTT 12900

TGCAGTGCCT ATTTTTTTAT TTATATCTGG TTTTTTATCT TTTTCTCGAG ATATACATGA 12960

TACACCATAC TATCTCAAGA AAAAATTATT AAGAATTATT ATACCTTATC TCTCATGGTC 13020

TTGTTTTTAC TTCTTTCTTG TATTCATATT TAATAACCAA AGTTTCGCTG TCAAAAATAT 13080

TATAGCTCAG TTGATTCTTG GTACGGGAAT AGGTGTGGGA TATTTTGTGA TCGTTTTAGT 13140

GCAATTTATT ATGCTGACAC CGTTAATTAA TGCGATTACA TCTATAAAAA TACATATTAT 13200

GATTATATGT GGTTTTACTA TCATAGGTGT GGGGATTAAT TATATGTCTG TGAAGATTGA 13260

TTGCTTGAAA CTGTTTTCAC AGTTTCCTTA TTCCGCCATT CCTTTTTTTG TATGGTATCC 13320

TTTTTATCAT TTGGGCTTTG TTTTCAATAA GTATAATATA TCTTGTTCAA AGTGGTCTAA 13380

TAGTTCCATC TTATTTTGGA TTTTATCTTT TTCACTTACA TTAGCCATTT CTGAAGGGAT 13440

ATTTTGGGGT TATACTGGGA ACTATTCATT TGCTGTGTCT CAACTTAAAC TATCATCTTT 13500

GGTTCTCTCT TTATGCGTTT GTATGGTTGT TTATTTTTAT CTAAACCATG ATATAAATTA 13560

TAAATACCTT TCTCAATTAG GGACAATGTC CTACGGGATT TATCTTACTC ATATGTTGTT 13620

TGTTTGGTTC TATCATTATC TCTTTTCGTA TACGAGTGTG TTTATAAGGC ATTTAGTTAT 13680

CTCGTGTGGC TTTATCTTTA TATTAAGTAC AATATCATCA GTTACCTTAG TTTTTTTTAT 13740

The termination of Orf12

AAGAAAAATT TTGGGGAGAT ATTCCGTATA TATTGTAGGG TAGGTGATTG CTTGATATTT 13800

ATACTGTTTT AAGAAGAAGC CTAATTTATC TCAATACTGG GCATTTCACT AGAGCTGTAT 13860

TAGTATATTT GTCACATTCT CATCCGTATT TCTAATGTAT TTTTCAGAGC AGGAGACTTA 13920

AATGTCAAAG CAACAGATCG GCGTCGTCGG TATGGCAGTG ATGGGGCGCA ACCTTGCGCT 13980

CAACATCGAA AGCCGTGGTT ATACCGTCTC TATTTTCAAC CGTTCCCGTG AAAAGACGGA 14040

AGAAGTGATT GCCGAAAATC CAGGCAAAAA ACTGGTTCCT TACTATACGG TGAAAGAGTT 14100

TGTTGAATCT CTGGAAACGC CTCGTCGCAT CCTGTTAATG GTGAAAGCAG GTGCAGGCAC 14160

GGATGCTGCT ATTGATTCCC TCAAACCATA TCTCGATAAA GGCGACATCA TCATTGATGG 14220

TGGTAATACC TTCTTCCAGG ACACCATTCG TCGTAATCGT GAGCTTTCTG CAGAAGGTTT 14280

TAACTTCATT GGTACCGGTG TTTCCGGCGG TGAAGAAGGT GCGCTGAAAG GTCCTTCCAT 14340

TATGCCTGGT GGGCAGAAAG AAGCCTATGA ACTTGTTGCG CCGATCCTGA CCAAAATCGC 14400

CGCAGTGGCT GAAGACGGGG AGCCATGCGT TACCTATATT GGTGCCGATG GTGCAGGTCA 14460

CTATGTGAAG ATGGTTCACA ACGGTATTGA ATACGGTGAT ATGCAGCTGA TTGCTGAAGC 14520

CTATTCTCTC CTGAAAGGCG GCCTGAATCT CTGTAACGAA GAACTGGCAC AGACCTTTAC 14580

CGAGTGGAAT AACGGTGAAC TGAGTAGCTA CCTGATCGAC ATCACCAAAG ATATCTTCAC 14640

CAAAAAAGAT GAAGACGGTA ACTACCTGGT TGATGTGATT CTGGATGAAG CCGCTAACAA 14700

AGGTACCGGT AAATGGACCA GCCAAAGTGC GCTGGATCTC GGCGAACCGC TGTCGCTGAT 14760

TACCGAGTCT GTGTTTGCAC GTTATATATC TTCTTTGAAA GATCAGCGTG TTGCCGCCTC 14820

TAAAGTACTG TCTGGTCCGC AAGGACAGCC AGCAGGCGAC AAAGCTGAGT TCATTGAAAA 14880

AGTTCGCCGT GCGCTGTATT TGGGCAAAAT CGTTTCTTAC GCCCAGGGCT TCTCTCAGCT 14940

GCGTGCGGCG TCTGAAGAGT ACAACTGGGA TCTGAACTAC GGCGAAATCG CGAAAATTTT 15000

CCGTGCTGGC TGTATTATCC GTGCGCAGTT CCTGCAGAAA ATCACCGATG CTTATGCCGA 15060

AAATCCGCAG ATCGCTAACC TGCTGCTGGC TCCGTACTTC AAGCAAATTG CCGATGACTA 15120

CCAGCAGGCG CTGCGTGATG TCGTTGCTTA TGCAGTACAG AACGGTATCC CGGTTCCGAC 15180

CTTCGCCGCT GCGGTTGCCT ATTATGACAG CTACCGTGCC GCTGTTCTGC CTGCGAACCT 15240

GATCCAGG 15248

Only being preferred embodiment of the present invention below, is not that the present invention is imposed any restrictions, all according to the technology of the present invention essence to above embodiment make an amendment, equivalent variations and modification, all belong in the scope of technical solution of the present invention.

Claims (4)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O36, it is characterized in that, its wzx sequence is the Nucleotide of 5982 to 7496 bases among the SEQ ID NO:1, the wzy sequence is the Nucleotide of 9583 to 10659 bases among the SEQ ID NO:1, the orf8 sequence is the Nucleotide of 8631 to 9515 bases among the SEQ ID NO:1 or has above-mentioned insertion, disappearance or replace one or more Nucleotide, but the oligonucleotide of the O-antigen function of maintenance wzx, wzy, orf8 Nucleotide specific detection intestinal bacteria O36.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O36, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 6084 to 6101 bases among the SEQ ID NO:1 and the Nucleotide of 7138 to 7155 bases, the Nucleotide of 6259 to 6277 bases among the SEQ ID NO:1 and the Nucleotide of 6967 to 6984 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 9697 to 9714 bases among the SEQ ID NO:1 and the Nucleotide of 10570 to 10587 bases, the Nucleotide of 9814 to 9832 bases among the SEQ ID NO:1 and the Nucleotide of 10428 to 10445 bases; Or the oligonucleotide that comes from the orf8 gene is right: the Nucleotide of 8656 to 8673 bases among the SEQ ID NO:1 and the Nucleotide of 9330 to 9347 bases, the Nucleotide of 8758 to 8775 bases among the SEQ ID NO:1 and the Nucleotide of 9475 to 9492 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 O36 of 2.

4, the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O36 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.