CN1569874A - Nucleotide specific for escherichia coli 0123 O-antigen - Google Patents

Abstract

The invention provides a nucleotide specific for Escherichia coli 0123 O-antigen, which is the total nucleotide sequence of the gene cluster for controlling the synthesis of O-antigens in Escherichia, e.g. isolated nucleotide represented by SEQ ID No:1, with overall length of 17084 bases, or nucleotide of SEQ ID No:1 including one or more inserted, deleted or substituted bases, and sustaining the functions of the isolated nucleotides, it also includes the oligonucleotides of glycosyl transferase genes and oligosaccharide unit treatment genes in the O-antigen gene cluster originated from Escherichia 0123.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O123 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O123 type (Escherichia coli O123), particularly relate in the intestinal bacteria O123 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O123 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.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 gene nomenclature " Trends inMicrobiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of fourShigella boydii O-antigen loci:implication for Escherichia coli and Shigellarelationships " .Infection and Immunity, 11:6923-6930 bunch between JUMPStart sequence and gnd gene; Lei Wang and Peter Reeves (2000) " TheEscherichia coli O111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose and paratosesynthase genes (rfb) by polymerase chain reaction for identification of S.enterica majorserogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecularmicrobiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dryfermented sausage contaminated with Shiga-like toxin producing Escherichiacoli " .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 antigengene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specific selection andbacterial populations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' sidentification of the Enterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasiveEscherichia coli antigens O28ac; O112ac; O124; O136, O143, O144; O152 and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O123 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O123 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O123 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O123 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf9, orf11, orf15 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are high specials to the O-antigen of intestinal bacteria O123 type; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O123 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O123 type of these methods detections and identification of escherichia coli O123 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O123 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

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

The present invention is characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O123 type: it is the isolating Nucleotide shown in SEQ ID NO:1,17084 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type is comprising called after rmlB, rmlA, wzx, vioA, orf5, orf6, orf7, orf8, orf9, wzy, orf11, fnlA, qnlA, qnlB, orf15,16 genomic constitutions of orf16 are all between JUMPStart sequence and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf9, orf11, orf15 gene; Wherein said gene: wzx is the Nucleotide of 2169 to 3614 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 8478 to 9728 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 7469 to 8494 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 9725 to 10846 bases among the SEQ ID NO:1; Orf15 is the Nucleotide of 13884 to 15077 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 2627 to 2646 bases among the SEQ ID NO:1 and the Nucleotide of 3275 to 3294 bases; The Nucleotide of 2806 to 2825 bases among the SEQ ID NO:1 and the Nucleotide of 3376 to 3395 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8986 to 9005 bases among the SEQ IDNO:1 and the Nucleotide of 9484 to 9503 bases; The Nucleotide of 8603 to 8622 bases among the SEQ IDNO:1 and the Nucleotide of 9392 to 9409 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type is providing the O-antigen of expressing intestinal bacteria O123 type by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O123 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 O123 type bunch: with the genome of intestinal bacteria O123 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 1ong 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 O123 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O123 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O123 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O123, 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 O123.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O123 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O123 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O123 type bunch: with the genome of intestinal bacteria O123 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JUMPStart sequences Design upstream primer #wl-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTAAGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, Annealed 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, at last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product, merge 5 pipe long PCR products, and with the WizardPCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9 μ l 0.1MMnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O123 type;

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

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical ResearchCouncil) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O123 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O123 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O123 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) Orffinder finds gene, find the reading frame of 16 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 O123 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O123 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O123 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O123 type all is high special.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O123 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ l supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 2627 to 2646 bases among the SEQ ID NO:1 and the Nucleotide of 3275 to 3294 bases; The Nucleotide of 2806 to 2825 bases among the SEQ ID NO:1 and the Nucleotide of 3376 to 3395 bases; The Nucleotide of 8986 to 9005 bases among the SEQ ID NO:1 and the Nucleotide of 9484 to 9503 bases; The Nucleotide of 8603 to 8622 bases among the SEQ ID NO:1 and the Nucleotide of 9392 to 9409 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O123 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 O123 type, its complete sequence shown in SEQ ID NO:1,17084 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 O123 type by method of the present invention, as shown in table 3, it comprises called after rmlB, rmlA, wzx, vioA, orf5, orf6, orf7, orf8, orf9, wzy, orf11, fnlA, qnlA, qnlB, orf15,16 genomic constitutions of orf16 are all between JUMPStart sequence and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O123 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf9, orf11, orf15 gene).Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to intestinal bacteria O123 type O-antigen.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O123 type is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O123 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O123 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O123 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As used herein, " oligonucleotide " mainly refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 2169 to 3614 bases from SEQ ID NO:1), wzy gene (nucleotide position is the Nucleotide of 8478 to 9728 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O123 type.

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

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O123 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that coming from coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O123 type.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O123 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.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 O123 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O123 type first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O123 type by inserting to express, and become useful vaccine.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).

Embodiment 1: genomic extraction:

37 ℃ of incubated overnight intestinal bacteria O123 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds l0 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30 μ l TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O123 type bunch:

With the genome of intestinal bacteria O123 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JUMPStart sequences Design upstream primer (#wl-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C) 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 was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then,

Annealed 15 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 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

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

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

Be electric transformed competence colibacillus cell at last: get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O123 type with the EcoRI enzyme.

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

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

Embodiment 5: the splicing of nucleotide sequence and analysis:

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O123 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O123 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O123 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffinder finds gene, find the reading frame of 16 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 O123 type at last, as shown in table 3.

By retrieving and comparing, find the dTDP-D-glucose 4 of rmlB coding among orf1 encoded protein and the Escherichia coli, the aminoacid sequence of 6-dehydratase (AAK60448) has 92% consistence and 95% similarity, by search, find that the homology desired value of orf1 encoded protein and known NAD dependent epimerase/dehydratase family consensus sequence is 1.3e to Pfam protein-based order sequenced data storehouse ^-210Therefore we can determine that this gene is rmlB.The aminoacid sequence of the glucose-1-phosphate thymidylyltransferase (AAO68468) of rmlA coding has 80% consistence and 90% similarity among orf2 encoded protein and the Salmonella enterica subsp.enterica serovar Typhi Ty2, by search, find that the homology desired value of the consensus sequence of orf2 encoded protein and known Nucleotidyl transferase is 2.6e to Pram protein-based order sequenced data storehouse ^-109Therefore we can determine that this gene is rmlA.The proteic aminoacid sequence of VioA (AAD44154) of vioA coding has 60% consistence and 76% similarity among orf4 encoded protein and the Escherichia coli, by search, find that the homology desired value of orf4 encoded protein and the proteic consensus sequence of known DegT/DnrJ/EryC1/StrS aminotransferase is 1.7e to Pfam protein-based order sequenced data storehouse ^-87Therefore we can determine that this gene is vioA.The proteic aminoacid sequence of the WbnG that encodes among orf5 encoded protein and the Shigelladysenteriae (AAR97959) has 38% consistence and 60% similarity, because the definite function of this gene can't be determined, so we are with its temporary called after orf5.The aminoacid sequence of conserved hypothetical protein (AAQ61692) of coding has 26% consistence and 45% similarity among orf6 encoded protein and the Chromobacterium violaceum ATCC 12472, because the definite function of this gene can't be determined, so we are with the temporary called after orf6 of this gene.The aminoacid sequence of Acetyltransferase (AAP10400) of coding has 33% consistence and 56% similarity among orf7 encoded protein and the Bacillus cereus ATCC 14579, by search, find that the homology desired value of the consensus sequence of orf7 encoded protein and known Acetyltransferase (GNAT) family is 5.6e to Pfam protein-based order sequenced data storehouse ^-07Because the definite function of this gene can't be determined, so we are with the temporary called after orf7 of this gene.The aminoacid sequence of the MaoC familyprotein (AAK22689) that encodes among orf8 encoded protein and the Caulobacter crescentus CB15 has 48% consistence and 70% similarity, by search, find that the homology desired value of the consensus sequence of orf8 encoded protein and known MaoC like domain is 5.5e to Pfam protein-based order sequenced data storehouse ^-29Because the definite function of this gene can't be determined, so we are with the temporary called after orf8 of this gene.The aminoacid sequence of the L-QuiNAc synthase (AAR24274) of fnlA coding has 84% consistence and 92% similarity among orf12 encoded protein and the Shigella boydii, by search, find that the homology desired value of the consensus sequence of orf12 encoded protein and known Polysaccharide biosynthesis protein is 1.7e to Pfam protein-based order sequenced data storehouse ^-42Therefore we can determine that this gene is fnlA.The aminoacid sequence of the L-QuiNAc synthase (AAR24275) of qnlA coding has 54% consistence and 71% similarity among orf13 encoded protein and the Shigella boydii.Therefore we can determine that this gene is qnlA.The aminoacid sequence of the L-QuiNAc synthase (AAR24276) that encodes among orf14 encoded protein and the Shigella boydii has 72% consistence and 86% similarity, by search, find that the homology desired value of the consensus sequence of orf14 encoded protein and known UDP-N-acetylglucosamine 2-epimerase is 1.3e to Pfam protein-based order sequenced data storehouse ^-123Therefore we can determine that this gene is qnlB.The aminoacid sequence of the WbuC protein (AAN60465) that encodes among the albumen of orf16 sign indicating number and the Escherichia coli has 42% consistence and 62% similarity.Because the definite function of this gene can't be determined, so we are with its temporary called after orf16.

Orf3 and orf10 are the proteic genes that there is transmembrane segment in only two codings among the intestinal bacteria O123.The aminoacid sequence of the O-antigen transferring enzyme (AAD44153) of orf3 encoded protein and Escherichia coli coding has 44% consistence and 66% similarity, it contains 11 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf1 is wzx.The O-antigen polysaccharase (AAO39700) of orf10 encoded protein and Escherichia coli has 23% consistence and 48% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf10 is wzy.

Orf9, orf11, the albumen of three genes encodings of orf15 and other known glycosyltransferases have the sequence identity of 31-55% and the sequence similarity of 50-71%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the albumen of these three genes encodings and the consensus sequence of known glycosyltransferase is very high, so we infer this three genes encoding glycosyltransferases.Because the definite function of these three genes can't be determined, so we are with these three genes temporary called after orf9, orf11 and orf15.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O123 type bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O123 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O123 type all is high special; The position of these genes in nucleotide sequence sees Table 1.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O123 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ l supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 2627 to 2646 bases among the SEQ ID NO:1 and the Nucleotide of 3275 to 3294 bases; The Nucleotide of 2806 to 2825 bases among the SEQ ID NO:1 and the Nucleotide of 3376 to 3395 bases; The Nucleotide of 8986 to 9005 bases among the SEQ IDNO:1 and the Nucleotide of 9484 to 9503 bases; The Nucleotide of 8603 to 8622 bases among the SEQ ID NO:1 and the Nucleotide of 9392 to 9409 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 1, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O123 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 intestinal bacteria O123 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O123 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O-antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O123 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O123 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

The genomic dna that contains intestinal bacteria O123 type in the 13rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25 μ l.Template is the 1:20 dilution, gets 1 μ l.After reaction finishes, get 10 μ lPCR products and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O123 type and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O123 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O123 type.These all oligonucleotide all can be used for the intestinal bacteria O123 type in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O123 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O123 type, altogether by 16 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are JUMPStart sequence and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O123 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O123 type in the drawings, at initial code of each open reading frame with stop the underscoring of code.Initial code of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria 0123 type

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria 0123 type

<160>1

<170>PatentIn?version?3.2

<210>1

<211>17084

<212>DNA

<213>Escherichia?coli

<400>1

attggtagct?gtaagccaag?ggcggtagcg?tgcattaata?cctctattaa?tcaaactaag?????60

agccgctaat?ttaacagcat?gctctgaagt?aatatggaat?aaattaagtg?aaaatacttg????120

ttactggtgg?cgcaggattt?attggttctg?ctgtagttcg?tcacattata?aataatacgc????180

aggatagtgt?tgttaatgtc?gataaattaa?cgtacgccgg?aaacctggaa?tcacttgctg????240

atgtttctga?ttctgaacgc?tatgtttttg?aacatgcgga?tatttgcgat?gctgctgcaa????300

tggcgcggat?ttttgctcag?catcagccgg?atgcagtgat?gcacctggct?gctgaaagcc????360

atgtggatcg?ttctatcact?ggccctgcgg?catttattga?aaccaatatt?gttggtactt????420

atgtcctttt?agaagccgct?cgcaattact?ggtctgctct?tgatagcgac?aagaaaaata????480

gtttccgttt?tcatcatatt?tctactgacg?aagtatatgg?cgacttaccc?catcctgatg????540

aagtaaatag?taatgaagcg?ttaccgctat?ttacggaaat?gacagcttat?gcaccaagta????600

gcccatattc?tgcttctaaa?gcatccagcg?atcatttagt?ccgcgcgtgg?aaacgtacct????660

atggtttacc?gaccattgtg?actaattgtt?ctaacaatta?tggtccttat?catttcccgg????720

aaaaactgat?tccattggtt?attcttaatg?ctctggaagg?taaggcatta?cctatttatg????780

gtaaggggga?ccaaattcgc?gactggttgt?atgtagaaga?tcatgcccgg?gcattgtaca????840

ccgttatgac?ccagggtgta?gtaggtgaaa?cctacaatat?cggtggtcgt?aacgagaaaa????900

aaaacctgga?cgtggtaaat?actatttgcg?atctgcttga?tgagattgta?cctaaacaag????960

ggtcttatcg?cgatcaaatt?atctacgtta?ctgaccgtcc?tgggcacgat?cgccgctatg???1020

cgattgatgc?atcaaaaatc?agcgatgagc?tgggctggaa?accgcaggaa?acctttgaat???1080

cgggaattcg?aaaaactatt?ggttggtatt?taaataattt?agagtggtgc?cgtcgtgtgc???1140

aagacggtag?ctatcatcgt?gaacgtttag?gattacagta?aatgaaaggt?atcatacttg???1200

caggtggttc?aggaactcgt?ttatatccag?tcacaatggc?tgtaagtaaa?caattgttgc???1260

cagtttatga?taaaccaatg?atttattacc?cattaagtac?attaatgttg?gctggtatac???1320

gaaatatact?tattataagt?acacctcaag?atattcctcg?ttttatgggg?ttattaggtg???1380

atggtagtca?atggggactt?cagttagaat?acaaaataca?gaatagtcct?gatggattag???1440

cgcaagcatt?tatattaggt?gaggatttta?ttggaaacga?taattgtgca?ttgattttag???1500

gggataatat?attctatgga?catgatctcc?agaaacacct?agaaatagcg?ctctcaaaag???1560

ataaaggtgc?tacagttttc?gcttatcatg?ttaaagaccc?tatgagatac?ggcgttgtag???1620

agtttgataa?acaaggtaaa?gccatttcac?ttgaagagaa?acctgaaatt?ccaaaaagta???1680

attatgcagt?aacgggattg?tatttttatg?ataataatgt?tgttgagata?gcgaaatcat???1740

taaaaccctc?gaagcgagga?gagctagaaa?taacggatgt?taatcgactc?tatttggagc???1800

ggggtgagct?ttctgtagct?atgatggggc?gaggttatgc?ttggctagat?actggaactc???1860

atgaaagtct?aattgaagct?agcaatttca?tccagacaat?tgaagctaga?cagggtttaa???1920

aagtatcctg?ccctgaagaa?atagcattta?ataaaaaatt?tattgataaa?acgcagttaa???1980

taaaattagc?taagcctttg?gagaaaaatt?catacggaaa?atatttaata?aaactggcag???2040

agtcgaattg?attttttatg?aatgattaat?ttgttcatag?aataaaaagt?gaatgcctcc???2100

atggttaatg?agttaattta?acaactaaga?aggtgagtat?ctatctctaa?gtgttattga???2160

gaaggtatat?gaattcaaat?gtaaaaaaaa?atattagcgc?tgtaaatgga?ttgaagtgga???2220

gtgcgatcga?aagaatatgt?tcacaaggta?tccagctact?tttaatgata?gtcttggcta???2280

gacaattagg?gcctggtgca?tttggcctta?ttggaatgct?gacaatattt?attacaatag???2340

gtcaggtctt?tattgatagt?ggttttagtg?ccgctctcat?tcgaaaaaat?gaaagaacag???2400

aatcagacta?tgcaactgtt?ttttacttta?acatgacagt?tgccattctg?ttttatgcag???2460

tgttattttt?ttgcgcccca?ttcatagctg?aattttataa?gcgtaacgaa?ttaattgaat???2520

taacaagagt?tctgggttta?acaataataa?taagtgcttt?tattattgtt?caacgaatac???2580

aattaagtgt?cattttggat?ttcaaaactc?aagctatatc?gtcattatcc?agtgtcataa???2640

tctcaggagg?gtgtgcacta?ttaatggcat?ataatggttt?tggtgtatgg?tcgttagtta???2700

tacagactat?taccatgggg?cttgttaatt?tagttatttt?aaatatatat?aatccatggt???2760

taccgaagag?gagtttttca?aaaaaatcat?ttcatggatt?tttttctttt?ggctccagac???2820

ttctgatttc?atcactgata?gattcaatat?acactaatat?ttatttggta?gttataggga???2880

agtcctttag?cgctagcaca?ctgggccaat?ttacacaagc?taatttatta?tcaaatacgc???2940

cggccatgac?gttaacgaca?gttgtacaaa?gagttaccta?tccattatta?agtaatgtga???3000

ataatgctaa?ggggaatatt?gacgagatat?atcttaggat?attaaggctt?actgccgcag???3060

ccgtttttcc?agtaatgttc?ctattggcaa?taattgctaa?accttttgtc?gttctatttc???3120

ttggccaaca?atgggaacct?gttgcggaat?taatgagcat?attatgtata?ggatactgtt???3180

tatatccagt?acatgctatt?aatctaaact?tattacaggt?aaaaggacga?actgatttat???3240

ttttgaagtt?agaaataata?aaaaaaactc?tcatcacggt?tattctaata?gtaacaatac???3300

catatggtgt?taaaataata?tgtataggta?tttttgcaca?gtattatata?tccttgttga???3360

taaatacata?ctatacaggg?aaactcagca?gcttaagtgc?aattgcacag?ataaaggcat???3420

tattgccaat?ttggttaatg?gcatctatca?gttcggcaat?tagttggttc?ttaataccaa???3480

gagagatatt?ttcggaatta?tatcaaataa?taggaatatt?gataaccaat?atttcattat???3540

atggaatagg?aatgtatctt?ttccaaaaag?atatttatga?aatggtaaag?tttttattta???3600

taaaaacaaa?ataattttat?gaacaaagag?atgaaaatgt?taaatggcaa?gattttagta???3660

acgcaaccat?ttttacctga?actaagagag?tttattccct?atctggaaaa?aatatgggaa???3720

aataaatggt?taacaaacaa?tggtccattt?catcagcaat?tagaaaatga?tttgtgccgt???3780

tatttaggcg?tggagtacgt?ctccttattt?aataatgcta?caattgctct?gattacggca???3840

gtccaatcat?tagaattgac?tggtgaagta?attacaaccc?cgtattcatt?tgtggcaaca???3900

actcactcat?tgatgtggaa?taatctaaat?ccagtatttg?tcgatgtcag?tagagataca???3960

tttaatatca?atccgtctca?aattgaagcg?gcgataacag?aaaaaacaac?agccataatg???4020

gcagtccatt?gttatggtaa?tccttgtgat?gtgattgcaa?tagaaaaaat?agccaaaaag???4080

tataaactta?aagtgatata?tgatgctgct?catgcttttg?gggtaaattt?taagggggaa???4140

agtttattaa?aatacggaga?tttatcggtt?gttagttttc?atgcaactaa?agtatttaat???4200

acatttgaag?gtggcgtgat?tatttgccca?aatgcagaaa?ctaagctcaa?aatagatcag???4260

ctaaaaaact?ttggttttga?agatgagcta?acaataaaat?caattggtat?taatggaaaa???4320

atgagtgagg?tcaatgcagc?atttggcctt?gtacagttga?aacatgttaa?tgaagctata???4380

agtaaaagaa?aagaaattaa?tgatttatat?ggcaagttat?tagggaatgt?gaaaggcata???4440

tcattagcaa?aatttgataa?actagctacg?aaaaattttt?catactaccc?gattcttatt???4500

gaagatgact?atgggatgag?tcgagatgaa?ttatgtcatt?tacttcaaaa?aaataatata???4560

tttgctagga?aatattttta?tcctttaata?agcgatatgg?atttatataa?aaatatggag???4620

tcagcgagaa?aagaaaatct?acacattgct?cgagatattt?ccaacaaagt?actttgctta???4680

ccaatttatg?cagatcttga?tttagacatt?gtgagattta?tagcgagagt?aataggtaat???4740

aaaaaatgaa?attagcaata?atgcaaccct?atctatttcc?ttatctaggt?tattatcaat???4800

taatgtcatc?agttgataag?tttattattt?atgatgatgt?ttcatacatc?aaaaatggtt???4860

ggataaacag?aaatagaatt?cttgttaatg?gtaatgctca?ttattttact?gtgccagtta???4920

taggtgggag?ttgtaataat?aaaataaata?ctgttaaaat?tgacaagaca?aagaaaaaag???4980

ctatcaataa?aatcattatt?acaattgaac?aagcctataa?aaaatccgtt?ttttttgatg???5040

aggtctttcc?agtcatttat?ggtgtgttat?ctaaagagta?tgatttcata?tccgatctgg???5100

caataacgtc?attattgtca?ataaaaaaca?aacttgatat?tggagcagaa?gttgttttga???5160

cttcgaccaa?ctatggtaac?aataatttaa?cttcgcaaga?tcgcgtcatt?gatattaatg???5220

taaaagagca?tgcgtccact?tatattaatt?ctgaaggcgg?gagattactt?tatgataaaa???5280

agacattcaa?actaaatggc?gtaaatttga?aatttataca?tccagaaatt?ttaccttaca???5340

aacaactttg?taatggtgag?tttgtaccat?ccttatcaat?tatagatgta?gtaatgaata???5400

atggttggga?tactacaaag?cagttagtaa?atagctttga?actgaaggat?tgaacaatgc???5460

gtgagcataa?ttatgcgatc?ggaggatatt?tttcactaga?attgcaggcg?cttaaaaata???5520

acactgcaaa?tgaaaaagta?tatttacaat?cagcgagggc?atgttttcaa?ttactgctag???5580

aaagcattga?agttagtaga?gtatggttac?catattatat?atgtgatgtc?gtcgttgata???5640

caataaatga?aattgggatt?gaaatcttgt?attatagtat?ttctaaagat?tttattccgc???5700

agacgtttcc?agttttagaa?gaaaatgatg?tttttgtcta?tgttaattat?tttggggtgt???5760

gtgatgagca?aacaaagtta?attttgcaaa?aatatcctcc?agagaaagtt?atcttagata???5820

actcacaagc?tttttatagc?ggacacaata?ataatttagg?aacaatttat?tcacctagga???5880

aattttttgg?cgtccctgat?ggtggtattc?tgataactaa?tcagactata?atattacctt???5940

cttcacagga?taatgattca?tcacagtata?taaatcatct?cattgggcgt?ctaatatccc???6000

atcccagcga?atactatact?gattatatta?aagctgaaga?acgattaaaa?aaaattaaaa???6060

aagccaaagt?gatgtcttat?ttaactcgga?aattattaga?ctccatcaat?tatcatgaga???6120

ttaaaaaaat?aagggatgat?aattttaaat?tccttcacca?tgccctcgaa?aagataaatg???6180

gaataaaaat?tccggagata?gtcaatggtc?cattatgcta?tccattgctt?tcaaaaaaca???6240

acaaactgaa?agatatttta?atcaagaatg?aaatttatgt?gcctacatat?tggaaagatg???6300

tattgaatcg?agtagatata?aactcgacag?aatttgaatt?tgtctcaaac?ttaatacctc???6360

tgccatgtga?tcaacgttac?tcatctatcc?agatgaagaa?aataatcaat?attgtacttg???6420

aggaaaacta?atgaatatca?taggaaaaac?agttaagttg?cgtgctgtgg?aaattgatga???6480

tttagaattg?ttaaataaat?gggctaacga?tccggaaatt?tggtatatgc?ttggtggttg???6540

gcatttcccc?tattccaaaa?ataatactga?aaaatggata?aaaaatattg?ataataatga???6600

ttcgaaaaat?caaatattcg?ctattgaaac?tgaagagcat?ggattgattg?gcactgctaa???6660

cttagtaaat?attgattgga?aaaataaaaa?tgcatttcat?gggataatgt?tgggaaatgt???6720

tgaaacacga?ggtaaagggt?acgcgcaaga?cgttgtaatg?agtctgatga?gatatgcctt???6780

tgatgaatta?ggattaaatc?gacttgatgg?tgatatgatt?gaatataata?aattatcaat???6840

taatttttac?atcaaaagat?gtggttggaa?aatcgaggga?attaagaaag?agtggttctt???6900

tagaaagggg?caatattttg?ataaagtagt?tgttggaata?accaaaaaag?aatatttaga???6960

acatattgag?aaaagcaagt?attgggagac?aaaatgatgg?aacgttttaa?attgggggac???7020

acagcaactt?atactcaaac?tataactgat?gcagatatta?aaagcttcgc?aggtatatct???7080

ggggataata?atccagtaca?tatgagtgac?gaatatgcag?aaggttcaag?gtttaaaaag???7140

cgtatagctc?atggtctgat?atctgctagt?tttttttcag?ctctatttgg?tacaaaatta???7200

cctgggcctg?gatgtgtata?tgtaaatcaa?agcttgaaat?ttcttagacc?tgtttatatt???7260

aatgatacgg?ttacagcgcg?tgtagtctta?acagacattg?atgttgtaaa?aagaagactc???7320

ttttttgata?cgatttgtga?agttaaccga?aaaaaagtta?tcacggggaa?ggctgaaatt???7380

tacttgccag?aataaagcgc?tttaaatttg?atgttaactt?gctacatatt?gatgttttat???7440

ctctttttat?ctttgaagtt?tatcaaaaat?gaatgtttcc?atgtcagata?taaaagttag???7500

tgtatgtatt?atttctttta?accaacaaaa?ctatatacga?cagtgcttgg?atggtgtctt???7560

ttctcagaaa?acaaattttg?agtatgaggt?tattatacgt?gatgattgca?gtaccgataa???7620

tacatattta?acaataatgg?aatatattga?cactttagat?gaagagaaaa?aaaagaacat???7680

aaaaataacg?gtacttgatg?gcacgaaaaa?tattggagca?aataataatt?ttatcgaaac???7740

atttaagact?tcagtaggtc?aatggctagc?tatatgtgag?ggagatgatt?attggtgtga???7800

tcaggggaaa?ttacaaaaac?agtacgacta?cgctatttca?catagtgatt?gctcattagt???7860

cgtgcatccc?gctctgataa?gtgaaaataa?tgtaatacgt?aaaacatctt?gggcgtgtat???7920

gaataaaaca?ataaatcagc?ttagcgatgt?aatacgtgca?aaaggacagt?tctcaccaac???7980

tggctcctat?tttttcaaac?gagaaatctt?aaatgttcta?ccattgtggt?tttcaacagc???8040

tcctgttggt?gactattaca?tggaaatctt?tgctacatcg?cttggatctt?gtcatacaat???8100

tcctgatgct?atgtcagttt?atagaatcaa?ttcaacgggg?tcttggtctg?acctgttaaa???8160

aaaagataga?aatggccaac?ggattattaa?tacttatctt?tctcaattgg?agtatttgga???8220

taaacttgcc?gaaatctttc?cttcttgtgt?cgatgatatc?acaataaaaa?gatcgcatgc???8280

acaatatgca?gcagcaatgg?gttatcttgc?aaatggtgat?tatactaatt?ttcgtatatt???8340

aatggataag?tcagatacaa?acggctggta?tgataatatg?cattctattt?tttattattt???8400

aagaaatagc?agggcgcttt?ccatgttaat?gttcagattc?aagcctgcaa?ttaaatctgc???8460

tgtcatgtgg?gggcgtcgtg?tatttaacaa?ctaattattg?cgactttttg?tcccgaactc???8520

gaatcgttag?gcgcatattt?ttgttagttt?atttgatttc?aaatgtttac?gcctatatag???8580

tgtttactga?aacaggagta?ttaatagggg?attatcaagg?agtactcata?atatataaag???8640

atcaggttgt?gtatttattg?atgttaacaa?taatgtcata?ttatattgtt?tgtggtcctg???8700

tttttaactg?tctatcaaaa?gtaaaaatta?gactaaggac?atttaaaggc?ttaaactctt???8760

ttgcttatat?cctgtttttt?tttcaaatcc?tatttgctgt?ttttaatatt?tcaacaggtt???8820

cgaatgccgc?gggcactgat?tttcaaactg?gtggagtcat?aagactatta?tggctttttt???8880

taccagttga?ctatctattt?tatatatatt?atttcgtagg?ccgagagaaa?aaagtaagta???8940

aaatatatct?ggccaatgtg?gttattttta?ttctatcaat?gttaagtaga?ggatggcttg???9000

ggtggacttt?ggttttgctt?tatgcagagt?tatgtttctt?tttttattca?caaaaaaaaa???9060

ttaaaataaa?atatcttatc?ttattgtttt?tcttgcttat?tgttgcacca?ttagcattta???9120

gtctaaaaat?tcaattgcgt?gctgatttgt?attccagtgg?tattggtggg?gttatatcaa???9180

ctttaagtaa?tattgattac?attcaatctt?ataataattt?catcgctggt?ttcttatcca???9240

gaatacaaca?gttatcaaat?attgttttct?tttatgacca?tcaacaagaa?ttatataaat???9300

ttgtttcttc?agatattgta?tcaaactatg?cttgggaagg?attacctcaa?caaactgtag???9360

ctaaacttct?aggattagat?cctggtgttg?atatgcatat?ttttctttat?agtcattata???9420

tttcttcaac?ttcagaggct?gtaactacat?tgcaagttgg?attcatttct?tggctctttt???9480

taggcaccct?gtcatccgta?ttttatccac?tgtttgtctt?tgctattata?tgcatatcct???9540

tatttttatc?taaaaaactt?ggaggggaaa?aactatgtgc?acttacgtgg?attatgattt???9600

ttctttctat?tatgtgcggg?tggtataatg?cttatttggt?atatatgcag?gcattaatta???9660

cattttattt?tatcatgggt?tttttgaatt?taattactct?agaaaaaaca?aaaattaatc???9720

atacatgatg?gatgacgaga?aatatatgag?taatttagta?atagttaacg?caacagcttt???9780

agcgtctagc?ggcgctttga?cgattttgaa?ccagttttta?gaacacgcac?taaatgattc???9840

taagcataag?tatctttgtt?ttatacatga?gagtgttaaa?aaaaagagtg?ttaataatgt???9900

caccgttatt?actataaaaa?aacaaagttt?tattcaacga?gtatggtggg?atctatatgg???9960

attgaataaa?tatataaaga?aaaataattt?gaaacctaaa?aaaatagttt?ccttgcagaa??10020

tacatctgta?aatagtaatt?tcgagcagat?aatatatcta?caccagtcga?ttccctttag??10080

caattttaga?ataaaaatta?aatttcaata?tgctatattt?ttcatgtata?aatacgtgta??10140

tccttttttt?atattcttta?gaactaaaaa?caccactttc?gttgttcaga?ccgattggat??10200

gaaagatgct?atcgtagcaa?agaaaaaaat?tgccaaagaa?cgagttcatg?taattaaacc??10260

tgatataata?ttaccggtta?ataatcttgc?ttaccctgat?gaacatgaca?aagataattc??10320

atctgaagtg?ttttttttat?atccggccac?gccacttttc?tataaaaacc?atttgattat??10380

cttagatgca?atgaggattc?taaaaacaga?aggaatactt?gctaatacaa?aattccaagt??10440

gacttttaaa?caagatgata?atgatgagtt?agcaattaaa?atagccaaat?atgatttagt??10500

tgataatata?agcttcttgg?gagtattatc?ctatgaagaa?ttgtttcata?aatacttaaa??10560

agcagatgct?atactttttc?ctagctactt?ggaaagcttt?ggtttaccgt?tagcagaggg??10620

cgcaatgctt?gggaaatata?taatttgtag?cgatttgcca?tatgcaagag?atgtgcttaa??10680

caattattca?aatgtagaat?atataaatca?tgatgatgcc?agtaaatggg?cattagctat??10740

gaaggaaata?atattaaaga?atagaaataa?tactttatct?gaaagtgaaa?atagctatat??10800

gtattgtccg?aaaacaagct?gggcagattt?cttcgagtta?atctaattga?ggtatacgat??10860

gtttaaaaat?aaggtacttc?tcattactgg?tggcacaggt?tcctttggga?atgccgtttt??10920

acaacgcttt?ttagattcag?atattggcga?aattcgaatc?tttagtcgtg?atgagaaaaa??10980

acaagatgat?atgcgtaaaa?aatacgcaag?tgataagtta?aaattttata?taggggatgt??11040

gagggactat?agcagtgttt?taacagcgac?tcgtggggtt?gattttattt?atcatgcagc??11100

agcattaaag?caagtacctt?catgtgagtt?ttatccaatg?gaagctgtca?agactaacgt??11160

tattggaact?gataacgtac?ttgaagctgc?aattgcaaac?aaagtttcac?gtattgtttg??11220

tttgagtacc?gataaagctg?tttatccaat?taatgctatg?ggtacatcta?aggctatgat??11280

ggaaaaagtt?atcgtagcta?agtcaagaaa?cttacctaaa?gatattacta?tttgtgcgac??11340

gcgatatgga?aatgtaatgg?cttcccgtgg?ttcggtcatc?cctctgttta?ttaatcaaat??11400

acttgaagga?cgcccaatca?ctatcaccga?tccgtgtatg?actcgattca?tgatgacttt??11460

ggatgatgct?gtagatcttg?tattacatgc?ttttgaacat?ggtactaatg?gtgatatttt??11520

tgttcaaaag?gcaccagctg?caacaataga?tacacttacc?aagtcattac?ttaagttaac??11580

taaacaacat?tctcacccaa?ttaatatcat?tggcactcgc?catggtgaaa?aactatttga??11640

ggtactctgc?agtcgggaag?aaatgttggt?tgcagaagat?caaggtaatt?actatcgtat??11700

acctagtgat?aaacgtgatc?taaattatga?aaaattcttc?gacaaaggga?ctaaagaaat??11760

tcagtttgtt?gaagactata?attctcataa?tacccgtcgt?ttagatgttg?atgaaatggt??11820

tgctcttctt?agaaaattag?attacatcaa?taaaattgag?gctggtgaaa?aggcagatcc??11880

cgatgcgtaa?aaaaattcta?attgttggcg?caaatggcat?gttaggtagt?agtttactac??11940

gctacttttc?atcaattgga?gattatgaag?ttcttggtac?gacaagaagt?atggtcgttg??12000

caaaacaact?tgagcaaaag?cacaatgtga?aaattattga?caacgttgat?gttattgatt??12060

ttaaacgatt?agagactgta?gtagtagagc?ataagccaaa?tattgttttc?aactgcgttg??12120

ggataattaa?acaacttgat?gcagcaaaaa?acaatatatt?atctattgaa?attaactcat??12180

tacttccaca?taaattagct?caattatgtt?cagctcatag?tgctaaactc?atacattttt??12240

caacagattg?catttttaaa?ggtaccaaag?gtaattatgt?tgaggatgat?gagtctgatg??12300

caattgattt?atatggtaaa?tctaagttct?tgggtgaagt?tgaatataat?gggcatttaa??12360

ctttgcggac?ttctattatt?ggccatgagt?tgggatcaaa?tcatagtctt?gttgactggt??12420

ttttatcaca?gaaaaaatcg?gtgaaaggat?ttactaatgc?gatattctca?ggcctcccaa??12480

cttgttatat?ggcagaggtt?atccataaat?atgttcttcc?caacaatctt?gctggtttat??12540

ttcatttaag?tgtagagcca?attagtaaat?atgatttgtt?aaatattatt?aaaatagtat??12600

atggagtaag?tactgatata?gagccaacca?acgaatttaa?aatagaccgc?agcttgaatt??12660

ctacgctatt?tcgtaataag?acaaattttg?tcccagaatc?ttgggataaa?ttaattgaaa??12720

agatgaaaga?tgaatacaat?aaatatttct?aattcaaaaa?tacgggttgt?tactgtagtt??12780

ggcactaggc?ctgaaatcat?tcgtttatcg?agggtaattg?ctgttctcga?tgagtacact??12840

gaacattttc?ttgttcatac?cggacaaaac?tatgattacg?agcttaatga?ggtattcttt??12900

aatgagcttg?aaatcagaaa?acctgatttt?ttcatgaatg?ccgcgggcca?gaatgctgct??12960

gagaccatcg?gaaatgtaat?tattgaagct?gataagatat?ttgataaatt?gcaacctgaa??13020

gctttactta?ttcttggtga?tacaaatagt?gctctagttt?ccattgcagc?taaaagacga??13080

aaaattccta?tctttcatat?ggaagcgggc?aaccgatgct?ttgattacag?agttccggag??13140

gaaataaata?ggaagattgt?tgatcatatt?tcagatatta?atttaacata?tagtgagata??13200

gctagggatt?atcttttgag?ggaagggctt?cctgctgatc?aaataatcaa?aaccggtagc??13260

cctatgcgag?aggttctaaa?tttttataag?gataaaattt?catcttcttc?tattcttgaa??13320

aaacttaacc?ttcaagcatc?tagttatttc?cttgttagta?gtcatcgaga?agagaatgtt??13380

gattcgcctg?agaagttgcg?ctctcttatt?gagacgttaa?atatagtatc?tgagaaatat??13440

aaactgccag?taatagtatc?aacgcatcct?cgtactcgta?atagaattga?tgctttaggt??13500

atcacggtaa?gtaataatat?cattttttct?aagccattcg?gatttttaga?ttatataaaa??13560

ctgcagcaaa?atgctcgtgt?cgtgctctct?gatagtggaa?ctataactga?agaatcatca??13620

gttttgaatt?ttccagcctt?aaacctgcga?gaagtacatg?agagaccaga?aggatttgaa??13680

gaagctgttg?ttatgtttgt?cgggctagat?agaaacagaa?ttattcaggg?aatcgatatt??13740

ttgcagggtc?aaaaacgtgg?tgataatgat?cgtgaccttc?atatggttac?agattatcag??13800

gcagacaatg?tttcaattaa?aattcttaga?attattatga?gctatacaaa?ctttattaat??13860

caaaaggttt?ggaaaaaatt?ctaatgcgta?ttgcgttaat?atgtgatgac?tatttgcctg??13920

atagtactcg?tgttagtgct?aagatgatgc?atgaattggc?ttgtgaactt?ttagaaaaag??13980

gacatgagcc?aatcgttatt?tgtccttgca?ataaaataca?gacgctcgaa?attttgaatt??14040

tggatggtgt?cgtcgtttat?aagttcccta?atggtgctat?aaagaatgta?tctaaaatat??14100

caagagcaat?aaacgaatcc?atgctatcat?tcaacgcatg?gcgatttatt?ggaaaatata??14160

ttcgagaaac?taaaattgat?ggtgttgtgt?actactcacc?atctattttt?tttggaaaat??14220

tagcaaataa?aatcaaagaa?aattggcatt?gtaaatcata?tttaatctta?agggattctt??14280

ttcctcagtg?gctggtagat?caggggataa?ttaaggaggg?agggcttgcg?gaacgatatt??14340

ttaggtactt?cgagcagatc?aattatgatg?ctgcagatta?tattggttta?atgtcggata??14400

gaaacaaaga?tattttcatt?aataaatatc?agaataaata?caaggtgcaa?actttattca??14460

attgggctga?ctttaaaggt?atagataata?ttcccagtgc?gactctacga?tcaaaattag??14520

cacttcaaaa?caaagtaatt?tttttttatg?gaggcaatat?tggtcatgct?caggatatga??14580

tgaatttaat?gaggttggtt?agatcagcat?cttatcgtga?tgatgtgcat?tttttattga??14640

ttggacaagg?tgatgaagtt?tcccttgtaa?agcagtttat?tatagataat?tctttaaaaa??14700

actgtacgta?tttaccatcc?ataacacaat?cagagtttaa?gtctgtatta?aagattgttg??14760

atgttggttt?gtttagcctt?gctaaaaatc?atactgttca?taacttccct?gataaattgt??14820

tagggtatat?ggcaaataaa?ctccctatat?taggtagtgt?taatgcaagc?aatgatgtta??14880

tggagattat?caatggggca?aaggccgggt?ttgtttttgt?taacggaaat?gacgaggctt??14940

tacttaatgc?tgcaataaat?cttgcggatg?atactcagct?aagaaaaaac?ctgggatgta??15000

atgcttactc?tttattacaa?gaaaaattct?ctgtggaaat?ggctgctgaa?aagatattaa??15060

gcagcttatt?ttcctaacag?taatatggtg?gttttatgaa?agtgtctttt?gtaagtcagg??15120

atgatttcga?gaatttaatt?caggaagcat?ttgcatccgt?tcgcttgaga?tctcacttac??15180

tcctccatga?aagccctaat?gatgctgtcc?agagaataat?gataggactt?gtcaaaggaa??15240

cgtatatccc?gcctcatttt?catgaatttc?agcatcaatg?ggaacatttc?catgtgtttc??15300

aaggtgaggt?tgaattaatg?ctatttgata?gtaacggctg?tttaaataaa?aaggttattc??15360

ttggtgggca?gagtaagaat?attattgcac?aaatttcacc?actcacccca?catacactag??15420

tttgtagatc?tcctacagca?gttatcatgg?agattaaaga?aggtcctttt?gatgaaaaat??15480

gcgctaaagt?catcccctca?tggtcatata?gtgaagatta?ttccatttta?tccagagata??15540

ggatcatcgc?aatgatgggc?caattatcaa?ttggagatag?atttagtctc?taaacttcac??15600

agcaattagc?gaagtaatct?tctttttcat?accttctata?ttatatttat?ccactactat??15660

tatagctttg?aatactgagt?taacatctac?actacattca?agccgtgcat?acgtcgcggt??15720

gaccacccct?gacaggagta?aataatgtca?aagcaacaga?ttggcgtagt?cggtatggca??15780

gtgatggggc?gcaaccttgc?gctcaacatc?gaaagccgtg?gttataccgt?ctctattttc??15840

aaccgttccc?gtgaaaagac?ggaagaagtg?attgccgaaa?atccgggcaa?gaaactggtt??15900

ccttactata?cggtgaaaga?gtttgttgaa?tctctggaaa?cgcctcgtcg?catcctgtta??15960

atggtgaaag?caggtgcagg?cacggatgct?gctattgatt?ccctcaaacc?atatctcgat??16020

aaaggcgaca?tcatcatcga?tggtggtaac?accttcttcc?aggacaccat?tcgtcgtaac??16080

cgtgagcttt?ccgcagaagg?ctttaacttc?atcggtaccg?gtgtttccgg?tggtgaggag??16140

ggcgcactaa?aaggtccttc?cattatgcct?ggtgggcaga?aagaagccta?tgaacttgtt??16200

gcgccgatcc?tgaccaaaat?cgccgcagtg?gctgaagacg?gtgagccatg?cgttacctat??16260

attggtgccg?atggcgcagg?tcactatgtg?aagatggttc?acaacggtat?tgaatacggc??16320

gatatgcagc?tgattgctga?agcctattct?ctgcttaaag?gtggtctgaa?cctcaccaac??16380

gaagaactgg?cgcagacctt?taccgagtgg?aataacggtg?aactgagcag?ctacctgatc??16440

gacatcacca?aagatatctt?caccaaaaaa?gatgaagacg?gtaactatct?ggttgatgtg??16500

atcctggatg?aagcagcaaa?caaaggtacg?ggcaaatgga?ccagccagag?tgcgctggat??16560

cttggcgaac?cgctgtcgct?gattaccgaa?tctgtgtttg?cacgttatat?ctcttctctg??16620

aaagatcagc?gtgttgccgc?gtctaaagtt?ctctctggtc?cgcaagcgca?gccagcaggc??16680

gacaaagctg?agttcatcga?aaaagttcgt?cgtgctctgt?atctgggcaa?aatcgtttct??16740

tacgctcagg?gcttctctca?gctgcgagcg?gcgtctgaag?agtacaactg?ggatctgaac??16800

tacggcgaaa?tcgcgaaaat?tttccgtgct?ggctgcatca?tccgtgcgca?gttcctgcag??16860

aaaatcaccg?atgcttatgc?cgaaaatccg?cagatcgcta?acctgctgct?ggctccgtac??16920

ttcaagcaaa?ttgccgatga?ctaccagcag?gcgctgcgtg?atgtcgttgc?ttatgcagta??16980

cagaacggta?tcccggttcc?gaccttcgcg?gctgcggttg?cctattatga?cagctaccgc??17040

gccgcagttc?tgcctgcgaa?cctaatccag?gcacagcgcg?acta???????????????????17084

Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O123 type bunch and primer and PCR data wherein

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								??wzx	The transhipment enzyme	2169-3614	??2627-2646	??3275-3294	??668bp	????0 *	?????55
			??2806-2825	??3376-3395	??590bp	????0 *	?????60
								??wzy	Polysaccharase	8478-9728	??8986-9005	??9484-9503	??518bp	????0 *	?????55
			??8603-8622	??9392-9409	??888bp	????0 *	?????50

^*Only in intestinal bacteria O123 type, obtain a correct band

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

The bacterium source that contains in this group of group number

1, wild-type e. coli 01,02,05,07,08,09,012,013,014,015,016,017,018,019ab, IMVS ^a

020，021，022，023，024

2, wild-type e. coli 04,010,025,026,027,028,029,030,032,033,034,035, IMVS ^a

036，037，038，040，041，042，043

3, wild-type e. coli 06,044,045,046,048,049,050,051,052,054,055,056, IMVS ^a

057，058，060，061，062，053

4, wild-type e. coli 063,065,066,069,070,071,074,075,076,077,078, IMVS ^a

079，080，081，082，083，068

5, wild-type e. coli 084,085,086,087,088,089,090,091,092,098,099, IMVS ^a

0101，0102，0103，0104，0105，0106，097

6, wild-type e. coli 0107,0108,0109,0110,0111,0112ab, 0112ac, 0113, IMVS ^a

0115，0116，0118，0120，0125，0126，0128，0117

7, wild-type e. coli 0129,0130,0131,0132,0133,0134,0135,0136,0137, IMVS ^a

0138，0139，0141，0142，0143，0144，0145，0140

8, wild-type e. coli 0146,0147,0148,0150,0152,0154,0156,0157,0158, IMVS ^a

0159，0160，0161，0163，0164，0165，0166，0153????????????????????b

9, wild-type e. coli 0168,0169,0170,0171,0172,0173, c

Shigella dysenteriae D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 d

10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d

B16，B17，B18

11, shigella flexneri F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, d

DS，DR

12, wild-type e. coli 03,011,039,059,064,073,096,095,0100,0114,0151,0155, IMVS ^a

0124，0162，0167，0121，0127，0149，0119

13, the 6th group of bacterial strain adds intestinal bacteria reference culture 0123 IMVS ^a

^*For the convenience that detects, we are divided into one group with every 13-19 bacterium, 12 groups altogether

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

b.??Statens?Serum?Institut，Copenhagen，Denmark

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

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

Table 3 intestinal bacteria O123 type O-antigen gene structure iron

Escherichia?coli?O123?O?antigen?gene?cluster

orf#??rmlB??rmlA??wzx???vioA?orf5??orf6?orf7?orf8?orf9??wzy???orf11??fnlA??qnlA??qnlB?orf15?orf16??gnd

G+C％?43.0???35.3?31.9??30.7?29.4??31.2?31.2?35.3?34.1??29.8??30.0???36.4??31.8??34.9??33.7????37.6

Table 4 intestinal bacteria O123 type O-antigen gene cluster gene position

ATTGGTAGCT?GTAAGCCAAG?GGCGGTAGCG?TGCATTAATA?CCTCTATTAA?TCAAACTAAG??????60

Orf1's is initial

AGCCGCTAAT?TTAACAGCAT?GCTCTGAAGT?AATATGGAAT?AAATTAA GTG?AAAATACTTG???120

TTACTGGTGG?CGCAGGATTT?ATTGGTTCTG?CTGTAGTTCG?TCACATTATA?AATAATACGC?????180

AGGATAGTGT?TGTTAATGTC?GATAAATTAA?CGTACGCCGG?AAACCTGGAA?TCACTTGCTG?????240

ATGTTTCTGA?TTCTGAACGC?TATGTTTTTG?AACATGCGGA?TATTTGCGAT?GCTGCTGCAA?????300

TGGCGCGGAT?TTTTGCTCAG?CATCAGCCGG?ATGCAGTGAT?GCACCTGGCT?GCTGAAAGCC?????360

ATGTGGATCG?TTCTATCACT?GGCCCTGCGG?CATTTATTGA?AACCAATATT?GTTGGTACTT?????420

ATGTCCTTTT?AGAAGCCGCT?CGCAATTACT?GGTCTGCTCT?TGATAGCGAC?AAGAAAAATA?????480

GTTTCCGTTT?TCATCATATT?TCTACTGACG?AAGTATATGG?CGACTTACCC?CATCCTGATG?????540

AAGTAAATAG?TAATGAAGCG?TTACCGCTAT?TTACGGAAAT?GACAGCTTAT?GCACCAAGTA?????600

GCCCATATTC?TGCTTCTAAA?GCATCCAGCG?ATCATTTAGT?CCGCGCGTGG?AAACGTACCT?????660

ATGGTTTACC?GACCATTGTG?ACTAATTGTT?CTAACAATTA?TGGTCCTTAT?CATTTCCCGG?????720

AAAAACTGAT?TCCATTGGTT?ATTCTTAATG?CTCTGGAAGG?TAAGGCATTA?CCTATTTATG?????780

GTAAGGGGGA?CCAAATTCGC?GACTGGTTGT?ATGTAGAAGA?TCATGCCCGG?GCATTGTACA?????840

CCGTTATGAC?CCAGGGTGTA?GTAGGTGAAA?CCTACAATAT?CGGTGGTCGT?AACGAGAAAA?????900

AAAACCTGGA?CGTGGTAAAT?ACTATTTGCG?ATCTGCTTGA?TGAGATTGTA?CCTAAACAAG?????960

GGTCTTATCG?CGATCAAATT?ATCTACGTTA?CTGACCGTCC?TGGGCACGAT?CGCCGCTATG????1020

CGATTGATGC?ATCAAAAATC?AGCGATGAGC?TGGGCTGGAA?ACCGCAGGAA?ACCTTTGAAT????1080

CGGGAATTCG?AAAAACTATT?GGTTGGTATT?TAAATAATTT?AGAGTGGTGC?CGTCGTGTGC????1140

The termination orf2's of orf1 is initial

AAGACGGTAG?CTATCATCGT?GAACGTTTAG?GATTACAG TA?AATGAAAGGT?ATCATACTTG??1200

CAGGTGGTTC?AGGAACTCGT?TTATATCCAG?TCACAATGGC?TGTAAGTAAA?CAATTGTTGC????1260

CAGTTTATGA?TAAACCAATG?ATTTATTACC?CATTAAGTAC?ATTAATGTTG?GCTGGTATAC????1320

GAAATATACT?TATTATAAGT?ACACCTCAAG?ATATTCCTCG?TTTTATGGGG?TTATTAGGTG????1380

ATGGTAGTCA?ATGGGGACTT?CAGTTAGAAT?ACAAAATACA?GAATAGTCCT?GATGGATTAG????1440

CGCAAGCATT?TATATTAGGT?GAGGATTTTA?TTGGAAACGA?TAATTGTGCA?TTGATTTTAG????1500

GGGATAATAT?ATTCTATGGA?CATGATCTCC?AGAAACACCT?AGAAATAGCG?CTCTCAAAAG????1560

ATAAAGGTGC?TACAGTTTTC?GCTTATCATG?TTAAAGACCC?TATGAGATAC?GGCGTTGTAG????1620

AGTTTGATAA?ACAAGGTAAA?GCCATTTCAC?TTGAAGAGAA?ACCTGAAATT?CCAAAAAGTA????1680

ATTATGCAGT?AACGGGATTG?TATTTTTATG?ATAATAATGT?TGTTGAGATA?GCGAAATCAT????1740

TAAAACCCTC?GAAGCGAGGA?GAGCTAGAAA?TAACGGATGT?TAATCGACTC?TATTTGGAGC????1800

GGGGTGAGCT?TTCTGTAGCT?ATGATGGGGC?GAGGTTATGC?TTGGCTAGAT?ACTGGAACTC????1860

ATGAAAGTCT?AATTGAAGCT?AGCAATTTCA?TCCAGACAAT?TGAAGCTAGA?CAGGGTTTAA????1920

AAGTATCCTG?CCCTGAAGAA?ATAGCATTTA?ATAAAAAATT?TATTGATAAA?ACGCAGTTAA????1980

TAAAATTAGC?TAAGCCTTTG?GAGAAAAATT?CATACGGAAA?ATATTTAATA?AAACTGGCAG????2040

The termination of orf2

AGTCGAAT TG?ATTTTTTATG?AATGATTAAT?TTGTTCATAG?AATAAAAAGT?GAATGCCTCC??2100

ATGGTTAATG?AGTTAATTTA?ACAACTAAGA?AGGTGAGTAT?CTATCTCTAA?GTGTTATTGA????2160

Orf3's is initial

GAAGGTAT AT?GAATTCAAAT?GTAAAAAAAA?ATATTAGCGC?TGTAAATGGA?TTGAAGTGGA??2220

GTGCGATCGA?AAGAATATGT?TCACAAGGTA?TCCAGCTACT?TTTAATGATA?GTCTTGGCTA????2280

GACAATTAGG?GCCTGGTGCA?TTTGGCCTTA?TTGGAATGCT?GACAATATTT?ATTACAATAG????2340

GTCAGGTCTT?TATTGATAGT?GGTTTTAGTG?CCGCTCTCAT?TCGAAAAAAT?GAAAGAACAG????2400

AATCAGACTA?TGCAACTGTT?TTTTACTTTA?ACATGACAGT?TGCCATTCTG?TTTTATGCAG????2460

TGTTATTTTT?TTGCGCCCCA?TTCATAGCTG?AATTTTATAA?GCGTAACGAA?TTAATTGAAT????2520

TAACAAGAGT?TCTGGGTTTA?ACAATAATAA?TAAGTGCTTT?TATTATTGTT?CAACGAATAC????2580

AATTAAGTGT?CATTTTGGAT?TTCAAAACTC?AAGCTATATC?GTCATTATCC?AGTGTCATAA????2640

TCTCAGGAGG?GTGTGCACTA?TTAATGGCAT?ATAATGGTTT?TGGTGTATGG?TCGTTAGTTA????2700

TACAGACTAT?TACCATGGGG?CTTGTTAATT?TAGTTATTTT?AAATATATAT?AATCCATGGT????2760

TACCGAAGAG?GAGTTTTTCA?AAAAAATCAT?TTCATGGATT?TTTTTCTTTT?GGCTCCAGAC????2820

TTCTGATTTC?ATCACTGATA?GATTCAATAT?ACACTAATAT?TTATTTGGTA?GTTATAGGGA????2880

AGTCCTTTAG?CGCTAGCACA?CTGGGCCAAT?TTACACAAGC?TAATTTATTA?TCAAATACGC????2940

CGGCCATGAC?GTTAACGACA?GTTGTACAAA?GAGTTACCTA?TCCATTATTA?AGTAATGTGA????3000

ATAATGCTAA?GGGGAATATT?GACGAGATAT?ATCTTAGGAT?ATTAAGGCTT?ACTGCCGCAG????3060

CCGTTTTTCC?AGTAATGTTC?CTATTGGCAA?TAATTGCTAA?ACCTTTTGTC?GTTCTATTTC????3120

TTGGCCAACA?ATGGGAACCT?GTTGCGGAAT?TAATGAGCAT?ATTATGTATA?GGATACTGTT????3180

TATATCCAGT?ACATGCTATT?AATCTAAACT?TATTACAGGT?AAAAGGACGA?ACTGATTTAT????3240

TTTTGAAGTT?AGAAATAATA?AAAAAAACTC?TCATCACGGT?TATTCTAATA?GTAACAATAC????3300

CATATGGTGT?TAAAATAATA?TGTATAGGTA?TTTTTGCACA?GTATTATATA?TCCTTGTTGA????3360

TAAATACATA?CTATACAGGG?AAACTCAGCA?GCTTAAGTGC?AATTGCACAG?ATAAAGGCAT????3420

TATTGCCAAT?TTGGTTAATG?GCATCTATCA?GTTCGGCAAT?TAGTTGGTTC?TTAATACCAA????3480

GAGAGATATT?TTCGGAATTA?TATCAAATAA?TAGGAATATT?GATAACCAAT?ATTTCATTAT????3540

ATGGAATAGG?AATGTATCTT?TTCCAAAAAG?ATATTTATGA?AATGGTAAAG?TTTTTATTTA????3600

The termination orf4 of orf3 initial

TAAAAACAAA?A TAATTTT AT?GAACAAAGAG?ATGAAAATGT?TAAATGGCAA?GATTTTAGTA3660

ACGCAACCAT?TTTTACCTGA?ACTAAGAGAG?TTTATTCCCT?ATCTGGAAAA?AATATGGGAA????3720

AATAAATGGT?TAACAAACAA?TGGTCCATTT?CATCAGCAAT?TAGAAAATGA?TTTGTGCCGT????3780

TATTTAGGCG?TGGAGTACGT?CTCCTTATTT?AATAATGCTA?CAATTGCTCT?GATTACGGCA????3840

GTCCAATCAT?TAGAATTGAC?TGGTGAAGTA?ATTACAACCC?CGTATTCATT?TGTGGCAACA????3900

ACTCACTCAT?TGATGTGGAA?TAATCTAAAT?CCAGTATTTG?TCGATGTCAG?TAGAGATACA????3960

TTTAATATCA?ATCCGTCTCA?AATTGAAGCG?GCGATAACAG?AAAAAACAAC?AGCCATAATG????4020

GCAGTCCATT?GTTATGGTAA?TCCTTGTGAT?GTGATTGCAA?TAGAAAAAAT?AGCCAAAAAG????4080

TATAAACTTA?AAGTGATATA?TGATGCTGCT?CATGCTTTTG?GGGTAAATTT?TAAGGGGGAA????4140

AGTTTATTAA?AATACGGAGA?TTTATCGGTT?GTTAGTTTTC?ATGCAACTAA?AGTATTTAAT????4200

ACATTTGAAG?GTGGCGTGAT?TATTTGCCCA?AATGCAGAAA?CTAAGCTCAA?AATAGATCAG????4260

CTAAAAAACT?TTGGTTTTGA?AGATGAGCTA?ACAATAAAAT?CAATTGGTAT?TAATGGAAAA????4320

ATGAGTGAGG?TCAATGCAGC?ATTTGGCCTT?GTACAGTTGA?AACATGTTAA?TGAAGCTATA????4380

AGTAAAAGAA?AAGAAATTAA?TGATTTATAT?GGCAAGTTAT?TAGGGAATGT?GAAAGGCATA????4440

TCATTAGCAA?AATTTGATAA?ACTAGCTACG?AAAAATTTTT?CATACTACCC?GATTCTTATT????4500

GAAGATGACT?ATGGGATGAG?TCGAGATGAA?TTATGTCATT?TACTTCAAAA?AAATAATATA????4560

TTTGCTAGGA?AATATTTTTA?TCCTTTAATA?AGCGATATGG?ATTTATATAA?AAATATGGAG????4620

TCAGCGAGAA?AAGAAAATCT?ACACATTGCT?CGAGATATTT?CCAACAAAGT?ACTTTGCTTA????4680

CCAATTTATG?CAGATCTTGA?TTTAGACATT?GTGAGATTTA?TAGCGAGAGT?AATAGGTAAT????4740

The termination of the initial orf4 of orf

AAAAA ATGAA?ATTAGCAATA?ATGCAACCCT?ATCTATTTCC?TTATCTAGGT?TATTATCAAT??4800

TAATGTCATC?AGTTGATAAG?TTTATTATTT?ATGATGATGT?TTCATACATC?AAAAATGGTT????4860

GGATAAACAG?AAATAGAATT?CTTGTTAATG?GTAATGCTCA?TTATTTTACT?GTGCCAGTTA????4920

TAGGTGGGAG?TTGTAATAAT?AAAATAAATA?CTGTTAAAAT?TGACAAGACA?AAGAAAAAAG????4980

CTATCAATAA?AATCATTATT?ACAATTGAAC?AAGCCTATAA?AAAATCCGTT?TTTTTTGATG????5040

AGGTCTTTCC?AGTCATTTAT?GGTGTGTTAT?CTAAAGAGTA?TGATTTCATA?TCCGATCTGG????5100

CAATAACGTC?ATTATTGTCA?ATAAAAAACA?AACTTGATAT?TGGAGCAGAA?GTTGTTTTGA????5160

CTTCGACCAA?CTATGGTAAC?AATAATTTAA?CTTCGCAAGA?TCGCGTCATT?GATATTAATG????5220

TAAAAGAGCA?TGCGTCCACT?TATATTAATT?CTGAAGGCGG?GAGATTACTT?TATGATAAAA????5280

AGACATTCAA?ACTAAATGGC?GTAAATTTGA?AATTTATACA?TCCAGAAATT?TTACCTTACA????5340

AACAACTTTG?TAATGGTGAG?TTTGTACCAT?CCTTATCAAT?TATAGATGTA?GTAATGAATA????5400

The termination orf6's of orf5 is initial

ATGGTTGGGA?TACTACAAAG?CAGTTAGTAA?ATAGCTTTGA?ACTGAAGGAT? TGAACA ATGC5460

GTGAGCATAA?TTATGCGATC?GGAGGATATT?TTTCACTAGA?ATTGCAGGCG?CTTAAAAATA????5520

ACACTGCAAA?TGAAAAAGTA?TATTTACAAT?CAGCGAGGGC?ATGTTTTCAA?TTACTGCTAG????5580

AAAGCATTGA?AGTTAGTAGA?GTATGGTTAC?CATATTATAT?ATGTGATGTC?GTCGTTGATA????5640

CAATAAATGA?AATTGGGATT?GAAATCTTGT?ATTATAGTAT?TTCTAAAGAT?TTTATTCCGC????5700

AGACGTTTCC?AGTTTTAGAA?GAAAATGATG?TTTTTGTCTA?TGTTAATTAT?TTTGGGGTGT????5760

GTGATGAGCA?AACAAAGTTA?ATTTTGCAAA?AATATCCTCC?AGAGAAAGTT?ATCTTAGATA????5820

ACTCACAAGC?TTTTTATAGC?GGACACAATA?ATAATTTAGG?AACAATTTAT?TCACCTAGGA????5880

AATTTTTTGG?CGTCCCTGAT?GGTGGTATTC?TGATAACTAA?TCAGACTATA?ATATTACCTT????5940

CTTCACAGGA?TAATGATTCA?TCACAGTATA?TAAATCATCT?CATTGGGCGT?CTAATATCCC????6000

ATCCCAGCGA?ATACTATACT?GATTATATTA?AAGCTGAAGA?ACGATTAAAA?AAAATTAAAA????6060

AAGCCAAAGT?GATGTCTTAT?TTAACTCGGA?AATTATTAGA?CTCCATCAAT?TATCATGAGA????6120

TTAAAAAAAT?AAGGGATGAT?AATTTTAAAT?TCCTTCACCA?TGCCCTCGAA?AAGATAAATG????6180

GAATAAAAAT?TCCGGAGATA?GTCAATGGTC?CATTATGCTA?TCCATTGCTT?TCAAAAAACA????6240

ACAAACTGAA?AGATATTTTA?ATCAAGAATG?AAATTTATGT?GCCTACATAT?TGGAAAGATG????6300

TATTGAATCG?AGTAGATATA?AACTCGACAG?AATTTGAATT?TGTCTCAAAC?TTAATACCTC????6360

TGCCATGTGA?TCAACGTTAC?TCATCTATCC?AGATGAAGAA?AATAATCAAT?ATTGTACTTG????6420

The termination orf7's of orf6 is initial

AGGAAAAC TA?ATGAATATCA?TAGGAAAAAC?AGTTAAGTTG?CGTGCTGTGG?AAATTGATGA??6480

TTTAGAATTG?TTAAATAAAT?GGGCTAACGA?TCCGGAAATT?TGGTATATGC?TTGGTGGTTG????6540

GCATTTCCCC?TATTCCAAAA?ATAATACTGA?AAAATGGATA?AAAAATATTG?ATAATAATGA????6600

TTCGAAAAAT?CAAATATTCG?CTATTGAAAC?TGAAGAGCAT?GGATTGATTG?GCACTGCTAA????6660

CTTAGTAAAT?ATTGATTGGA?AAAATAAAAA?TGCATTTCAT?GGGATAATGT?TGGGAAATGT????6720

TGAAACACGA?GGTAAAGGGT?ACGCGCAAGA?CGTTGTAATG?AGTCTGATGA?GATATGCCTT????6780

TGATGAATTA?GGATTAAATC?GACTTGATGG?TGATATGATT?GAATATAATA?AATTATCAAT????6840

TAATTTTTAC?ATCAAAAGAT?GTGGTTGGAA?AATCGAGGGA?ATTAAGAAAG?AGTGGTTCTT????6900

TAGAAAGGGG?CAATATTTTG?ATAAAGTAGT?TGTTGGAATA?ACCAAAAAAG?AATATTTAGA????6960

The termination of the initial orf7 of orf8

ACATATTGAG?AAAAGCAAGT?ATTGGGAGAC?AAA ATGATGG?AACGTTTTAA?ATTGGGGGAC??7020

ACAGCAACTT?ATACTCAAAC?TATAACTGAT?GCAGATATTA?AAAGCTTCGC?AGGTATATCT????7080

GGGGATAATA?ATCCAGTACA?TATGAGTGAC?GAATATGCAG?AAGGTTCAAG?GTTTAAAAAG????7140

CGTATAGCTC?ATGGTCTGAT?ATCTGCTAGT?TTTTTTTCAG?CTCTATTTGG?TACAAAATTA????7200

CCTGGGCCTG?GATGTGTATA?TGTAAATCAA?AGCTTGAAAT?TTCTTAGACC?TGTTTATATT????7260

AATGATACGG?TTACAGCGCG?TGTAGTCTTA?ACAGACATTG?ATGTTGTAAA?AAGAAGACTC????7320

TTTTTTGATA?CGATTTGTGA?AGTTAACCGA?AAAAAAGTTA?TCACGGGGAA?GGCTGAAATT????7380

The termination of orf8

TACTTGCCAG?AA TAAAGCGC?TTTAAATTTG?ATGTTAACTT?GCTACATATT?GATGTTTTAT??7440

Orf9's is initial

CTCTTTTTAT?CTTTGAAGTT?TATCAAAA AT?GAATGTTTCC?ATGTCAGATA?TAAAAGTTAG??7500

TGTATGTATT?ATTTCTTTTA?ACCAACAAAA?CTATATACGA?CAGTGCTTGG?ATGGTGTCTT????7560

TTCTCAGAAA?ACAAATTTTG?AGTATGAGGT?TATTATACGT?GATGATTGCA?GTACCGATAA????7620

TACATATTTA?ACAATAATGG?AATATATTGA?CACTTTAGAT?GAAGAGAAAA?AAAAGAACAT????7680

AAAAATAACG?GTACTTGATG?GCACGAAAAA?TATTGGAGCA?AATAATAATT?TTATCGAAAC????7740

ATTTAAGACT?TCAGTAGGTC?AATGGCTAGC?TATATGTGAG?GGAGATGATT?ATTGGTGTGA????7800

TCAGGGGAAA?TTACAAAAAC?AGTACGACTA?CGCTATTTCA?CATAGTGATT?GCTCATTAGT????7860

CGTGCATCCC?GCTCTGATAA?GTGAAAATAA?TGTAATACGT?AAAACATCTT?GGGCGTGTAT????7920

GAATAAAACA?ATAAATCAGC?TTAGCGATGT?AATACGTGCA?AAAGGACAGT?TCTCACCAAC????7980

TGGCTCCTAT?TTTTTCAAAC?GAGAAATCTT?AAATGTTCTA?CCATTGTGGT?TTTCAACAGC????8040

TCCTGTTGGT?GACTATTACA?TGGAAATCTT?TGCTACATCG?CTTGGATCTT?GTCATACAAT????8100

TCCTGATGCT?ATGTCAGTTT?ATAGAATCAA?TTCAACGGGG?TCTTGGTCTG?ACCTGTTAAA????8160

AAAAGATAGA?AATGGCCAAC?GGATTATTAA?TACTTATCTT?TCTCAATTGG?AGTATTTGGA????8220

TAAACTTGCC?GAAATCTTTC?CTTCTTGTGT?CGATGATATC?ACAATAAAAA?GATCGCATGC????8280

ACAATATGCA?GCAGCAATGG?GTTATCTTGC?AAATGGTGAT?TATACTAATT?TTCGTATATT????8340

AATGGATAAG?TCAGATACAA?ACGGCTGGTA?TGATAATATG?CATTCTATTT?TTTATTATTT????8400

AAGAAATAGC?AGGGCGCTTT?CCATGTTAAT?GTTCAGATTC?AAGCCTGCAA?TTAAATCTGC????8460

The termination of the initial orf9 of orf10

TGTCATGTGG?GGGCGTC GTG?TATTTAACAA?C TAATTATTG?CGACTTTTTG?TCCCGAACTC8520

GAATCGTTAG?GCGCATATTT?TTGTTAGTTT?ATTTGATTTC?AAATGTTTAC?GCCTATATAG????8580

TGTTTACTGA?AACAGGAGTA?TTAATAGGGG?ATTATCAAGG?AGTACTCATA?ATATATAAAG????8640

ATCAGGTTGT?GTATTTATTG?ATGTTAACAA?TAATGTCATA?TTATATTGTT?TGTGGTCCTG????8700

TTTTTAACTG?TCTATCAAAA?GTAAAAATTA?GACTAAGGAC?ATTTAAAGGC?TTAAACTCTT????8760

TTGCTTATAT?CCTGTTTTTT?TTTCAAATCC?TATTTGCTGT?TTTTAATATT?TCAACAGGTT????8820

CGAATGCCGC?GGGCACTGAT?TTTCAAACTG?GTGGAGTCAT?AAGACTATTA?TGGCTTTTTT????8880

TACCAGTTGA?CTATCTATTT?TATATATATT?ATTTCGTAGG?CCGAGAGAAA?AAAGTAAGTA????8940

AAATATATCT?GGCCAATGTG?GTTATTTTTA?TTCTATCAAT?GTTAAGTAGA?GGATGGCTTG????9000

GGTGGACTTT?GGTTTTGCTT?TATGCAGAGT?TATGTTTCTT?TTTTTATTCA?CAAAAAAAAA????9060

TTAAAATAAA?ATATCTTATC?TTATTGTTTT?TCTTGCTTAT?TGTTGCACCA?TTAGCATTTA????9120

GTCTAAAAAT?TCAATTGCGT?GCTGATTTGT?ATTCCAGTGG?TATTGGTGGG?GTTATATCAA????9180

CTTTAAGTAA?TATTGATTAC?ATTCAATCTT?ATAATAATTT?CATCGCTGGT?TTCTTATCCA????9240

GAATACAACA?GTTATCAAAT?ATTGTTTTCT?TTTATGACCA?TCAACAAGAA?TTATATAAAT????9300

TTGTTTCTTC?AGATATTGTA?TCAAACTATG?CTTGGGAAGG?ATTACCTCAA?CAAACTGTAG????9360

CTAAACTTCT?AGGATTAGAT?CCTGGTGTTG?ATATGCATAT?TTTTCTTTAT?AGTCATTATA????9420

TTTCTTCAAC?TTCAGAGGCT?GTAACTACAT?TGCAAGTTGG?ATTCATTTCT?TGGCTCTTTT????9480

TAGGCACCCT?GTCATCCGTA?TTTTATCCAC?TGTTTGTCTT?TGCTATTATA?TGCATATCCT????9540

TATTTTTATC?TAAAAAACTT?GGAGGGGAAA?AACTATGTGC?ACTTACGTGG?ATTATGATTT????9600

TTCTTTCTAT?TATGTGCGGG?TGGTATAATG?CTTATTTGGT?ATATATGCAG?GCATTAATTA????9660

CATTTTATTT?TATCATGGGT?TTTTTGAATT?TAATTACTCT?AGAAAAAACA?AAAATTAATC????9720

The termination of the initial orf10 of orf11

ATAC ATGATG?GATGACGAGA?AATATATGAG?TAATTTAGTA?ATAGTTAACG?CAACAGCTTT??9780

AGCGTCTAGC?GGCGCTTTGA?CGATTTTGAA?CCAGTTTTTA?GAACACGCAC?TAAATGATTC????9840

TAAGCATAAG?TATCTTTGTT?TTATACATGA?GAGTGTTAAA?AAAAAGAGTG?TTAATAATGT????9900

CACCGTTATT?ACTATAAAAA?AACAAAGTTT?TATTCAACGA?GTATGGTGGG?ATCTATATGG????9960

ATTGAATAAA?TATATAAAGA?AAAATAATTT?GAAACCTAAA?AAAATAGTTT?CCTTGCAGAA???10020

TACATCTGTA?AATAGTAATT?TCGAGCAGAT?AATATATCTA?CACCAGTCGA?TTCCCTTTAG???10080

CAATTTTAGA?ATAAAAATTA?AATTTCAATA?TGCTATATTT?TTCATGTATA?AATACGTGTA???10140

TCCTTTTTTT?ATATTCTTTA?GAACTAAAAA?CACCACTTTC?GTTGTTCAGA?CCGATTGGAT???10200

GAAAGATGCT?ATCGTAGCAA?AGAAAAAAAT?TGCCAAAGAA?CGAGTTCATG?TAATTAAACC???10260

TGATATAATA?TTACCGGTTA?ATAATCTTGC?TTACCCTGAT?GAACATGACA?AAGATAATTC???10320

ATCTGAAGTG?TTTTTTTTAT?ATCCGGCCAC?GCCACTTTTC?TATAAAAACC?ATTTGATTAT???10380

CTTAGATGCA?ATGAGGATTC?TAAAAACAGA?AGGAATACTT?GCTAATACAA?AATTCCAAGT???10440

GACTTTTAAA?CAAGATGATA?ATGATGAGTT?AGCAATTAAA?ATAGCCAAAT?ATGATTTAGT???10500

TGATAATATA?AGCTTCTTGG?GAGTATTATC?CTATGAAGAA?TTGTTTCATA?AATACTTAAA???10560

AGCAGATGCT?ATACTTTTTC?CTAGCTACTT?GGAAAGCTTT?GGTTTACCGT?TAGCAGAGGG???10620

CGCAATGCTT?GGGAAATATA?TAATTTGTAG?CGATTTGCCA?TATGCAAGAG?ATGTGCTTAA???10680

CAATTATTCA?AATGTAGAAT?ATATAAATCA?TGATGATGCC?AGTAAATGGG?CATTAGCTAT???10740

GAAGGAAATA?ATATTAAAGA?ATAGAAATAA?TACTTTATCT?GAAAGTGAAA?ATAGCTATAT???10800

The termination orf12's of orf11 is initial

GTATTGTCCG?AAAACAAGCT?GGGCAGATTT?CTTCGAGTTA?ATC TAATTGA?GGTATACG AT10860

GTTTAAAAAT?AAGGTACTTC?TCATTACTGG?TGGCACAGGT?TCCTTTGGGA?ATGCCGTTTT???10920

ACAACGCTTT?TTAGATTCAG?ATATTGGCGA?AATTCGAATC?TTTAGTCGTG?ATGAGAAAAA????10980

ACAAGATGAT?ATGCGTAAAA?AATACGCAAG?TGATAAGTTA?AAATTTTATA?TAGGGGATGT????11040

GAGGGACTAT?AGCAGTGTTT?TAACAGCGAC?TCGTGGGGTT?GATTTTATTT?ATCATGCAGC????11100

AGCATTAAAG?CAAGTACCTT?CATGTGAGTT?TTATCCAATG?GAAGCTGTCA?AGACTAACGT????11160

TATTGGAACT?GATAACGTAC?TTGAAGCTGC?AATTGCAAAC?AAAGTTTCAC?GTATTGTTTG????11220

TTTGAGTACC?GATAAAGCTG?TTTATCCAAT?TAATGCTATG?GGTACATCTA?AGGCTATGAT????11280

GGAAAAAGTT?ATCGTAGCTA?AGTCAAGAAA?CTTACCTAAA?GATATTACTA?TTTGTGCGAC????11340

GCGATATGGA?AATGTAATGG?CTTCCCGTGG?TTCGGTCATC?CCTCTGTTTA?TTAATCAAAT????11400

ACTTGAAGGA?CGCCCAATCA?CTATCACCGA?TCCGTGTATG?ACTCGATTCA?TGATGACTTT????11460

GGATGATGCT?GTAGATCTTG?TATTACATGC?TTTTGAACAT?GGTACTAATG?GTGATATTTT????11520

TGTTCAAAAG?GCACCAGCTG?CAACAATAGA?TACACTTACC?AAGTCATTAC?TTAAGTTAAC????11580

TAAACAACAT?TCTCACCCAA?TTAATATCAT?TGGCACTCGC?CATGGTGAAA?AACTATTTGA????11640

GGTACTCTGC?AGTCGGGAAG?AAATGTTGGT?TGCAGAAGAT?CAAGGTAATT?ACTATCGTAT????11700

ACCTAGTGAT?AAACGTGATC?TAAATTATGA?AAAATTCTTC?GACAAAGGGA?CTAAAGAAAT????11760

TCAGTTTGTT?GAAGACTATA?ATTCTCATAA?TACCCGTCGT?TTAGATGTTG?ATGAAATGGT????11820

TGCTCTTCTT?AGAAAATTAG?ATTACATCAA?TAAAATTGAG?GCTGGTGAAA?AGGCAGATCC????11880

The termination of the initial orf12 of orf13

CG ATGCG TAA?AAAAATTCTA?ATTGTTGGCG?CAAATGGCAT?GTTAGGTAGT?AGTTTACTAC11940

GCTACTTTTC?ATCAATTGGA?GATTATGAAG?TTCTTGGTAC?GACAAGAAGT?ATGGTCGTTG????12000

CAAAACAACT?TGAGCAAAAG?CACAATGTGA?AAATTATTGA?CAACGTTGAT?GTTATTGATT????12060

TTAAACGATT?AGAGACTGTA?GTAGTAGAGC?ATAAGCCAAA?TATTGTTTTC?AACTGCGTTG????12120

GGATAATTAA?ACAACTTGAT?GCAGCAAAAA?ACAATATATT?ATCTATTGAA?ATTAACTCAT????12180

TACTTCCACA?TAAATTAGCT?CAATTATGTT?CAGCTCATAG?TGCTAAACTC?ATACATTTTT????12240

CAACAGATTG?CATTTTTAAA?GGTACCAAAG?GTAATTATGT?TGAGGATGAT?GAGTCTGATG????12300

CAATTGATTT?ATATGGTAAA?TCTAAGTTCT?TGGGTGAAGT?TGAATATAAT?GGGCATTTAA????12360

CTTTGCGGAC?TTCTATTATT?GGCCATGAGT?TGGGATCAAA?TCATAGTCTT?GTTGACTGGT????12420

TTTTATCACA?GAAAAAATCG?GTGAAAGGAT?TTACTAATGC?GATATTCTCA?GGCCTCCCAA????12480

CTTGTTATAT?GGCAGAGGTT?ATCCATAAAT?ATGTTCTTCC?CAACAATCTT?GCTGGTTTAT????12540

TTCATTTAAG?TGTAGAGCCA?ATTAGTAAAT?ATGATTTGTT?AAATATTATT?AAAATAGTAT????12600

ATGGAGTAAG?TACTGATATA?GAGCCAACCA?ACGAATTTAA?AATAGACCGC?AGCTTGAATT????12660

CTACGCTATT?TCGTAATAAG?ACAAATTTTG?TCCCAGAATC?TTGGGATAAA?TTAATTGAAA????12720

The termination of the initial orf13 of orf14

AGATGAAAG A?TGAATACAAT?AAATATTTC T?AATTCAAAAA?TACGGGTTGT?TACTGTAGTT12780

GGCACTAGGC?CTGAAATCAT?TCGTTTATCG?AGGGTAATTG?CTGTTCTCGA?TGAGTACACT????12840

GAACATTTTC?TTGTTCATAC?CGGACAAAAC?TATGATTACG?AGCTTAATGA?GGTATTCTTT????12900

AATGAGCTTG?AAATCAGAAA?ACCTGATTTT?TTCATGAATG?CCGCGGGCCA?GAATGCTGCT????12960

GAGACCATCG?GAAATGTAAT?TATTGAAGCT?GATAAGATAT?TTGATAAATT?GCAACCTGAA????13020

GCTTTACTTA?TTCTTGGTGA?TACAAATAGT?GCTCTAGTTT?CCATTGCAGC?TAAAAGACGA????13080

AAAATTCCTA?TCTTTCATAT?GGAAGCGGGC?AACCGATGCT?TTGATTACAG?AGTTCCGGAG????13140

GAAATAAATA?GGAAGATTGT?TGATCATATT?TCAGATATTA?ATTTAACATA?TAGTGAGATA????13200

GCTAGGGATT?ATCTTTTGAG?GGAAGGGCTT?CCTGCTGATC?AAATAATCAA?AACCGGTAGC????13260

CCTATGCGAG?AGGTTCTAAA?TTTTTATAAG?GATAAAATTT?CATCTTCTTC?TATTCTTGAA????13320

AAACTTAACC?TTCAAGCATC?TAGTTATTTC?CTTGTTAGTA?GTCATCGAGA?AGAGAATGTT????13380

GATTCGCCTG?AGAAGTTGCG?CTCTCTTATT?GAGACGTTAA?ATATAGTATC?TGAGAAATAT????13440

AAACTGCCAG?TAATAGTATC?AACGCATCCT?CGTACTCGTA?ATAGAATTGA?TGCTTTAGGT????13500

ATCACGGTAA?GTAATAATAT?CATTTTTTCT?AAGCCATTCG?GATTTTTAGA?TTATATAAAA????13560

CTGCAGCAAA?ATGCTCGTGT?CGTGCTCTCT?GATAGTGGAA?CTATAACTGA?AGAATCATCA????13620

GTTTTGAATT?TTCCAGCCTT?AAACCTGCGA?GAAGTACATG?AGAGACCAGA?AGGATTTGAA????13680

GAAGCTGTTG?TTATGTTTGT?CGGGCTAGAT?AGAAACAGAA?TTATTCAGGG?AATCGATATT????13740

TTGCAGGGTC?AAAAACGTGG?TGATAATGAT?CGTGACCTTC?ATATGGTTAC?AGATTATCAG????13800

GCAGACAATG?TTTCAATTAA?AATTCTTAGA?ATTATTATGA?GCTATACAAA?CTTTATTAAT????13860

The termination orf15's of orf14 is initial

CAAAAGGTTT?GGAAAAAATT?C TAATGCGTA?TTGCGTTAAT?ATGTGATGAC?TATTTGCCTG??13920

ATAGTACTCG?TGTTAGTGCT?AAGATGATGC?ATGAATTGGC?TTGTGAACTT?TTAGAAAAAG????13980

GACATGAGCC?AATCGTTATT?TCTCCTTGCA?ATAAAATACA?GACGCTCGAA?ATTTTGAATT????14040

TGGATGGTGT?CGTCGTTTAT?AAGTTCCCTA?ATGGTGCTAT?AAAGAATGTA?TCTAAAATAT????14100

CAAGAGCAAT?AAACGAATCC?ATGCTATCAT?TCAACGCATG?GCGATTTATT?GGAAAATATA????14160

TTCGAGAAAC?TAAAATTGAT?GGTGTTGTGT?ACTACTCACC?ATCTATTTTT?TTTGGAAAAT????14220

TAGCAAATAA?AATCAAAGAA?AATTGGCATT?GTAAATCATA?TTTAATCTTA?AGGGATTCTT????14280

TTCCTCAGTG?GCTGGTAGAT?CAGGGGATAA?TTAAGGAGGG?AGGGCTTGCG?GAACGATATT????14340

TTAGGTACTT?CGAGCAGATC?AATTATGATG?CTGCAGATTA?TATTGGTTTA?ATGTCGGATA????14400

GAAACAAAGA?TATTTTCATT?AATAAATATC?AGAATAAATA?CAAGGTGCAA?ACTTTATTCA????14460

ATTGGGCTGA?CTTTAAAGGT?ATAGATAATA?TTCCCAGTGC?GACTCTACGA?TCAAAATTAG????14520

CACTTCAAAA?CAAAGTAATT?TTTTTTTATG?GAGGCAATAT?TGGTCATGCT?CAGGATATGA????14580

TGAATTTAAT?GAGGTTGGTT?AGATCAGCAT?CTTATCGTGA?TGATGTGCAT?TTTTTATTGA????14640

TTGGACAAGG?TGATGAAGTT?TCCCTTGTAA?AGCAGTTTAT?TATAGATAAT?TCTTTAAAAA????14700

ACTGTACGTA?TTTACCATCC?ATAACACAAT?CAGAGTTTAA?GTCTGTATTA?AAGATTGTTG????14760

ATGTTGGTTT?GTTTAGCCTT?GCTAAAAATC?ATACTGTTCA?TAACTTCCCT?GGTAAATTGT????14820

TAGGGTATAT?GGCAAATAAA?CTCCCTATAT?TAGGTAGTGT?TAATGCAAGC?AATGATGTTA????14880

TGGAGATTAT?CAATGGGGCA?AAGGCCGGGT?TTGTTTTTGT?TAACGGAAAT?GACGAGGCTT????14940

TACTTAATGC?TGCAATAAAT?CTTGCGGATG?ATACTCAGCT?AAGAAAAAAC?CTGGGATGTA????15000

ATGCTTACTC?TTTATTACAA?GAAAAATTCT?CTGTGGAAAT?GGCTGCTGAA?AAGATATTAA????15060

The termination orf16's of orf15 is initial

GCAGCTTATT?TTCC TAACAG?TAATATGGTG?GTTTT ATGAA?AGTGTCTTTT?GTAAGTCAGG15120

ATGATTTCGA?GAATTTAATT?CAGGAAGCAT?TTGCATCCGT?TCGCTTGAGA?TCTCACTTAC????15180

TCCTCCATGA?AAGCCCTAAT?GATGCTGTCC?AGAGAATAAT?GATAGGACTT?GTCAAAGGAA????15240

CGTATATCCC?GCCTCATTTT?CATGAATTTC?AGCATCAATG?GGAACATTTC?CATGTGTTTC????15300

AAGGTGAGGT?TGAATTAATG?CTATTTGATA?GTAACGGCTG?TTTAAATAAA?AAGGTTATTC????15360

TTGGTGGGCA?GAGTAAGAAT?ATTATTGCAC?AAATTTCACC?ACTCACCCCA?CATACACTAG????15420

TTTGTAGATC?TCCTACAGCA?GTTATCATGG?AGATTAAAGA?AGGTCCTTTT?GATGAAAAAT????15480

GCGCTAAAGT?CATCCCCTCA?TGGTCATATA?GTGAAGATTA?TTCCATTTTA?TCCAGAGATA????15540

The termination of orf16

GGATCATCGC?AATGATGGGC?CAATTATCAA?TTGGAGATAG?ATTTAGTCTC? TAAACTTCAC??15600

AGCAATTAGC?GAAGTAATCT?TCTTTTTCAT?ACCTTCTATA?TTATATTTAT?CCACTACTAT????15660

TATAGCTTTG?AATACTGAGT?TAACATCTAC?ACTACATTCA?AGCCGTGCAT?ACGTCGCGGT????15720

GACCACCCCT?GACAGGAGTA?AATAATGTCA?AAGCAACAGA?TTGGCGTAGT?CGGTATGGCA????15780

GTGATGGGGC?GCAACCTTGC?GCTCAACATC?GAAAGCCGTG?GTTATACCGT?CTCTATTTTC????15840

AACCGTTCCC?GTGAAAAGAC?GGAAGAAGTG?ATTGCCGAAA?ATCCGGGCAA?GAAACTGGTT????15900

CCTTACTATA?CGGTGAAAGA?GTTTGTTGAA?TCTCTGGAAA?CGCCTCGTCG?CATCCTGTTA????15960

ATGGTGAAAG?CAGGTGCAGG?CACGGATGCT?GCTATTGATT?CCCTCAAACC?ATATCTCGAT????16020

AAAGGCGACA?TCATCATCGA?TGGTGGTAAC?ACCTTCTTCC?AGGACACCAT?TCGTCGTAAC????16080

CGTGAGCTTT?CCGCAGAAGG?CTTTAACTTC?ATCGGTACCG?GTGTTTCCGG?TGGTGAGGAG????16140

GGCGCACTAA?AAGGTCCTTC?CATTATGCCT?GGTGGGCAGA?AAGAAGCCTA?TGAACTTGTT????16200

GCGCCGATCC?TGACCAAAAT?CGCCGCAGTG?GCTGAAGACG?GTGAGCCATG?CGTTACCTAT????16260

ATTGGTGCCG?ATGGCGCAGG?TCACTATGTG?AAGATGGTTC?ACAACGGTAT?TGAATACGGC????16320

GATATGCAGC?TGATTGCTGA?AGCCTATTCT?CTGCTTAAAG?GTGGTCTGAA?CCTCACCAAC????16380

GAAGAACTGG?CGCAGACCTT?TACCGAGTGG?AATAACGGTG?AACTGAGCAG?CTACCTGATC????16440

GACATCACCA?AAGATATCTT?CACCAAAAAA?GATGAAGACG?GTAACTATCT?GGTTGATGTG????16500

ATCCTGGATG?AAGCAGCAAA?CAAAGGTACG?GGCAAATGGA?CCAGCCAGAG?TGCGCTGGAT????16560

CTTGGCGAAC?CGCTGTCGCT?GATTACCGAA?TCTGTGTTTG?CACGTTATAT?CTCTTCTCTG????16620

AAAGATCAGC?GTGTTGCCGC?GTCTAAAGTT?CTCTCTGGTC?CGCAAGCGCA?GCCAGCAGGC????16680

GACAAAGCTG?AGTTCATCGA?AAAAGTTCGT?CGTGCTCTGT?ATCTGGGCAA?AATCGTTTCT????16740

TACGCTCAGG?GCTTCTCTCA?GCTGCGAGCG?GCGTCTGAAG?AGTACAACTG?GGATCTGAAC????16800

TACGGCGAAA?TCGCGAAAAT?TTTCCGTGCT?GGCTGCATCA?TCCGTGCGCA?GTTCCTGCAG????16860

AAAATCACCG?ATGCTTATGC?CGAAAATCCG?CAGATCGCTA?ACCTGCTGCT?GGCTCCGTAC????16920

TTCAAGCAAA?TTGCCGATGA?CTACCAGCAG?GCGCTGCGTG?ATGTCGTTGC?TTATGCAGTA????16980

CAGAACGGTA?TCCCGGTTCC?GACCTTCGCG?GCTGCGGTTG?CCTATTATGA?CAGCTACCGC????17040

GCCGCAGTTC?TGCCTGCGAA?CCTAATCCAG?GCACAGCGCG?ACTA?????????????????????17084

Claims (10)

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O123 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,17084 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O123 type of claim 1, it is characterized in that: it comprises called after rmlB, rmlA, wzx, vioA, orf5, orf6, orf7, orf8, orf9, wzy, orf11, fnlA, qnlA, qnlB, orf15,16 genomic constitutions of orf16 are all between JUMPStart sequence and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O123 type of claim 2, it is characterized in that the gene that has high degree of specificity in the described gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf9, orf11, orf15 gene; Wherein said gene: wzx is the Nucleotide of 2169 to 3614 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 8478 to 9728 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 7469 to 8494 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 9725 to 10846 bases among the SEQ ID NO:1; Orf15 is the Nucleotide of 13884 to 15077 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O123 type, it is characterized in that: it also comprises and comes from described wzx gene, wzy gene and their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O123 type of claim 4, it is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 2627 to 2646 bases among the SEQ ID NO:1 and the Nucleotide of 3275 to 3294 bases; The Nucleotide of 2806 to 2825 bases among the SEQ ID NO:1 and the Nucleotide of 3376 to 3395 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8986 to 9005 bases among the SEQ ID NO:1 and the Nucleotide of 9484 to 9503 bases; The Nucleotide of 8603 to 8622 bases among the SEQ ID NO:1 and the Nucleotide of 9392 to 9409 bases.

6, the application of Nucleotide in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium of the described O-antigen-specific to intestinal bacteria O123 type of claim 1.

7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O123 type of claim 1 is providing the O-antigen of expressing intestinal bacteria O123 type by inserting to express, and the application in the preparation bacterial vaccine.

8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O123 type of claim 1, 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, for bacterial detection.

9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O123 type of claim 1 is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O123 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 O123 type bunch: with the genome of intestinal bacteria O123 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 O123 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O123 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O123 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O123, 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 O123.

10, the separation method of the Nucleotide of the O-antigen-specific to intestinal bacteria O123 type according to claim 9 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O123 types in the LB of 5mL substratum, centrifugal collecting cell; With the pH value is 8.0 500 μ l 50mM Tris-HCl and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, and the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes; The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is 25: 24: 1; Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O123 type bunch: with the genome of intestinal bacteria O123 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, be #wl-1098-ATTGGT AGC TGT AAG CCA AGG GCG GTA GCG T according to the JUMPStart sequences Design upstream primer that often is found in O-antigen gene bunch promoter region at first, the gnd gene design downstream primer according to O-antigen gene bunch downstream is #1524-TAG TCG CGT GNG CCT GGA TTA AGTTCG C again; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 55 annealing 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCRPreps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9 μ l 0.1M MnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions; Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: the mixing solutions extracting of primary isoamyl alcohol once, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is after using isopyknic ether extracting once again at 25: 24: 1, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water, in this mixture, add 2.5 μ ldNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mMDTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail; This mixture is through the equal-volume chloroform: after the mixing solutions extracting of primary isoamyl alcohol and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l, chloroform: the mixed volume ratio of primary isoamyl alcohol is 24: 1; 10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, be that 5.2 the 3M NaAc and the dehydrated alcohol precipitation of 2 times of volumes are connected mixture with the pH value of 1/10 volume at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O123 type;

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

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O123 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O123 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O123 type O-antigen gene bunch, Orffinder with American National biotechnology information science center finds gene, find the reading frame of 16 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 O123 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O123 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O123 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O123 type all is high special;

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby; The frozen bacterium liquid of 10 μ l intestinal bacteria O123 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ l supernatant as pcr template; To carrying out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg with oligonucleotide ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations; Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative; Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction; The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of primer reaction; Illustrate that primer is 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O123 in the pork filling when using aforesaid method.