CN1234864C - Nucleotide peculiar to 0-antigen of -164 type bacillus oil - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O164. The specific nucleotide is the complete nucleotide sequence of gene clusters in the Escherichia coli O164 for controlling the synthesis of O-antigens, including a separated nucleotide with the total length of 14006 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotide of IS genes and wzx genes stemmed from the O-antigen gene clusters of the Escherichia coli O164. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of the Escherichia coli O164 through PCR. The present invention also discloses a method for using the oligonucleotide to detect and identify the Escherichia coli O164 in the human body and the environment.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O164

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster among the intestinal bacteria O164 (Escherichia coli O164), particularly relate among the intestinal bacteria O164 oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O164 in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Intestinal bacteria O164 is a kind of stronger pathogenic bacterium, belongs to the invasive intestinal bacteria of intestines (EIEC), causes diarrhoea.Therefore the detection to intestinal bacteria O164 is important, needs the method that can detect intestinal bacteria O164 quickly and accurately.

The lipopolysaccharides that is positioned at the intestinal bacteria surface is the morbific inducements of intestinal bacteria, and O-antigen is lipopolysaccharides outermost layer structure, is the target of immune system recognition and the site of phage absorption.The antigenic disappearance of O-can cause the serum sensitivity of many pathogenic agent, perhaps seriously undermines the virulence [Frank etal (1987) " The function of antibody and complement in the lysis ofbacteria " .Rev Infect Dis 177:1750-1753.Pluschke G et al " Role of thecapsule and the O-antigen in resistance of O18:K1 Escherichia coli tocomplement-mediated king " .J Bacteriol 42:907-913] of pathogenic agent.Intestinal bacteria are kinds, and the bacterial strain in planting is generally identified by O-antigen and H-antigen (sometimes by K-antigen).Wherein O-antigen has the height diversity, intestinal bacteria have 166 kinds of different O-antigens, the antigenic variation of O-may be colibacillary origin and keep its multifarious major cause [Reeves, P.R (1992) " Variation in antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516].

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by o-antigen transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In intestinal bacteria, Shigellae and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichiacoli O111 and Salmonella enterica O35 gene clusters:gene clusters encodingthe same colitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises o-antigen transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

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

Shigella dysenteriae 3 types are the same with the antigenic structure of the O-of intestinal bacteria O124, and intestinal bacteria O124 and the antigenic structure of their O-are very similar.As follows:

GlcLA＝4-O-[(R)-1-carboxyethyl]-b-D-glucopyranose

The antigenic structure of O-of shigella dysenteriae 3 types and intestinal bacteria O124

Their serology of the antigenic structure of the O-of intestinal bacteria O164 has cross reaction.Therefore identify that by serological method it is difficult distinguishing them, also has above-mentioned said shortcoming.This research is identified fast and accurately intestinal bacteria O164 and shigella dysenteriae 3 types by the modern molecular biology method and is distinguished.

Summary of the invention

The purpose of this invention is to provide Nucleotide to the O-antigen-specific of intestinal bacteria O164.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O164, is the special Nucleotide that comes from IS sequence and adjacent wzx gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O164.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O164: IS sequence (transposase of encoding), transhipment enzyme gene are the wzx gene or with wzx the gene of identity function are arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf4, orf5, orf7, orf9 gene; Sugar synthesis path gene orf2, orf3, glf.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.In addition, in the O-of intestinal bacteria O164 antigen gene bunch, also have a complete insertion sequence (IS) and orf3 inside that the sudden change of a terminator is arranged.

Another purpose of the present invention has provided oligonucleotide, and they come from described IS sequence and adjacent wzx gene respectively; Length is at 18-30nt; They are special to the O-antigen of intestinal bacteria O164; Especially the oligonucleotide of listing in the table 1 is right, and they are high specials to the O-antigen of intestinal bacteria O164, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O164.

The above-mentioned oligonucleotide that another object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect O-antigen and detection and identification of escherichia coli O164 with identification of escherichia coli O164 by these methods.

A further object of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O164.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

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

The present invention is characterized in that to the Nucleotide of the O-antigen-specific of intestinal bacteria O164 it is the isolating Nucleotide shown in SEQ ID NO:1,14006 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O164 type, comprising called after IS sequence and wzx, orf2, orf3, orf4, orf5, wzy, orf7, glf, 9 genomic constitutions of orf9 are all between galF gene and gnd gene.

The antigenic nucleotide sequence of the O-of aforesaid intestinal bacteria O164 is characterized in that, gene comprises following gene: transhipment enzyme gene comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf4, orf5, orf7, orf9 gene.Wherein said transhipment enzyme gene is the Nucleotide of 2518 to 4011 bases among the SEQ ID NO:1; Described pol gene is the Nucleotide of 8424 to 9527 bases among the SEQ ID NO:1; Wherein said transhipment enzyme gene is the Nucleotide of 2518 to 4011 bases among the SEQ ID NO:1; Described pol gene is the Nucleotide of 8424 to 9527 bases among the SEQ ID NO:1; The orf4 gene is the Nucleotide of 6561 to 7565 bases among the SEQ ID NO:1; The orf5 gene is the Nucleotide of 7562 to 8350 bases among the SEQ ID NO:1; The orf7 gene is the Nucleotide of 9556 to 10605 bases among the SEQ ID NO:1; The orf9 gene is the Nucleotide of 11845 to 12618 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O164 is characterized in that it comes from described IS sequence and adjacent wzx gene respectively; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to intestinal bacteria O164 is characterized in that,

The described oligonucleotide that comes from the IS sequence is as upstream primer, and they are: the Nucleotide of 1868 to 1885 bases among the SEQ ID NO:1, the Nucleotide of 2265 to 2284 bases among the SEQ ID NO:1;

The described oligonucleotide that comes from the wzx gene is as downstream primer, and they are: the Nucleotide of 3020 to 3050 bases among the SEQ ID NO:1, the Nucleotide of 2809 to 2827 bases among the SEQ ID NO:1.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O164 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 O164 type is providing the O-antigen of expressing intestinal bacteria O164 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 O164 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 O164 is characterized in that it obtains with walking method.Comprise the steps:

(1) genomic extraction: the genome that extracts intestinal bacteria O164;

(2) primer design: design upstream and downstream primer in two genes adjacent or that be separated by in the O-antigen gene of shigella dysenteriae 3 types bunch; In the gene that requires the upstream and downstream primer to lay respectively at two adjacent genes or be separated by.Each is tested as follows to primer.

(3) corresponding sequence by the O-antigen gene among the pcr amplification intestinal bacteria O164 bunch: being distributed in adjacent intragenic upstream and downstream primer with every pair, is that template is carried out PCR with the genome of intestinal bacteria O164 type.Every pair of primer will obtain the PCR product of different sizes.With the PCR product that obtains, detect the size and the specificity thereof of PCR product by agarose gel electrophoresis.Merge 4 pipe PCR products and deliver to order-checking company, carry out the sequencing of Nucleotide from the corresponding PCR product of both direction after, and design primer and order-checking again and lead to up to the sequence of each PCR product being surveyed from two ends to purifying with their corresponding upstream and downstream primer.

(4) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit the above-mentioned all nucleotide sequences that obtain.

(5) inaccurate sequence checks order once more: the sequence with the both sides of inaccurate sequence is a template design primer, and is that template is carried out PCR with the genome of intestinal bacteria O164 type.Detect the size and the specificity thereof of PCR product by agarose gel electrophoresis, merge 4 pipe PCR products and deliver to order-checking company, carry out the sequencing of Nucleotide with their corresponding upstream and downstream primer from the corresponding PCR product of both direction after purifying.

(6) carry out the splicing and the analysis of nucleotide sequence once more: the sequence that will newly obtain joins splices once more in original sequence and edits all nucleotide sequences.Finally obtain the complete sequence accurately of the O-antigen gene bunch of intestinal bacteria O164 type.With information biology software the sequence that obtains is carried out the comparison and the analysis of sequence, and make the note of gene of Artemis software.

(7) screening of specific gene: behind the O-antigen gene of O-antigen gene that has compared intestinal bacteria O164 type bunch and shigella dysenteriae 3 types bunch, be IS sequence and adjacent wzx gene design primer at the different sequences of the O-antigen gene of the O-antigen gene of intestinal bacteria O164 type bunch and shigella dysenteriae 3 types bunch; Upstream primer is distributed in the IS sequence, and downstream primer is distributed in IS sequence and the adjacent wzx gene, to guarantee its specificity to intestinal bacteria O164 type; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines their antigenic high degree of specificity of O-to intestinal bacteria O164 type;

(8) detection of primer sensitivity: cultivate intestinal bacteria O164, 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 O164

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O164 is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O164 in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, use isopyknic phenol again: chloroform: twice of the solution extracting of primary isoamyl alcohol (25: 24: 1), get supernatant liquor again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) primer design: design upstream and downstream primer in two genes adjacent or that be separated by in the O-antigen gene of shigella dysenteriae 3 types bunch; In the gene that requires the upstream and downstream primer to lay respectively at two adjacent genes or be separated by.The primer of design is as follows:

The primer numbering

Primer sequence

Residing gene

The annealing temperature of PCR reaction (℃)

The extension time (branch/second) of PCR reaction

The size of PCR product (base)

Primer is in the O-antigen gene of shigella dysenteriae 3 types bunch

#1523	5’-ATT GTG GCT GCA GGG ATC AAA GAA ATC-3’	galF	56	3 minutes/30 seconds	About 3500	1
							#82	3’-GGCACTTACGTCGTATA-5′	wzx				1903
#100	5’-TGTGCAGAGTTCAGGGAG-3’	wzx	50	2 minutes	1321	1835
							#hp-2	3′-GCAAAGTCCTCACCGTA-5′	Orf2				3156
#hp- 1	5′-AGCGTGTTTGTCCCTTTA-3′	Orf2	55	2 minutes	1873	2773
							#hp- 4	3′-ATTAGAAGTATTCCCTTTCC-5′	Orf3				4646
#hp- 3	5′-AGGACGCATCATCAATCT-3′	Orf3	50	2 minutes	1644	4199
							#72	3′-ACACCTATCCGAAAGACTT-5′	Orf4				5843
#204	5′-AAGGAGTTGCCTATACACG-3′	Orf4	50	2 minutes	755	5377
							#209	3′-CTAACCACGTCCGAAATA-5′	Orf5				6132
#208	5′-TGGAGTATGAGCCAAAGAA-3′	Orf5	58	2 minutes 30 seconds	2395	5575
							#58	3′-GCTTATTTCACGCTGGATCT-5′	wzy				7970
#57	5′-TTTGTTTCTGATATATGCGGT-3′	wzy	50	2 minutes	1028	7277
							#214	5′-CGAAATTCAATGGTGGTC-3′	Orf7				8305
#75	5′-TTCAATGGTGGTCTCTCA-3′	Orf7	50	3 minutes 30 seconds	2731	8410
							#78	3′-TGAAGGGCAATATACCGC-5′	Orf9				11141
#213	5′-TGGACGATGATTGTATGC-3′	Orf9	52	2 minutes 30 seconds	2064	10565
							#1524	5’-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3’	gnd				12629

(3) corresponding sequence by the O-antigen gene among the pcr amplification intestinal bacteria O164 bunch: being distributed in adjacent intragenic upstream and downstream primer with every pair, is that template is carried out PCR with the genome of intestinal bacteria O164 type.The PCR response procedures was as follows: 95 ℃ of pre-sex change 3 minutes, 95 ℃ of sex change are 15 seconds then, and annealing temperature is because of the difference different (in seeing Table) of primer, and annealing time is 50 seconds, 72 ℃ of extension time is carried out 30 circulations like this because of the difference different (in seeing Table) of primer; At last, continue to extend 10 minutes at 72 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product; Merge 4 pipe PCR products and deliver to order-checking company, carry out the sequencing of Nucleotide from the corresponding PCR product of both direction after, and design primer and order-checking again and lead to up to the sequence of each PCR product being surveyed from two ends to purifying with their corresponding upstream and downstream primer.

(4) splicing of nucleotide sequence and editor: 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 full length sequence of Nucleotide of the O-antigen gene bunch of intestinal bacteria O164.

(5) inaccurate sequence checks order once more: the sequence with the both sides of inaccurate sequence is a template design primer, and is that template is carried out PCR with the genome of intestinal bacteria O164 type.Detect the size and the specificity thereof of PCR product by agarose gel electrophoresis, merge 4 pipe PCR products and deliver to order-checking company, carry out the sequencing of Nucleotide with their corresponding upstream and downstream primer from the corresponding PCR product of both direction after purifying.

(6) carry out the splicing and the analysis of nucleotide sequence once more: the sequence that will newly obtain with the Pregap4 of Staden package software package joins splicing once more in original sequence, and with all nucleotide sequences of Gap4 software editing.Finally obtain the complete sequence accurately of the O-antigen gene bunch of intestinal bacteria O164 type.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O164 bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 openings, determine also that with the function of finding these open reading frame what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O164 at last; And with the texture ratio of shigella dysenteriae 3 types, find that they have identical gene.These genes have identical putting in order.(5 ' end) has an insertion sequence (IS) before first open reading frame of unique O-antigen gene that is not both intestinal bacteria O164 bunch.

(7) screening of specific gene: behind the O-antigen gene of O-antigen gene that has compared intestinal bacteria O164 type bunch and shigella dysenteriae 3 types bunch, be IS sequence and adjacent wzx gene design primer at the different sequences of the O-antigen gene of the O-antigen gene of intestinal bacteria O164 type bunch and shigella dysenteriae 3 types bunch; Upstream primer is distributed in the IS sequence, and downstream primer is distributed in IS sequence and the adjacent wzx gene, to guarantee its specificity to intestinal bacteria O164 type; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines that the IS sequence is a high special to the O-antigen of intestinal bacteria O164 type; Can carry out PCR with above-mentioned primer, distinguish and differentiate intestinal bacteria O164 type and shigella dysenteriae 3 types in the environment.

(8) detection of primer sensitivity: the frozen bacterium liquid of intestinal bacteria O164 is inoculated in the triangular flask of LB substratum, 30 ℃ of-40 ℃ of cultivations, 180 to 250 rev/mins, cultivate a few hours to saturated, get cultured bacterium liquid dilution, get dilution bacterium liquid coating LB agar plate, 30 ℃ to 40 ℃, cultivate a few hours counting, calculate viable bacteria concentration in the stoste; In being the live pig meat stuffing of 20g, 5 parts of weight mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add the LB substratum, filter, and filtered liquid 180 to 250 rev/mins, is cultivated a few hours in 30 ℃ of-40 ℃ of cultivations; Peek ml is in 6 from cultured bacterium liquid, and centrifugal several minutes of 000g removes supernatant, adds the MQ ultrapure water and blows precipitation and mixing open, puts into 100 ℃ of boiling water and boils several minutes, and lysate is in 12, and centrifugal several minutes of 000g gets supernatant as pcr template; As upstream primer, they are with the described oligonucleotide that comes from the IS sequence: the Nucleotide of 1868 to 1885 bases among the SEQ IDNO:1; The Nucleotide of 2265 to 2284 bases among the SEQ ID NO:1.The described oligonucleotide that comes from the wzx gene is as downstream primer, and they are: the Nucleotide of 3020 to 3050 bases among the SEQ ID NO:1; The Nucleotide of 2809 to 2827 bases among the SEQ ID NO:1 carries 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 2 pairs of primers reaction; The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 2 pairs of primers reaction; Illustrate that these 2 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O164 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 O164, its complete sequence shown in SEQ ID NO:1,14006 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 O164 by method of the present invention, as described in Table 3, it comprises called after IS sequence and wzx, orf2, orf3, orf4, orf5, wzy, orf7, glf, 9 genomic constitutions of orf9 are all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O164, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf4, orf5, orf7, orf9 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises orf2, orf3, glf.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.

The 3rd aspect of the present invention provides the IS sequence in the O-antigen gene bunch that comes from intestinal bacteria O164 and the oligonucleotide of adjacent wzx gene, and they are any one section oligonucleotide in IS sequence and the adjacent wzx gene.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O164 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, so, can determine these primers promptly the listed oligonucleotide of table 1 be high special to intestinal bacteria O164 and their O-antigen.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O164 comprises the steps: 1) genomic extraction; 2) primer design; 3) corresponding sequence by the O-antigen gene among the pcr amplification intestinal bacteria O164 bunch; 4) splicing of nucleotide sequence and editor; 5) inaccurate sequence checks order once more; 6) carry out the splicing and the analysis of nucleotide sequence once more, finally obtain the structure of O-antigen gene bunch; 7) screening of specific gene; 8) detection of primer sensitivity.

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

As described herein, " oligonucleotide " mainly is meant any one section oligonucleotide in the IS sequence that derives from the O-antigen gene bunch and the adjacent wzx gene, and they can change on length, generally change in 10 to 20 Nucleotide scopes.Especially come from IS sequence (nucleotide position is 1258 to 2277 bases from SEQ ID NO:1), the oligonucleotide in the wzx gene (nucleotide position is 2518 to 4011 bases from SEQ ID NO:1) all is a high special to intestinal bacteria O164.

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 IS sequence and adjacent wzx gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is the combinations that come from the oligonucleotide in IS sequence and the adjacent wzx gene respectively.

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) (ii) the encode gene of translocase of the IS sequence of coding transposase comprises the wzx gene or with wzx the gene of identity function is arranged.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 O164.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

The present inventor considers following situation: when 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 IS sequence that comes from the coding transposase and the gene of coding translocase, comprises the wzx gene or with wzx the oligonucleotide of the gene of identity function is arranged.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O164.

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) (ii) the encode gene of translocase of the IS sequence of coding transposase comprises the wzx gene or with wzx the gene of identity function is arranged.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 O164.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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) (ii) the encode gene of translocase of the IS sequence of coding transposase comprises the wzx gene or with wzx the gene of identity function is arranged.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 O164.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.

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O164 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 O164 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 O164 in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution extracting twice, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: primer design: design upstream and downstream primer in two genes adjacent or that be separated by in the O-antigen gene of shigella dysenteriae 3 types bunch; In the gene that requires the upstream and downstream primer to lay respectively at two adjacent genes or be separated by.The primer of design is as follows:

The primer numbering	Primer sequence	Residing gene	The annealing temperature of PCR reaction (℃)	The extension time (branch/second) of PCR reaction	The size of PCR product (base)	Primer is in the O-antigen gene of shigella dysenteriae 3 types bunch
							#1523	5’-ATT GTG GCT GCA GGG ATC AAA GAA ATC-3’	galF	56	3 minutes/30 seconds	About 3500	1
#82	3’-GGCACTTACGTCGTATA-5′	wzx				1903
							#100	5’-TGTGCAGAGTTCAGGGAG-3’	wzx	50	2 minutes	1321	1835
#hp-2	3′-GCAAAGTCCTCACCGTA-5′	Orf2				3156
							#hp- 1	5′-AGCGTGTTTGTCCCTTTA-3′	Orf2	55	2 minutes	1873	2773
#hp- 4	3′-ATTAGAAGTATTCCCTTTCC-5′	Orf3				4646
							#hp-3	5′-AGGACGCATCATCAATCT-3′	Orf3	50	2 minutes	1644	4199
#72	3′-ACACCTATCCGAAAGACTT-5′	Orf4				5843
							#204	5′-AAGGAGTTGCCTATACACG-3′	Orf4	50	2 minutes	755	5377
#209	3′-CTAACCACGTCCGAAATA-5′	Orf5				6132

#208	5′-TGGAGTATGAGCCAAAGAA-3′	Orf5	58	2 minutes 30 seconds	2395	5575
							#58	3′-GCTTATTTCACGCTGGATCT-5′	wzy				7970
#57	5′-TTTGTTTCTGATATATGCGGT-3′	wzy	50	2 minutes	1028	7277
							#214	5′-CGAAATTCAATGGTGGTC-3′	Orf7				8305
#75	5′-TTCAATGGTGGTCTCTCA-3′	Orf7	50	3 minutes 30 seconds	2731	8410
							#78	3′-TGAAGGGCAATATACCGC-5′	Orf9				11141
#213	5′-TGGACGATGATTGTATGC-3′	Orf9	52	2 minutes 30 seconds	2064	10565
							#1524	5’-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3’	gnd				12629

Embodiment 3: by the corresponding sequence of the O-antigen gene among the pcr amplification intestinal bacteria O164 bunch: being distributed in adjacent intragenic upstream and downstream primer with every pair, is that template is carried out PCR with the genome of intestinal bacteria O164 type.The PCR response procedures was as follows: 95 ℃ of pre-sex change 3 minutes, 95 ℃ of sex change are 15 seconds then, and annealing temperature is because of the difference different (in seeing Table) of primer, and annealing time is 50 seconds, 72 ℃ of extension time is carried out 30 circulations like this because of the difference different (in seeing Table) of primer; At last, continue to extend 10 minutes at 72 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product; Merge 4 pipe PCR products and deliver to order-checking company, carry out the sequencing of Nucleotide from the corresponding PCR product of both direction after, and design primer and order-checking again and lead to up to the sequence of each PCR product being surveyed from two ends to purifying with their corresponding upstream and downstream primer.

Embodiment 4: the splicing of nucleotide sequence and editor: 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 full length sequence of Nucleotide of the O-antigen gene bunch of intestinal bacteria O164.

Embodiment 5: inaccurate sequence checks order once more: the sequence with the both sides of inaccurate sequence is a template design primer, and is that template is carried out PCR with the genome of intestinal bacteria O164 type.Detect the size and the specificity thereof of PCR product by agarose gel electrophoresis, merge 4 pipe PCR products and deliver to order-checking company, carry out the sequencing of Nucleotide with their corresponding upstream and downstream primer from the corresponding PCR product of both direction after purifying.

Embodiment 6: carry out the splicing and the analysis of nucleotide sequence once more

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 O164 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O164 is done 6 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O164 bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O164 at last, as shown in table 3.

By retrieval and relatively discovery, between the galF of the O-of intestinal bacteria O164 antigen gene bunch and wzx, one section sequence (1258bp-2277bp) is arranged, comparison shows that the albumen of tnpA genes encoding of it and Pseudomonas putida has 68% similarity in 341 amino acid.With the transposase of Caulobacter crescentusIS1111A/IS1328/IS1533 family 60% similarity is arranged also, inverted repeats is also arranged at its two ends.Therefore this section sequence is an insertion sequence.Find that it is the same that orf1 and the wzx gene of E.coli O157:H7 have 17% aminoacid sequence, promptly has 17% homogeny, 42% similarity in 440 amino acid; And and the wzx gene of Bacteroidesfragilis in 506 amino acid, 20% homogeny is arranged, 42% similarity, this wzx gene has 13 potential transmembrane domains, and the algorithm [Eisenberg by Eisenberg etc., D, Schwarz, E.etal (1984) " Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf1 also has 13 potential transmembrane domains; and with many wzx protein similars; orf2 has about 50 amino acid whose conservative motifs at the proteic aminoterminal of wzx; so can determine orf1 is the wzx gene, called after wzx.The albumen of the hypothesis of 0rf2 and Sinorhizobium meliloti has 34% homogeny in 363 amino acid, 52% similarity is arranged, temporarily with orf2 called after orf2 gene.Orf3 also has 23% homogeny with the albumen of the hypothesis of Sinorhizobium meliloti in 226 amino acid, 42% similarity is temporarily with orf4 called after orf4 gene.Orf2 and Orf3 participate in synthetic and the transfer pyruvic acid, and a terminator codon is arranged in orf3, make it lose function, so do not have the side chain of pyruvic acid in the O-antigen of intestinal bacteria O164, with shigella dysenteriae 3 types.The eps9I gene of off4 and Streptococcus thernophilus has 30% homogeny in 239 amino acid, 54% similarity, and the eps9I gene is a glycosyltransferase gene; The CpsIaI gene of off4 and Streptococcus agalactiae has 29% homogeny in 390 amino acid in addition, 47% similarity, the CpsIaI gene is a N-acetyl glycosyltransferase gene, so think that orf4 is a glycosyltransferase gene, called after orf4.Orf5 and colibacillary cap8J gene show higher homology, this genes encoding galactosyltransferase, so orf5 is the galactosyltransferase gene, and called after orf5.Orf6 is the albumen that a prediction has 10 transmembrane segments, the topological framework of its inner membrance has the characteristic feature of well-known O-antigen polysaccharase, in addition, the wzy gene of the coding O-antigen polysaccharase of it and E.coli has 23% homogeny, 44% similarity in 355 amino acid; In 353 amino acid 22% homogeny is arranged with the wzy gene of Streptococcus thermophilus, 44% similarity is so name orf6 is the wzy gene.Among orf7 and the Streptococcus pneumoniae among coding cpsI gene of galactosyltransferase and the Streptococcus pneumoniae cps23FU gene of coding galactosyltransferase 34.5% and 33% homogeny is arranged respectively on amino acid levels, and semi-lactosi is arranged in the O-antigenic structure of shigella dysenteriae 3 types, so determine that orf7 also is a galactosyltransferase gene, called after orf7.Orf8 has the aminoacid sequence of glf gene of O-antigen gene bunch of 63% aminoacid sequence and E.coli K12 identical, shows the homology that height is arranged between them.In addition, sugared Galf is synthesized in the enzyme catalysis of glf genes encoding, and this is the composition in the shigella dysenteriae 3 type O-antigenic structures just, so infer that orf8 is the glf gene, called after glf.The gene BL0045 of orf9 and Bifidobacterium longum and the wcfN gene of Bacteoides fragilis have 33% homogeny on amino acid levels, and these two genes are the glycosyltransferase genes in the exocellular polysaccharide gene cluster.Therefore orf9 also is a glycosyltransferase gene, called after orf9 gene.

Infer that according to the structure of intestinal bacteria O164 it should have four glycosyltransferase genes, shift five sugar respectively with the synthetic antigenic oligosaccharide unit of O-.Need the wzx gene that oligosaccharide unit is transferred to outside the film, the wzy gene aggregates into polysaccharide with oligosaccharide unit, special monose in the oligosaccharide unit is also synthetic by O-antigen gene bunch, and is synthetic by the glf gene as Galf, and these genes all are found in the O-of intestinal bacteria O164 antigen gene bunch.But in the antigenic structure of intestinal bacteria O164 O-, also has GalNAc.In Yersinaenterocolitica O8, UDP-GalNAc is generated by the semi-lactosi mutase catalysis UDP-GlcNAc of gne genes encoding, in the O-of intestinal bacteria O164 antigen gene bunch, also do not find the gne gene, but Peter Reeves etc. are by having the gne gene in PCR discovery other gene clusters in some bacterium such as E.coli O124, that is to say that an O-antigen gene bunch outer gne gene transformation UDP-GlcNAc generates UDP-GalNAc and provides O-antigen to synthesize essential sugar, so we think that the gne gene may be in other gene clusters of intestinal bacteria O164.

Above-mentioned analysis revealed, the O-antigen gene of intestinal bacteria O164 type bunch has identical gene with the O-antigen gene cocooning tool of shigella dysenteriae 3 types.These genes have identical putting in order.Unique different be that (5 ' end) has an insertion sequence (IS) before first open reading frame of O-antigen gene bunch of intestinal bacteria O164.This is that the O-antigen gene of shigella dysenteriae 3 types bunch does not have.

(7) screening of specific gene: behind the O-antigen gene of O-antigen gene that has compared intestinal bacteria O164 type bunch and shigella dysenteriae 3 types bunch, be IS sequence and adjacent wzx gene design primer at the different sequences of the O-antigen gene of the O-antigen gene of intestinal bacteria O164 type bunch and shigella dysenteriae 3 types bunch; Upstream primer is distributed in the IS sequence, and downstream primer is distributed in the intersection or wzx gene of IS sequence and adjacent wzx gene, and to guarantee its specificity to intestinal bacteria O164 type, these gene positions in nucleotide sequence see Table 1.In table, also listed the size of each primer.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.Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines that the IS sequence is a high special to the O-antigen of intestinal bacteria O164 type; Can carry out PCR with above-mentioned primer, identification of escherichia coli O164 type is also distinguished intestinal bacteria O164 type and shigella dysenteriae 3 types in the environment.

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

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

In the 8th group, contain the genomic dna of intestinal bacteria O164 as positive control.In the 13rd group is the genomic dna that does not contain intestinal bacteria O164, as negative control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 2 minutes, 95 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For IS sequence and adjacent wzx gene, there are 4 pairs of primers detected, the every pair of primer is distributed in respectively in IS sequence and the adjacent wzx gene.Obtained except be PCR in the 8th group after the correct band of expection size, the correct band of any size does not all increase in other groups.So the IS sequence all is a high special to intestinal bacteria O164 and O-antigen thereof.

(8) detection of primer sensitivity: the frozen bacterium liquid of intestinal bacteria O164 is inoculated in the triangular flask of LB substratum, 30 ℃ of-40 ℃ of cultivations, 180 to 250 rev/mins, cultivate a few hours to saturated, get cultured bacterium liquid dilution, get dilution bacterium liquid coating LB agar plate, 30 ℃ to 40 ℃, cultivate a few hours counting, calculate viable bacteria concentration in the stoste; In being the live pig meat stuffing of 20g, 5 parts of weight mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add the LB substratum, filter, and filtered liquid 180 to 250 rev/mins, is cultivated a few hours in 30 ℃ of-40 ℃ of cultivations; Peek ml is in 6 from cultured bacterium liquid, and centrifugal several minutes of 000g removes supernatant, adds the MQ ultrapure water and blows precipitation and mixing open, puts into 100 ℃ of boiling water and boils several minutes, and lysate is in 12, and centrifugal several minutes of 000g gets supernatant as pcr template; As upstream primer, they are with the described oligonucleotide that comes from the IS sequence: the Nucleotide of 1868 to 1885 bases among the SEQ ID NO:1; The Nucleotide of 2265 to 2284 bases among the SEQ ID NO:1.The described oligonucleotide that comes from the wzx gene is as downstream primer, and they are: the Nucleotide of 3020 to 3050 bases among the SEQ ID NO:1; The Nucleotide of 2809 to 2827 bases among the SEQ ID NO:1 carries 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 condition of PCR reaction 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 O164 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 O164 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 O164 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O164, has listed the structure of the O-antigen gene bunch of intestinal bacteria O164 in table, IS sequence and 9 genomic constitutions altogether, and each gene box indicating, and in square frame, write the title of gene.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O164, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O164, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in bacterium: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O164

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O164

<160>1

<170>PatentIn version 3.2

<210>1

<211>14006

<212>DNA

<213>Escherichia coli

<400>1

ctcctggtaa ctcacgcgtc caagaacgcg gtcgaaaacc acttcgacac ctcttatgaa 60

ttagaatctc tccttgagca gcgcgtgaag cgtcaactgt tggcggaagt acaatctatc 120

tgtccgccgg gcgtgaccat tatgaacgtg cgtcagggtg aacctttagg tttaggccac 180

tccatcttat gtgcgcgacc tgccattggt gacaacccat ttatcgtggt actgccagac 240

gtagtgatcg acgacgccag cgccgacccg ctgcgctaca accttgctgc catgattgcg 300

cgcttcaacg aaacgggccg tagccaggtg ctggcaaaac gtatgccggg tgacctctct 360

gaatactccg tcatccagac caaagaaccg ctggaccgtg aaggtaaagt cagccgcatt 420

gttgaattta tcgaaaaacc ggatcagccg cagacgctgg actcagacat catggccgta 480

ggtcgctatg tgctttctgc cgatatttgg ccggaactag aacgcactca gcctggtgca 540

tgggggcgta ttcagctgac tgatgctatt gccgaactgg cgaaaaaaca gtccgttgat 600

gccatgctga tgactggaga cagctacgac tgcggtaaaa aaatgggcta tatgcaggcg 660

tttgtgaagt acggactgcg taacctgaaa gaaggggcga agttccgcaa aggtattgag 720

aggttgttga atgattaaaa acatgaacaa atataacggt aaataagaga aattgtaacg 780

gcagtgagga tttgcggcga agctcaaacg tggcgaatat ccctgtcgtt atttattaat 840

aaatcatcgt actaacaatg gaataaaaaa gtgccttgtt ttaagtttta caggatattc 900

cttgtttctg gaggagattg ataagacaat cagcatctga atttatcaga acttgtgtgt 960

cttgctgatt acattgttat attcatgcag tgtactggta gctgtggagc caggggcggt 1020

agcgtgcatt aatattcaca atatattctt ttaaatgtaa gcagaattaa tcgaacctaa 1080

ataatgtcac gtaacaaaat aatcataaaa atgtaatgaa cgcatcccac acaattaaac 1140

acggagcagt atgatggtta aaagggtgaa actggcaggt gtggagcctc gcagattccg 1200

gcatcatagt gcccaatacg ggatgcataa agctccctca gactggagac tccacaaatg 1260

aaatatacac cggttggcgt tgatatcgca aaacatgtca ttcagattca cttcatcaat 1320

gagcacacag gtgaagtggt tgataaacag ttgcgtagac aggattttct gacgttcttc 1380

ggcaaccgtg agccatgcct gattggtatg gaggcctgtg gaggttctca gcactgggca 1440

cgggaactga caaaacttgg tcataaagtc cggttgttgc aggcccgctt cgttaaggca 1500

ttcgtcatgg gcaataagaa tgatgtgatg gatgcccggg ctatctggat ggcggttcag 1560

cagccgggta aagaaatcgc cgtaaaaaca gaagaacagc agtcggtact ggttctgcac 1620

cgtacccgca tgcaactggt gaagttccgg accgcacaaa ttaatgccct gcacgggacg 1680

ttactggagt ttggtgaaac catccacaaa ggccgggcag cgatggagcg ggagttcccc 1740

gaagcactgg aacggatgaa agagagactg ccaccgtatc tcattatggt tctggaaaac 1800

cagtacaacc gactgaatga gctggactca ctgatagagg atattgaaaa acagcttacc 1860

agcgtggcga ggcagaatga aacctgtaag cggttgctgg atattcctgg cgttggacca 1920

cttattgcga cggcagcggt ggccaccatg ggggaagcat cagcgtttaa atcggggcga 1980

gagttcgccg catatgttgg tctggttcca aaacaaactg gctccggagg gaaagtacgt 2040

ctgctgggga taagcaaacg tggtgacact tatctcagga cattatttat ccacggtgca 2100

agagcggtgg cattagtagc taaagagcct ggcccgtgga taaccgaact gaaaaaacgt 2160

cgtccagcca gtgtggcaat cgtcgccatg gcaaacaagc tggcacgaac agtatgggcg 2220

ataaccgccc atgaccgtaa gtatgacagg aaccacgtca gtatcagacc atattaatcg 2280

ctgataccat taaacaatga actcttaaca aaagggtgaa tgctgaaagg ttgctatggc 2340

ggccagagtg atgacaaaga caggtaagac cgtgactcac taaacctgaa cagtattttg 2400

ggcttgaagt ccgccgtgaa aataaggggt gagtcggcga attacatagg ggctcgcagc 2460

gttacggctg caataaagcc ggatataaag ctgcaaccta cccgtcatgt caaaacaatg 2520

gatgccttgc aaacgggatg cgttcatata aatactatgt ttttggcaac aagaacctta 2580

gtttctttaa gtgtttcatt ttacacaaca agaatagtta tacaacagtt gggggcggca 2640

gactatggct tatttaatat catttatgga gttgtaacat tttttacatt cgtagtcacc 2700

gcgatgaatg attctgtaca gcgttatatt gcaataggtg ttggttcaca aaaaatatcg 2760

gttattagag atgctgtaaa aaatagtatg tttatatttg ttatatctgc tttcatattg 2820

gcagtatgtc ttcttttggc aaggggggtg atcatacata atattttaaa tatacctaaa 2880

gattctattg aaaatgcaag tgtgttatat ttggttgcag tattttcaat aaccatactt 2940

ataattcaga cgccacttaa tgcgatggtg ttagcatacg agaaaatgtc attttacgca 3000

tacatgatga tatttgaaat ggtagctaaa atgagtatgg ctttgttact aactttactg 3060

gaaaaagata aggtcattgt atattcaata cttttaggtt ctatttcttt ttttaacttg 3120

ttggtttatt tatgctactg cttgatatgt tttaaaaaat caatgtttgg tgggagaata 3180

aaatttagag tgctgaaaga aatttcaact ttttcatttt ggaatatatt tggtaatttc 3240

tcttacatgt gcagagttca gggagtgaac attgtaatta atatgtttta cgctatagcc 3300

gtgaatgcag catatgcgat ttcgattact gttttaaatg caattaatac acttacgcaa 3360

tcattaatta cagcattaag gcctcaaata tttaaatcat atggcgaatg tgatttaaaa 3420

agatataatc atctagttct ctttggttca aaatatacat tttcaatatt gtttctgtta 3480

agtagccctg taattctatg tgcggatgag ttgttaaaga tatggcttga tattgtacca 3540

gattatacag ttgaatttgt gaggttggtc attgttgtgg cttttattga tagtttttca 3600

tatagcatga ttgctggtat tcaggctacg ggtaggatta aaacatatca actagttgtt 3660

agtttaattg tcctaattaa tttgccgtta acctttattt tgtttaaggc aggaaataat 3720

gttttaagta tgttttatcc atttattgtt actgccatta tcaatcaagg tttaaggttg 3780

tattttatct atatcaatgc aggttttgat tataagaagt atttcactgt tgtgatctat 3840

ccttgcttgt tagctgtatg cttatcgtta gtgacagata tttcaattaa aaaaatgctt 3900

ccatttaaca gtgttattga tgttttaatt gtgtgtattt ttattttttc attcaataca 3960

ataatttttt attgggttgt agtttctaaa aaggaaaaaa atggttatta gaaaccctga 4020

aaagaaaagt gaaataacaa taaaagatgt tatagacaat gaactctgta ctggttgtgg 4080

agtttgtatt tcggaagata gtagcaaaac ctcctttatg aaatggaatt ctgaaggttt 4140

ttatgagcca tgtttctctc ctgtatcaac attattcaat atgcagcgtg tttgtccctt 4200

taacttaagt agggatacat tagtgaatga ggatgaactt gcgcatgagt tttttgatgg 4260

taagggatat ttagattctg aggttgggtt ttataaaaaa atatatgtgg gttattctaa 4320

acattttaga gagacttcat cctccggtgg tatagctaca tatgtatttg aacaacttct 4380

tagacgtaag tatgttgatg ctctatttat agttagggag cttggtggtt catatggata 4440

tcaagtcttc gataatccag aacatattaa ggatatgtct aaaacaagat attatcctgt 4500

aacgctagaa aagttatttg ataatattta taagcttaat ggtagagtgg ccgtttcagg 4560

agtggcatgt tttattaaag ccattaggct caaacaacat taccatccag aactgaaaga 4620

gaaaatacca ttccttgttg gtataatttg tggaggactc aaaagtcgat attacactga 4680

ttatttatca cagtctgctg gttgtgtcag tgagtaccaa aatgctgagt atcgagtgaa 4740

aaagaaagat agtcatgctc ttgactatcg tttcacatgt gttgagaaat caaataatat 4800

aattcattct gttgatatgc aaagaatggg ggatatgtgg ggttcagggc tatttaaggc 4860

aaacgcatgt gactattgtg atgatgttac aaccgaactc gcagacatat cattgggtga 4920

tgcctggatt tcgccataca atatggatgg agctggtaat aatgtagttg tatgtcgctc 4980

caccacagct catgaaatta ttttatctgg gattgagaaa aaagacttag aattaacgga 5040

actcgaatta gagcagttga aattgtcaca acaaggaagt tttaaccaca ggcataaagg 5100

attattatat agaataaaaa atgcagagaa aaataatcgc ttagtgcctg ttaagaggaa 5160

aagattctta cgctctatat cttttttatt aaagttaatt caaaaacaac gatcagttac 5220

aaggagaaaa agcatagaga tatggatgga aactcaaaat tcagcaactt tcgataagaa 5280

aatgaaggga tatttattca cgttgcgttg gttaacagtg gtcaatagga aattaagtcg 5340

gatgttcaag attgtttctc taaatagaat gggtaaaaaa tgaaagttgc aatattaact 5400

caaccattac atacgaatta tggtggcaca ttacaagcct atgcattaca aaaagtcttg 5460

attaatttgg gacatgaacc agagactatt aattataggt cgaaaattaa gaggccacta 5520

tttatccgtg ttgtgttgtc aaaaattaaa aggatagtat tatgccgaaa aatcacattt 5580

gattttacaa cacaagatag aattaatatt aggaagcatc atcaatcttt cattgataca 5640

agattaaact attcagagga gattaacggt actgaggggt tgagagatta tattttaaaa 5700

aataattacg gtgctgtaat aataggtagt gatcagacat ggagaccaat atattcgcct 5760

agaatagatt cattttttct cgatttttta agtgatgtaa acgatataaa aaatggcata 5820

tgcagcatct tttggtactg ataaatggga gttcacggag tcacaaacta atttatttaa 5880

attgttactt tcaaaatttg actatgtttc agttagagag tcctcaggag tcaaattatg 5940

tagtgaaaaa tttggtttga aagcagagtt ggttttagat ccgacattgc tattgacatt 6000

cgaagattat actcatttat tagacaatga ctatattaat cttcataagg gaaaggtgtt 6060

tagttatgta cttgatgaga attacgacaa aaaaaacttt atagacacag tagcaagtac 6120

tctccgcaca acgtcatttt atacttaccc taaaaaaatc actaaagatg aatatgtgat 6180

tagaaattat tccgagtatg aatatccgcc aatagaatat tggatttctt cgtttaaggc 6240

cgctgaattt attgtaacag attcatttca tggaacagta ttttctatta tttttaacaa 6300

acccttcatc gccattgcaa atgaagagag aggaaaggct agatttacgt cactattgga 6360

aatgtttggt cttgaaagac gacttgtgag taatttagat gatgttgatt taaagcttgt 6420

aaatgaaaaa attaactatg gccccataaa tgaaaaaatt gcttatttta gagaagagtg 6480

ccttaaaaag ttgcagttaa tgctaataaa ataatcagat tatgtatgtc tctctacaat 6540

taaaatatga atgttgatcg atgaagaatt gtaaagtatc tgtaattatt ccggtctata 6600

atgcggaaaa atatatacaa cgttgtattt taagcttatt aaagcagaca ttagatgatg 6660

ttgaaattat catcattgat gatggttcta ctgataattc actttctata ataaaagaaa 6720

ctgttgcatt gcatactgct agtagagctc gccaaaagag aatcaatata atctctcgag 6780

aaaacaaagg agttgcctat acacgaagtc agggattgag actttcgcag ggggaattta 6840

tcattcattt tgatagtgat gattgggcaa aatccaatat gctggaggaa atgtataaga 6900

caattgtttc caataatgca gacatggtta tatgcgatta ttttttagtg aagaataata 6960

aagaaatact tataaaacag agagtggagt atgagccaaa gaaatgtatt agatatctac 7020

tgactggtga gctagaaggg tttacatgga acaaactaat tagaaaaaaa tatatcgata 7080

aaaataaaat tgattttgtt aataaaatta cttatatgga ggattttttg ttcatattaa 7140

gtgtgttact acacaatcct aaaattattt ttcaggattg tgcatattat tattatcaaa 7200

aaagcaatcc taaatctcta actagccatg ctagtgtgga taggctttct gaaatgatta 7260

aagccgtaag tgaaatagaa aagaagataa ataaatataa tttacaaaag tatctgaata 7320

acgagtttca attgtttaag ttaaaacaaa aaatttggtt tatatctata agtaagttaa 7380

atgttaatga taatgtatgg aatctctttc ctgaaacaaa tccatttata tcaaaggtaa 7440

atgtgctttt ttattataaa atagtgcttt ttcttgatag tattaaactg cgtttttttt 7500

caaataagat catttatctc attggattgg tgcaggcttt attacaagaa aggaaagaga 7560

agtgatacca aaaataattc attactgctg gttcggtcgc tctccattgt cggaattaac 7620

aaagcagtgc attgcaagct gggagaaata ttgtcctgaa tataaaatca taagatggga 7680

tgaaaataat gtcgatttga attcttgttc ttttgttaga caagcttata aagaaaaaaa 7740

atgggcgttc gtttctgatt atgtaaggct taaagtggtt aatgaatatg gcggtatcta 7800

tcttgataca gatgttgaat taataaagcc attagatgat ttgttaatat atcctgcata 7860

tataggcttt gaaattaaca aggaatggta tgtaaattct ggtttggggt tcggttctgt 7920

taataataat ccggttttag aatcattaat tatggagtat gaaaatatta attttgtaaa 7980

tgaggatggg actctaaata taacgccgtg ccccataaga gaaacaaaag cattaacaaa 8040

aataggttta attcctgatg gtcaatgtca atcttttgat aatatagtga ttttttctgc 8100

agattatttt tgtcctgtat ctataacagg tgagagaaat ttttctgata aaacatattc 8160

aatacatcat tacgatgcta gttggttttc tgagcaaaag agaaaaggcc tgcaacgtaa 8220

aaagagattt ataaaactat tcggcaacct aataggcaca tatattaata aaccttttat 8280

ttttgtagat gaatgtcgcg agttcggatt agttaaagca atcaagaata tgcgttccaa 8340

ttttccttaa tttttagtgt taaggaataa gtttcgttaa aatactttat tcatttaatt 8400

aataaaggtt taatggtgat tagatgtact ttcttttgtt cttctcaata ttttattttg 8460

tatacgtaat cttaatagga acattatgtc ataactataa agcccttttg attttttcac 8520

taatccctct gacaatagtg tcaggtataa gatataatgt cggctttgat tttatgtcat 8580

atgttgatta tttcaatcaa ctcaaatatt caaatgatat atatctagat tctaccttta 8640

agtatataag ctactttacc tattatatag gaggtaacga acagattttg tttctgatat 8700

atgcggtttt ttattcggtt gcactttatt ttttaataaa actggtattg gaaaattata 8760

taacggaaat aaataatttt tatactattg gacttgtgtt atcattttat tcgttttatt 8820

ttttattatc atttaatcaa attagggctg tattatcagc tctaatacta tgttatgggc 8880

tattaaaaaa taaaatagat ttttctttta taataacaat tacattaagt attctttttc 8940

actctgcagc aatgttcatt ttacctcttt attttgtcct aagaaaaata aatgtaacag 9000

ttctgttggt gatattccct tttttggttg ttgcttcatt ctttaatatt ttttcagata 9060

ttgtgagatt cattctgaca ttgctaaata gtcgtttttt aacatatttt aactctgaat 9120

attttgttcc aaggacggga atggaaaaaa tgtatagtat gatctcaatg attattgttt 9180

tgggaatggt tgtttgtttg tctaaattat tacctaaaaa gtttgattta atgataaagt 9240

ttgttattct ttttgttcta cttagagcga tgtcaattga tatattgatt tttgctaggt 9300

ttagtgattt tcttaaacca atggctataa ttcttatatt taccgctgtt tatttttctt 9360

cgaataaagt gcgacctaga attatccttc ctttttattt aattatgaca ttgtttctct 9420

gtatttttaa tataataatc ggatcgaata taactaaggg gaactattat acttatggtt 9480

ataacatttg cttttttggc aataaatgta tagataaatt ttattagtaa ttgtcatttg 9540

atacttctgg tatttatgga aaaatatatg aataactttt cttgtaaaga acccccttca 9600

tttattgaaa ataaaattat attatcaatt attcttcctg tatataacgt atcagagtat 9660

ttaattgagt gcctaaacag tttgctgtta gacgttcatg aacgttatct taatagatgt 9720

gaagtaattg tggttgatga tggttcaact gataattcat ttgaattaat gagagagtat 9780

tctttgaaat atcctgaatc tattaagatt tattcgaaat tcaatggtgg tctctcagat 9840

gcaagaaact atggattatt aaaatcttca ggtaaatata tttcttttgt tgattcagat 9900

gatgttgtta atagaggttt tataaaggaa atcatcaatt tcattgatgc ctatgatttt 9960

gatatattgt catttgactt catgaagttt tttaataata atgatgctct tattctttca 10020

caaatagatg aattctccaa aatggcagaa agagtagatt cggagttcta caagtccaag 10080

cctgtttttg cttggaataa agtttatcga cgttctcttt tcgacaatga agtatttcct 10140

aaggggtggt attatgaaga tgttgcatta attccgttat tattagatag agcaaaagtt 10200

ttataccata ttaatagcgt ttgttatttt tatagacaga gacaaggagc aatcactttt 10260

ttttatgaca ataaatatct tgatatttta aaaggagtgt cttttttata tgacaggtca 10320

caatcttcat tcattaaaac gataattata aatcagtttt ttactttaac cttactatct 10380

ttaagattgc cgactgataa ttatttcatg aatatgaggg ggattattga tacctattgt 10440

gaaaaatttg acttggattc ctttgagcct aaactaatat taaaacacat tcctttttta 10500

ttgcttaaaa aattgaagtc gctgtgtgtt tatccactgt atttatttaa acctgcagtt 10560

tttttacata aacaaattaa acattttaga ggaaaactaa agtgaaatat gattatataa 10620

ttataggtag tggtcttttt gggagtgtgt ttgcacatga attagggaaa atgaaaaaaa 10680

aggtcaaggt tattgagcgt cgtgatcatg ttggtggtaa tgtatattgt gagaatattg 10740

aaggaattaa tgttcataaa tatggcgcac atatatttca tactaacgat aaagaaatat 10800

gggattatgt aaatcaattt gtagagttta atagatatac aaattcacca ttagcttttt 10860

caaaaggtaa aatttataat ttacctttta acatgaatac ctttaatcaa ctctgggggg 10920

ctattactcc tcaacatgcg aaagaaatta ttaaaaagca aagcggtgaa ataagtggaa 10980

ataaaccaag gaatcttgaa gagcaagcta tttctttggt gggacgagat atttataatt 11040

gtttgattaa ggagtacact gagaaacaat ggggacgacc ttgcactgaa ttgccatcat 11100

ttattataaa acgactccct gttaggttta catacgataa taactatttt aatgaccagt 11160

atcaaggaat accagtcggt ggttacaata aattaatcga tgcattatta aatggtattg 11220

aagttcaaac atgcactgac tattttgaag agaagaaata ttgggattct attgcaaaga 11280

aaattattta tacagggcct attgataagt tttattgcta taaatttgga gctttggatt 11340

atcgatcatt aaaatttgag catgaattac tgaatgtcga taattttcaa gggaatgctg 11400

ttgtaaatta tatagacaaa aaattcccat tcactcgtat aatagaacat aaattttttg 11460

aatttggtga acagcataat actctcatta ctcgagagta cccaaaagaa ttcgaagaag 11520

gcgatgagta ttactatcct gtcaatgatg aaaaaaatat gcaaatctat aaaatgtatt 11580

ataatctttc taaacaggaa agtaatgtga tttttggtgg acgtcttgcg gaatataaat 11640

attatgacat gcatcaagtt atcaaatcag ctttaaatgc attccgaaaa gaggtacgtt 11700

taggtgaata aagaagtaat cgcagttatt gtaacctata atagaaaagc attattgctg 11760

aaagttatag atgctgttat taaccaaagt tatccactaa aaaaaatatt gataatagat 11820

aataatagta ctgatggtac ggagatgttt atttcaaata ggctctctga cgtggtgaag 11880

tataaaaata cgggggataa tttagggggg gctggcgggt tttatagagg attcatagag 11940

gcggaaaaat atggctatga ttatctttgg ttaatggacg atgattttat gcctacagtg 12000

aattgtcttg aaatattaat atcaaatagc cctgatggta ttgtccaacc tgttagatat 12060

aacttggatg aaacttgtgc agagctttct ccattaactt atgatttatc taacccattt 12120

aagttaaacc ctaaaggtac accattaaaa aattacttaa atacaattgg taataaatct 12180

gcgaaggtag atattgaagc tattccattt gaaggtccat tgatttcacg aaaggttgtt 12240

gaaaaaatag gctatccaga gcctaagttt tttatttttt gtgatgacat tgaatacgct 12300

attaaagcta aaagaaaggg gattcccatc cagtgcaatc ttaaagccaa ggcataccgc 12360

cttttggtca acaatcaagg taatgatctt ctttcctgga aaggatattt tatgcttcga 12420

aatcttttct acctgcataa aacgtatggg accaatttcc ttgttagaca aaaacctatc 12480

gtattgggat taggatatgc attgtcgtgt gtattgaaat gcaatttttc tcaacttccc 12540

gttatatggc gtgctttttg ggattcatct acgttaagaa ataccgaaaa attcaggccc 12600

gaaatgagaa gtaaataatt tattattatg tttttattaa ttaatgtcga tttttactaa 12660

aattcttaac acgatattta tccctgacag gagtaaacaa tgtcaaagca acagatcggc 12720

gtcgtcggta tggcagtgat ggggcgcaac cttgcgctca acatcgaaag ccgtggttat 12780

accgtctcta ttttcaaccg ttcccgtgaa aaaacggaag aagtgattgc cgaaaatcca 12840

ggcaaaaaac tggttcctta ctatacggtg aaagagtttg ttgaatctct ggaaacgcct 12900

cgtcgcatcc tgttaatggt gaaagcaggt gcaggcacgg atgctgctat tgattctctc 12960

aagceatacc tcgataaagg tgacatcatc attgatggtg gtaacacctt cttccaggac 13020

accattcgtc gtaaccgtga gctttctgca gaaggcttta acttcattgg taccggtgtt 13080

tccggcggtg aagagggggc gctgaaaggg ccgtccatca tgcctggtgg ccagaaagaa 13140

gcctatgaac tggttgcacc gatcctgacc aaaatcgccg cagtagctga agacggggag 13200

ccatgcgtta cctatattgg tgccgatggt gcaggtcact atgtgaagat ggttcacaac 13260

ggtattgaat acggtgatat gcaactgatt gctgaagcct attctctgct taaaggtggc 13320

ctgaacctca ccaacgaaga actggcgcag acctttaccg agtggaataa cggtgaactg 13380

agcagctacc tgatcgacat cactaaagac atcttcacta aaaaagatga agacggtaac 13440

tacctggttg atgtgattct ggatgaagca gcaaacaaag gtaccggtaa atggactagc 13500

cagagcgcgc tggatctcgg cgaaccgctg tcgctgatta ccgagtctgt gtttgcacgt 13560

tatatctctt ctctgaaaga tcagcgcgtt gccgcgtcta aagttctcac tggcccgaaa 13620

gcacagccag ctggcgaaaa ggctgagttc atcgaaaaag ttcgtcgtgc gctgtatctg 13680

ggcaaaatcg tttcttacgc tcagggcttc tctcagctac gcgccgcgtc tgaagagtac 13740

aactgggatc tgaactacgg cgaaatcgcg aagattttcc gtgctggttg catcatccgt 13800

gcgcagttcc tgcagaaaat caccgatgcc tatgccgaaa atccgcagat cgctaacctg 13860

ctgctggctc cgtacttcaa gcaaattgcc gatgactacc agcaggcgct gcgcgatgtc 13920

gtcgcttacg cagtacagaa cggtatcccg gttccgacct tcgccgctgc ggttgcctat 13980

tatgacagct accgttctgc tgttct 14006

Table 1 comes from the primer and the PCR data of IS sequence and wzx gene in the O antigen gene bunch of intestinal bacteria O164

Primer	Primer location	The residing gene of primer	PCR product length	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
						Article one, the 4th reverse primer of the 4th forward primer of forward primer article one reverse primer	1868 to 1885 bases, 3020 to 3050 bases, 2265 to 2284 bases 2809 to 2827	IS sequence O-antigen transhipment enzyme IS sequence O-antigen transhipment enzyme	1182bp 562	0 0	58 55

The intestinal bacteria of 166 kinds of serotypes of table 2 and 43 strain Shigellaes and their source

The bacterium source that contains in this group of group number

1, wild-type e. coli O1, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, IMVSa

O19ab，O20，O21，O22，O23，O24

2, wild-type e. coli O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, IMVSa

O36，O37，O38，O40，O41，O42，O43

3, wild-type e. coli O6, O44, O45, O46, O48, O49, O50, O164, O52, O54, O55, O56, IMVSa

O57，O58，O60，O61，O62，O53

4, wild-type e. coli O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, IMVSa

O79，O80，O81，O82，O83，O68

5, wild-type e. coli O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, IMVSa

O101，O102，O103，O104，O105，O106，O97，

6, wild-type e. coli O107, O108, O109, O110, O111, O112ab, O112ac, O113, IMVSa

O115，O116，O118，O120，O123，O125，O126，O128，O117

7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O164, O137, IMVSa

O138，O139，O141，O142，O143，O144，O145，O140

8, wild-type e. coli O146, O147, O148, O150, O152, O154, O156, O157, O158, IMVSa

O159，O160，O161，O163，O164，O165，O166，O153 b

9, wild-type e. coli O168, O169, O170, O171, O172, O173, 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 O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVSa

O124，O167，O162，O121，O127，O149，O119

13, wild-type e. coli is removed the 8th group of bacterium of intestinal bacteria O164

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

a.Institude of Medical and Veterinary Science(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 is structural tables of the O-antigen gene bunch of intestinal bacteria O164

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O164

CTCCTGGTAA CTCACGCGTC CAAGAACGCG GTCGAAAACC ACTTCGACAC CTCTTATGAA 60

TTAGAATCTC TCCTTGAGCA GCGCGTGAAG CGTCAACTGT TGGCGGAAGT ACAATCTATC 120

TGTCCGCCGG GCGTGACCAT TATGAACGTG CGTCAGGGTG AACCTTTAGG TTTAGGCCAC 180

TCCATCTTAT GTGCGCGACC TGCCATTGGT GACAACCCAT TTATCGTGGT ACTGCCAGAC 240

GTAGTGATCG ACGACGCCAG CGCCGACCCG CTGCGCTACA ACCTTGCTGC CATGATTGCG 300

CGCTTCAACG AAACGGGCCG TAGCCAGGTG CTGGCAAAAC GTATGCCGGG TGACCTCTCT 360

GAATACTCCG TCATCCAGAC CAAAGAACCG CTGGACCGTG AAGGTAAAGT CAGCCGCATT 420

GTTGAATTTA TCGAAAAACC GGATCAGCCG CAGACGCTGG ACTCAGACAT CATGGCCGTA 480

GGTCGCTATG TGCTTTCTGC CGATATTTGG CCGGAACTAG AACGCACTCA GCCTGGTGCA 540

TGGGGGCGTA TTCAGCTGAC TGATGCTATT GCCGAACTGG CGAAAAAACA GTCCGTTGAT 600

GCCATGCTGA TGACTGGAGA CAGCTACGAC TGCGGTAAAA AAATGGGCTA TATGCAGGCG 660

TTTGTGAAGT ACGGACTGCG TAACCTGAAA GAAGGGGCGA AGTTCCGCAA AGGTATTGAG 720

AGGTTGTTGA ATGATTAAAA ACATGAACAA ATATAACGGT AAATAAGAGA AATTGTAACG 780

GCAGTGAGGA TTTGCGGCGA AGCTCAAACG TGGCGAATAT CCCTGTCGTT ATTTATTAAT 840

AAATCATCGT ACTAACAATG GAATAAAAAA GTGCCTTGTT TTAAGTTTTA CAGGATATTC 900

CTTGTTTCTG GAGGAGATTG ATAAGACAAT CAGCATCTGA ATTTATCAGA ACTTGTGTGT 960

CTTGCTGATT ACATTGTTAT ATTCATGCAG TGTACTGGTA GCTGTGGAGC CAGGGGCGGT 1020

AGCGTGCATT AATATTCACA ATATATTCTT TTAAATGTAA GCAGAATTAA TCGAACCTAA 1080

ATAATGTCAC GTAACAAAAT AATCATAAAA ATGTAATGAA CGCATCCCAC ACAATTAAAC 1140

ACGGAGCAGT ATGATGGTTA AAAGGGTGAA ACTGGCAGGT GTGGAGCCTC GCAGATTCCG 1200

The IS initiation site

GCATCATAGT GCCCAATACG GGATGCATAA AGCTCCCTCA GACTGGAGAC TCCACAA ATG 1260

AAATATACAC CGGTTGGCGT TGATATCGCA AAACATGTCA TTCAGATTCA CTTCATCAAT 1320

GAGCACACAG GTGAAGTGGT TGATAAACAG TTGCGTAGAC AGGATTTTCT GACGTTCTTC 1380

GGCAACCGTG AGCCATGCCT GATTGGTATG GAGGCCTGTG GAGGTTCTCA GCACTGGGCA 1440

CGGGAACTGA CAAAACTTGG TCATAAAGTC CGGTTGTTGC AGGCCCGCTT CGTTAAGGCA 1500

TTCGTCATGG GCAATAAGAA TGATGTGATG GATGCCCGGG CTATCTGGAT GGCGGTTCAG 1560

CAGCCGGGTA AAGAAATCGC CGTAAAAACA GAAGAACAGC AGTCGGTACT GGTTCTGCAC 1620

CGTACCCGCA TGCAACTGGT GAAGTTCCGG ACCGCACAAA TTAATGCCCT GCACGGGACG 1680

TTACTGGAGT TTGGTGAAAC CATCCACAAA GGCCGGGCAG CGATGGAGCG GGAGTTCCCC 1740

GAAGCACTGG AACGGATGAA AGAGAGACTG CCACCGTATC TCATTATGGT TCTGGAAAAC 1800

CAGTACAACC GACTGAATGA GCTGGACTCA CTGATAGAGG ATATTGAAAA ACAGCTTACC 1860

AGCGTGGCGA GGCAGAATGA AACCTGTAAG CGGTTGCTGG ATATTCCTGG CGTTGGACCA 1920

CTTATTGCGA CGGCAGCGGT GGCCACCATG GGGGAAGCAT CAGCGTTTAA ATCGGGGCGA 1980

GAGTTCGCCG CATATGTTGG TCTGGTTCCA AAACAAACTG GCTCCGGAGG GAAAGTACGT 2040

CTGCTGGGGA TAAGCAAACG TGGTGACACT TATCTCAGGA CATTATTTAT CCACGGTGCA 2100

AGAGCGGTGG CATTAGTAGC TAAAGAGCCT GGCCCGTGGA TAACCGAACT GAAAAAACGT 2160

CGTCCAGCCA GTGTGGCAAT CGTCGCCATG GCAAACAAGC TGGCACGAAC AGTATGGGCG 2220

The IS termination site

ATAACCGCCC ATGACCGTAA GTATGACAGG AACCACGTCA GTATCAGACC ATAT TAATCG 2280

CTGATACCAT TAAACAATGA ACTCTTAACA AAAGGGTGAA TGCTGAAAGG TTGCTATGGC 2340

GGCCAGAGTG ATGACAAAGA CAGGTAAGAC CGTGACTCAC TAAACCTGAA CAGTATTTTG 2400

GGCTTGAAGT CCGCCGTGAA AATAAGGGGT GAGTCGGCGA ATTACATAGG GGCTCGCAGC 2460

The Wzx initiation site

GTTACGGCTG CAATAAAGCC GGATATAAAG CTGCAACCTA CCCGTCATGT CAAAACA ATG 2520

GATGCCTTGC AAACGGGATG CGTTCATATA AATACTATGT TTTTGGCAAC AAGAACCTTA 2580

GTTTCTTTAA GTGTTTCATT TTACACAACA AGAATAGTTA TACAACAGTT GGGGGCGGCA 2640

GACTATGGCT TATTTAATAT CATTTATGGA GTTGTAACAT TTTTTACATT CGTAGTCACC 2700

GCGATGAATG ATTCTGTACA GCGTTATATT GCAATAGGTG TTGGTTCACA AAAAATATCG 2760

GTTATTAGAG ATGCTGTAAA AAATAGTATG TTTATATTTG TTATATCTGC TTTCATATTG 2820

GCAGTATGTC TTCTTTTGGC AAGGGGGGTG ATCATACATA ATATTTTAAA TATACCTAAA 2880

GATTCTATTG AAAATGCAAG TGTGTTATAT TTGGTTGCAG TATTTTCAAT AACCATACTT 2940

ATAATTCAGA CGCCACTTAA TGCGATGGTG TTAGCATACG AGAAAATGTC ATTTTACGCA 3000

TACATGATGA TATTTGAAAT GGTAGCTAAA ATGAGTATGG CTTTGTTACT AACTTTACTG 3060

GAAAAAGATA AGGTCATTGT ATATTCAATA CTTTTAGGTT CTATTTCTTT TTTTAACTTG 3120

TTGGTTTATT TATGCTACTG CTTGATATGT TTTAAAAAAT CAATGTTTGG TGGGAGAATA 3180

AAATTTAGAG TGCTGAAAGA AATTTCAACT TTTTCATTTT GGAATATATT TGGTAATTTC 3240

TCTTACATGT GCAGAGTTCA GGGAGTGAAC ATTGTAATTA ATATGTTTTA CGCTATAGCC 3300

GTGAATGCAG CATATGCGAT TTCGATTACT GTTTTAAATG CAATTAATAC ACTTACGCAA 3360

TCATTAATTA CAGCATTAAG GCCTCAAATA TTTAAATCAT ATGGCGAATG TGATTTAAAA 3420

AGATATAATC ATCTAGTTCT CTTTGGTTCA AAATATACAT TTTCAATATT GTTTCTGTTA 3480

AGTAGCCCTG TAATTCTATG TGCGGATGAG TTGTTAAAGA TATGGCTTGA TATTGTACCA 3540

GATTATACAG TTGAATTTGT GAGGTTGGTC ATTGTTGTGG CTTTTATTGA TAGTTTTTCA 3600

TATAGCATGA TTGCTGGTAT TCAGGCTACG GGTAGGATTA AAACATATCA ACTAGTTGTT 3660

AGTTTAATTG TCCTAATTAA TTTGCCGTTA ACCTTTATTT TGTTTAAGGC AGGAAATAAT 3720

GTTTTAAGTA TGTTTTATCC ATTTATTGTT ACTGCCATTA TCAATCAAGG TTTAAGGTTG 3780

TATTTTATCT ATATCAATGC AGGTTTTGAT TATAAGAAGT ATTTCACTGT TGTGATCTAT 3840

CCTTGCTTGT TAGCTGTATG CTTATCGTTA GTGACAGATA TTTCAATTAA AAAAATGCTT 3900

CCATTTAACA GTGTTATTGA TGTTTTAATT GTGTGTATTT TTATTTTTTC ATTCAATACA 3960

The Orf2 initiation site

ATAATTTTTT ATTGGGTTGT AGTTTCTAAA AAGGAAAAAA ATGGTTAT TA GAAACCCTGA 4020

The wzx termination site

AAAGAAAAGT GAAATAACAA TAAAAGATGT TATAGACAAT GAACTCTGTA CTGGTTGTGG 4080

AGTTTGTATT TCGGAAGATA GTAGCAAAAC CTCCTTTATG AAATGGAATT CTGAAGGTTT 4140

TTATGAGCCA TGTTTCTCTC CTGTATCAAC ATTATTCAAT ATGCAGCGTG TTTGTCCCTT 4200

TAACTTAAGT AGGGATACAT TAGTGAATGA GGATGAACTT GCGCATGAGT TTTTTGATGG 4260

TAAGGGATAT TTAGATTCTG AGGTTGGGTT TTATAAAAAA ATATATGTGG GTTATTCTAA 4320

ACATTTTAGA GAGACTTCAT CCTCCGGTGG TATAGCTACA TATGTATTTG AACAACTTCT 4380

TAGACGTAAG TATGTTGATG CTCTATTTAT AGTTAGGGAG CTTGGTGGTT CATATGGATA 4440

TCAAGTCTTC GATAATCCAG AACATATTAA GGATATGTCT AAAACAAGAT ATTATCCTGT 4500

AACGCTAGAA AAGTTATTTG ATAATATTTA TAAGCTTAAT GGTAGAGTGG CCGTTTCAGG 4560

AGTGGCATGT TTTATTAAAG CCATTAGGCT CAAACAACAT TACCATCCAG AACTGAAAGA 4620

GAAAATACCA TTCCTTGTTG GTATAATTTG TGGAGGACTC AAAAGTCGAT ATTACACTGA 4680

TTATTTATCA CAGTCTGCTG GTTGTGTCAG TGAGTACCAA AATGCTGAGT ATCGAGTGAA 4740

AAAGAAAGAT AGTCATGCTC TTGACTATCG TTTCACATGT GTTGAGAAAT CAAATAATAT 4800

AATTCATTCT GTTGATATGC AAAGAATGGG GGATATGTGG GGTTCAGGGC TATTTAAGGC 4860

AAACGCATGT GACTATTGTG ATGATGTTAC AACCGAACTC GCAGACATAT CATTGGGTGA 4920

TGCCTGGATT TCGCCATACA ATATGGATGG AGCTGGTAAT AATGTAGTTG TATGTCGCTC 4980

CACCACAGCT CATGAAATTA TTTTATCTGG GATTGAGAAA AAAGACTTAG AATTAACGGA 5040

ACTCGAATTA GAGCAGTTGA AATTGTCACA ACAAGGAAGT TTTAACCACA GGCATAAAGG 5100

ATTATTATAT AGAATAAAAA ATGCAGAGAA AAATAATCGC TTAGTGCCTG TTAAGAGGAA 5160

AAGATTCTTA CGCTCTATAT CTTTTTTATT AAAGTTAATT CAAAAACAAC GATCAGTTAC 5220

AAGGAGAAAA AGCATAGAGA TATGGATGGA AACTCAAAAT TCAGCAACTT TCGATAAGAA 5280

AATGAAGGGA TATTTATTCA CGTTGCGTTG GTTAACAGTG GTCAATAGGA AATTAAGTCG 5340

The Orf2 termination site

GATGTTCAAG ATTGTTTCTC TAAATAGAAT GGGTAAAAA＿＿ AAAGTTGC AATATTAACT 5400

The Orf3 initiation site

CAACCATTAC ATACGAATTA TGGTGGCACA TTACAAGCCT ATGCATTACA AAAAGTCTTG 5460

ATTAATTTGG GACATGAACC AGAGACTATT AATTATAGGT CGAAAATTAA GAGGCCACTA 5520

TTTATCCGTG TTGTGTTGTC AAAAATTAAA AGGATAGTAT TATGCCGAAA AATCACATTT 5580

GATTTTACAA CACAAGATAG AATTAATATT AGGAAGCATC ATCAATCTTT CATTGATACA 5640

AGATTAAACT ATTCAGAGGA GATTAACGGT ACTGAGGGGT TGAGAGATTA TATTTTAAAA 5700

AATAATTACG GTGCTGTAAT AATAGGTAGT GATCAGACAT GGAGACCAAT ATATTCGCCT 5760

AGAATAGATT CATTTTTTCT CGATTTTTTA AGTGATGTAA ACGATATAAA AAATGGCATA 5820

TGCAGCATCT TTTGGTACTG ATAAATGGGA GTTCACGGAG TCACAAACTA ATTTATTTAA 5880

ATTGTTACTT TCAAAATTTG ACTATGTTTC AGTTAGAGAG TCCTCAGGAG TCAAATTATG 5940

TAGTGAAAAA TTTGGTTTGA AAGCAGAGTT GGTTTTAGAT CCGACATTGC TATTGACATT 6000

CGAAGATTAT ACTCATTTAT TAGACAATGA CTATATTAAT CTTCATAAGG GAAAGGTGTT 6060

TAGTTATGTA CTTGATGAGA ATTACGACAA AAAAAACTTT ATAGACACAG TAGCAAGTAC 6120

TCTCCGCACA ACGTCATTTT ATACTTACCC TAAAAAAATC ACTAAAGATG AATATGTGAT 6180

TAGAAATTAT TCCGAGTATG AATATCCGCC AATAGAATAT TGGATTTCTT CGTTTAAGGC 6240

CGCTGAATTT ATTGTAACAG ATTCATTTCA TGGAACAGTA TTTTCTATTA TTTTTAACAA 6300

ACCCTTCATC GCCATTGCAA ATGAAGAGAG AGGAAAGGCT AGATTTACGT CACTATTGGA 6360

AATGTTTGGT CTTGAAAGAC GACTTGTGAG TAATTTAGAT GATGTTGATT TAAAGCTTGT 6420

AAATGAAAAA ATTAACTATG GCCCCATAAA TGAAAAAATT GCTTATTTTA GAGAAGAGTG 6480

The orf3 termination site

CCTTAAAAAG TTGCAGTTAA TGCTAATAAA A TAATCAGAT TATGTATGTC TCTCTACAAT 6540

The Orf4 initiation site

TAAAATATGA ATGTTGATCG ATGAAGAATT GTAAAGTATC TGTAATTATT CCGGTCTATA 6600

ATGCGGAAAA ATATATACAA CGTTGTATTT TAAGCTTATT AAAGCAGACA TTAGATGATG 6660

TTGAAATTAT CATCATTGAT GATGGTTCTA CTGATAATTC ACTTTCTATA ATAAAAGAAA 6720

CTGTTGCATT GCATACTGCT AGTAGAGCTC GCCAAAAGAG AATCAATATA ATCTCTCGAG 6780

AAAACAAAGG AGTTGCCTAT ACACGAAGTC AGGGATTGAG ACTTTCGCAG GGGGAATTTA 6840

TCATTCATTT TGATAGTGAT GATTGGGCAA AATCCAATAT GCTGGAGGAA ATGTATAAGA 6900

CAATTGTTTC CAATAATGCA GACATGGTTA TATGCGATTA TTTTTTAGTG AAGAATAATA 6960

AAGAAATACT TATAAAACAG AGAGTGGAGT ATGAGCCAAA GAAATGTATT AGATATCTAC 7020

TGACTGGTGA GCTAGAAGGG TTTACATGGA ACAAACTAAT TAGAAAAAAA TATATCGATA 7080

AAAATAAAAT TGATTTTGTT AATAAAATTA CTTATATGGA GGATTTTTTG TTCATATTAA 7140

GTGTGTTACT ACACAATCCT AAAATTATTT TTCAGGATTG TGCATATTAT TATTATCAAA 7200

AAAGCAATCC TAAATCTCTA ACTAGCCATG CTAGTGTGGA TAGGCTTTCT GAAATGATTA 7260

AAGCCGTAAG TGAAATAGAA AAGAAGATAA ATAAATATAA TTTACAAAAG TATCTGAATA 7320

ACGAGTTTCA ATTGTTTAAG TTAAAACAAA AAATTTGGTT TATATCTATA AGTAAGTTAA 7380

ATGTTAATGA TAATGTATGG AATCTCTTTC CTGAAACAAA TCCATTTATA TCAAAGGTAA 7440

ATGTGCTTTT TTATTATAAA ATAGTGCTTT TTCTTGATAG TATTAAACTG CGTTTTTTTT 7500

CAAATAAGAT CATTTATCTC ATTGGATTGG TGCAGGCTTT ATTACAAGAA AGGAAAGAGA 7560

Orf5 initiation site orf4 termination site

A GTGATACCA AAAATAATTC ATTACTGCTG GTTCGGTCGC TCTCCATTGT CGGAATTAAC 7620

AAAGCAGTGC ATTGCAAGCT GGGAGAAATA TTGTCCTGAA TATAAAATCA TAAGATGGGA 7680

TGAAAATAAT GTCGATTTGA ATTCTTGTTC TTTTGTTAGA CAAGCTTATA AAGAAAAAAA 7740

ATGGGCGTTC GTTTCTGATT ATGTAAGGCT TAAAGTGGTT AATGAATATG GCGGTATCTA 7800

TCTTGATACA GATGTTGAAT TAATAAAGCC ATTAGATGAT TTGTTAATAT ATCCTGCATA 7860

TATAGGCTTT GAAATTAACA AGGAATGGTA TGTAAATTCT GGTTTGGGGT TCGGTTCTGT 7920

TAATAATAAT CCGGTTTTAG AATCATTAAT TATGGAGTAT GAAAATATTA ATTTTGTAAA 7980

TGAGGATGGG ACTCTAAATA TAACGCCGTG CCCCATAAGA GAAACAAAAG CATTAACAAA 8040

AATAGGTTTA ATTCCTGATG GTCAATGTCA ATCTTTTGAT AATATAGTGA TTTTTTCTGC 8100

AGATTATTTT TGTCCTGTAT CTATAACAGG TGAGAGAAAT TTTTCTGATA AAACATATTC 8160

AATACATCAT TACGATGCTA GTTGGTTTTC TGAGCAAAAG AGAAAAGGCC TGCAACGTAA 8220

AAAGAGATTT ATAAAACTAT TCGGCAACCT AATAGGCACA TATATTAATA AACCTTTTAT 8280

TTTTGTAGAT GAATGTCGCG AGTTCGGATT AGTTAAAGCA ATCAAGAATA TGCGTTCCAA 8340

The orf5 termination site

TTTTCCT TAA TTTTTAGTGT TAAGGAATAA GTTTCGTTAA AATACTTTAT TCATTTAATT 8400

The Orf6 initiation site

AATAAAGGTT TAATGGTGAT TAG ATGTACT TTCTTTTGTT CTTCTCAATA TTTTATTTTG 8460

TATACGTAAT CTTAATAGGA ACATTATGTC ATAACTATAA AGCCCTTTTG ATTTTTTCAC 8520

TAATCCCTCT GACAATAGTG TCAGGTATAA GATATAATGT CGGCTTTGAT TTTATGTCAT 8580

ATGTTGATTA TTTCAATCAA CTCAAATATT CAAATGATAT ATATCTAGAT TCTACCTTTA 8640

AGTATATAAG CTACTTTACC TATTATATAG GAGGTAACGA ACAGATTTTG TTTCTGATAT 8700

ATGCGGTTTT TTATTCGGTT GCACTTTATT TTTTAATAAA ACTGGTATTG GAAAATTATA 8760

TAACGGAAAT AAATAATTTT TATACTATTG GACTTGTGTT ATCATTTTAT TCGTTTTATT 8820

TTTTATTATC ATTTAATCAA ATTAGGGCTG TATTATCAGC TCTAATACTA TGTTATGGGC 8880

TATTAAAAAA TAAAATAGAT TTTTCTTTTA TAATAACAAT TACATTAAGT ATTCTTTTTC 8940

ACTCTGCAGC AATGTTCATT TTACCTCTTT ATTTTGTCCT AAGAAAAATA AATGTAACAG 9000

TTCTGTTGGT GATATTCCCT TTTTTGGTTG TTGCTTCATT CTTTAATATT TTTTCAGATA 9060

TTGTGAGATT CATTCTGACA TTGCTAAATA GTCGTTTTTT AACATATTTT AACTCTGAAT 9120

ATTTTGTTCC AAGGACGGGA ATGGAAAAAA TGTATAGTAT GATCTCAATG ATTATTGTTT 9180

TGGGAATGGT TGTTTGTTTG TCTAAATTAT TACCTAAAAA GTTTGATTTA ATGATAAAGT 9240

TTGTTATTCT TTTTGTTCTA CTTAGAGCGA TGTCAATTGA TATATTGATT TTTGCTAGGT 9300

TTAGTGATTT TCTTAAACCA ATGGCTATAA TTCTTATATT TACCGCTGTT TATTTTTCTT 9360

CGAATAAAGT GCGACCTAGA ATTATCCTTC CTTTTTATTT AATTATGACA TTGTTTCTCT 9420

GTATTTTTAA TATAATAATC GGATCGAATA TAACTAAGGG GAACTATTAT ACTTATGGTT 9480

The orf6 termination site

ATAACATTTG CTTTTTTGGC AATAAATGTA TAGATAAATT TTATTAG TAA TTGTCATTTG 9540

The orf7 initiation site

ATACTTCTGG TATTT ATGGA AAAATATATG AATAACTTTT CTTGTAAAGA ACCCCCTTCA 9600

TTTATTGAAA ATAAAATTAT ATTATCAATT ATTCTTCCTG TATATAACGT ATCAGAGTAT 9660

TTAATTGAGT GCCTAAACAG TTTGCTGTTA GACGTTCATG AACGTTATCT TAATAGATGT 9720

GAAGTAATTG TGGTTGATGA TGGTTCAACT GATAATTCAT TTGAATTAAT GAGAGAGTAT 9780

TCTTTGAAAT ATCCTGAATC TATTAAGATT TATTCGAAAT TCAATGGTGG TCTCTCAGAT 9840

GCAAGAAACT ATGGATTATT AAAATCTTCA GGTAAATATA TTTCTTTTGT TGATTCAGAT 9900

GATGTTGTTA ATAGAGGTTT TATAAAGGAA ATCATCAATT TCATTGATGC CTATGATTTT 9960

GATATATTGT CATTTGACTT CATGAAGTTT TTTAATAATA ATGATGCTCT TATTCTTTCA 10020

CAAATAGATG AATTCTCCAA AATGGCAGAA AGAGTAGATT CGGAGTTCTA CAAGTCCAAG 10080

CCTGTTTTTG CTTGGAATAA AGTTTATCGA CGTTCTCTTT TCGACAATGA AGTATTTCCT 10140

AAGGGGTGGT ATTATGAAGA TGTTGCATTA ATTCCGTTAT TATTAGATAG AGCAAAAGTT 10200

TTATACCATA TTAATAGCGT TTGTTATTTT TATAGACAGA GACAAGGAGC AATCACTTTT 10260

TTTTATGACA ATAAATATCT TGATATTTTA AAAGGAGTGT CTTTTTTATA TGACAGGTCA 10320

CAATCTTCAT TCATTAAAAC GATAATTATA AATCAGTTTT TTACTTTAAC CTTACTATCT 10380

TTAAGATTGC CGACTGATAA TTATTTCATG AATATGAGGG GGATTATTGA TACCTATTGT 10440

GAAAAATTTG ACTTGGATTC CTTTGAGCCT AAACTAATAT TAAAACACAT TCCTTTTTTA 10500

TTGCTTAAAA AATTGAAGTC GCTGTGTGTT TATCCACTGT ATTTATTTAA ACCTGCAGTT 10560

The orf7 termination site

TTTTTACATA AACAAATTAA ACATTTTAGA GGAAAACTAA AG TGAAATAT GATTATATAA 10620

The orf8 initiation site

TTATAGGTAG TGGTCTTTTT GGGAGTGTGT TTGCACATGA ATTAGGGAAA ATGAAAAAAA 10680

AGGTCAAGGT TATTGAGCGT CGTGATCATG TTGGTGGTAA TGTATATTGT GAGAATATTG 10740

AAGGAATTAA TGTTCATAAA TATGGCGCAC ATATATTTCA TACTAACGAT AAAGAAATAT 10800

GGGATTATGT AAATCAATTT GTAGAGTTTA ATAGATATAC AAATTCACCA TTAGCTTTTT 10860

CAAAAGGTAA AATTTATAAT TTACCTTTTA ACATGAATAC CTTTAATCAA CTCTGGGGGG 10920

CTATTACTCC TCAACATGCG AAAGAAATTA TTAAAAAGCA AAGCGGTGAA ATAAGTGGAA 10980

ATAAACCAAG GAATCTTGAA GAGCAAGCTA TTTCTTTGGT GGGACGAGAT ATTTATAATT 11040

GTTTGATTAA GGAGTACACT GAGAAACAAT GGGGACGACC TTGCACTGAA TTGCCATCAT 11100

TTATTATAAA ACGACTCCCT GTTAGGTTTA CATACGATAA TAACTATTTT AATGACCAGT 11160

ATCAAGGAAT ACCAGTCGGT GGTTACAATA AATTAATCGA TGCATTATTA AATGGTATTG 11220

AAGTTCAAAC ATGCACTGAC TATTTTGAAG AGAAGAAATA TTGGGATTCT ATTGCAAAGA 11280

AAATTATTTA TACAGGGCCT ATTGATAAGT TTTATTGCTA TAAATTTGGA GCTTTGGATT 11340

ATCGATCATT AAAATTTGAG CATGAATTAC TGAATGTCGA TAATTTTCAA GGGAATGCTG 11400

TTGTAAATTA TATAGACAAA AAATTCCCAT TCACTCGTAT AATAGAACAT AAATTTTTTG 11460

AATTTGGTGA ACAGCATAAT ACTCTCATTA CTCGAGAGTA CCCAAAAGAA TTCGAAGAAG 11520

GCGATGAGTA TTACTATCCT GTCAATGATG AAAAAAATAT GCAAATCTAT AAAATGTATT 11580

ATAATCTTTC TAAACAGGAA AGTAATGTGA TTTTTGGTGG ACGTCTTGCG GAATATAAAT 11640

ATTATGACAT GCATCAAGTT ATCAAATCAG CTTTAAATGC ATTCCGAAAA GAGGTACGTT 11700

The orf8 termination site

TAGGTGAA TA AAGAAGTAAT CGCAGTTATT GTAACCTATA ATAGAAAAGC ATTATTGCTG 11760

AAAGTTATAG ATGCTGTTAT TAACCAAAGT TATCCACTAA AAAAAATATT GATAATAGAT 11820

The orf9 initiation site

AATAATAGTA CTGATGGTAC GGAG ATGTTT ATTTCAAATA GGCTCTCTGA CGTGGTGAAG 11880

TATAAAAATA CGGGGGATAA TTTAGGGGGG GCTGGCGGGT TTTATAGAGG ATTCATAGAG 11940

GCGGAAAAAT ATGGCTATGA TTATCTTTGG TTAATGGACG ATGATTTTAT GCCTACAGTG 12000

AATTGTCTTG AAATATTAAT ATCAAATAGC CCTGATGGTA TTGTCCAACC TGTTAGATAT 12060

AACTTGGATG AAACTTGTGC AGAGCTTTCT CCATTAACTT ATGATTTATC TAACCCATTT 12120

AAGTTAAACC CTAAAGGTAC ACCATTAAAA AATTACTTAA ATACAATTGG TAATAAATCT 12180

GCGAAGGTAG ATATTGAAGC TATTCCATTT GAAGGTCCAT TGATTTCACG AAAGGTTGTT 12240

GAAAAAATAG GCTATCCAGA GCCTAAGTTT TTTATTTTTT GTGATGACAT TGAATACGCT 12300

ATTAAAGCTA AAAGAAAGGG GATTCCCATC CAGTGCAATC TTAAAGCCAA GGCATACCGC 12360

CTTTTGGTCA ACAATCAAGG TAATGATCTT CTTTCCTGGA AAGGATATTT TATGCTTCGA 12420

AATCTTTTCT ACCTGCATAA AACGTATGGG ACCAATTTCC TTGTTAGACA AAAACCTATC 12480

GTATTGGGAT TAGGATATGC ATTGTCGTGT GTATTGAAAT GCAATTTTTC TCAACTTCCC 12540

GTTATATGGC GTGCTTTTTG GGATTCATCT ACGTTAAGAA ATACCGAAAA ATTCAGGCCC 12600

The orf9 termination site

GAAATGAGAA GTAAA TAATT TATTATTATG TTTTTATTAA TTAATGTCGA TTTTTACTAA 12660

AATTCTTAAC ACGATATTTA TCCCTGACAG GAGTAAACAA TGTCAAAGCA ACAGATCGGC 12720

GTCGTCGGTA TGGCAGTGAT GGGGCGCAAC CTTGCGCTCA ACATCGAAAG CCGTGGTTAT 12780

ACCGTCTCTA TTTTCAACCG TTCCCGTGAA AAAACGGAAG AAGTGATTGC CGAAAATCCA 12840

GGCAAAAAAC TGGTTCCTTA CTATACGGTG AAAGAGTTTG TTGAATCTCT GGAAACGCCT 12900

CGTCGCATCC TGTTAATGGT GAAAGCAGGT GCAGGCACGG ATGCTGCTAT TGATTCTCTC 12960

AAGCCATACC TCGATAAAGG TGACATCATC ATTGATGGTG GTAACACCTT CTTCCAGGAC 13020

ACCATTCGTC GTAACCGTGA GCTTTCTGCA GAAGGCTTTA ACTTCATTGG TACCGGTGTT 13080

TCCGGCGGTG AAGAGGGGGC GCTGAAAGGG CCGTCCATCA TGCCTGGTGG CCAGAAAGAA 13140

GCCTATGAAC TGGTTGCACC GATCCTGACC AAAATCGCCG CAGTAGCTGA AGACGGGGAG 13200

CCATGCGTTA CCTATATTGG TGCCGATGGT GCAGGTCACT ATGTGAAGAT GGTTCACAAC 13260

GGTATTGAAT ACGGTGATAT GCAACTGATT GCTGAAGCCT ATTCTCTGCT TAAAGGTGGC 13320

CTGAACCTCA CCAACGAAGA ACTGGCGCAG ACCTTTACCG AGTGGAATAA CGGTGAACTG 13380

AGCAGCTACC TGATCGACAT CACTAAAGAC ATCTTCACTA AAAAAGATGA AGACGGTAAC 13440

TACCTGGTTG ATGTGATTCT GGATGAAGCA GCAAACAAAG GTACCGGTAA ATGGACTAGC 13500

CAGAGCGCGC TGGATCTCGG CGAACCGCTG TCGCTGATTA CCGAGTCTGT GTTTGCACGT 13560

TATATCTCTT CTCTGAAAGA TCAGCGCGTT GCCGCGTCTA AAGTTCTCAC TGGCCCGAAA 13620

GCACAGCCAG CTGGCGAAAA GGCTGAGTTC ATCGAAAAAG TTCGTCGTGC GCTGTATCTG 13680

GGCAAAATCG TTTCTTACGC TCAGGGCTTC TCTCAGCTAC GCGCCGCGTC TGAAGAGTAC 13740

AACTGGGATC TGAACTACGG CGAAATCGCG AAGATTTTCC GTGCTGGTTG CATCATCCGT 13800

GCGCAGTTCC TGCAGAAAAT CACCGATGCC TATGCCGAAA ATCCGCAGAT CGCTAACCTG 13860

CTGCTGGCTC CGTACTTCAA GCAAATTGCC GATGACTACC AGCAGGCGCT GCGCGATGTC 13920

GTCGCTTACG CAGTACAGAA CGGTATCCCG GTTCCGACCT TCGCCGCTGC GGTTGCCTAT 13980

TATGACAGCT ACCGTTCTGC TGTTCT 14006

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

Claims (3)

1, a kind of oligonucleotide is characterized in that, is selected from the Nucleotide of 1868 to 1885 bases among the SEQ ID NO:1; The Nucleotide of 2265 to 2284 bases among the SEQ ID NO:1; The Nucleotide of 3020 to 3050 bases among the SEQ ID NO:1; The Nucleotide of 2809 to 2827 bases among the SEQ ID NO:1.

2, a kind of oligonucleotide is right, it is characterized in that, is selected from the Nucleotide of 1868 to 1885 bases among the SEQ ID NO:1 and the Nucleotide of 3020 to 3050 bases among the SEQ ID NO:1; The Nucleotide of 2809 to 2827 bases among the Nucleotide of 2265 to 2284 bases among the SEQ ID NO:1 and the SEQ ID NO:1.

3, the application of the nucleotide pair of the Nucleotide of claim 1 or claim 2 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray as probe, for detecting intestinal bacteria O164 type.