CN1249235C - Nucleotide specific against o-antigen of colibacillus 0150 - Google Patents

Abstract

The present invention provides a kind of nucleotide specific to the O-antigen of Escherichia coli O150 type (Escherichia coli O150), it is the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O150 type, such as SEQ ID The isolated nucleotide shown in NO: 1, with a full length of 13552 bases; or SEQ ID NO: 1 with one or more inserted, deleted or substituted bases while maintaining the function of the isolated nucleotide Nucleotides; also include oligonucleotides derived from glycosyltransferase genes and oligosaccharide unit processing genes in the O-antigen gene cluster of Escherichia coli O150 type; All type O-antigens have high specificity; the invention also discloses a method for using the oligonucleotide of the invention to detect and identify Escherichia coli O150 type in livestock and environment.

Description

Nucleotides specific for the O-antigen of Escherichia coli type O150

technical field

The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O150 type (Escherichia coli O150), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O150 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O150 in livestock and the environment and identify the O-antigens in these pathogenic bacteria.

Background technique

Escherichia coli O150 was first detected in diseased animals in 1972 [Furowicz A J, Orskov F.Acta Pathol Microbiol Scand Microbiol Immunol, 1972,80 (3): 441-444.], it was confirmed that it can cause bovine et al. Enterotoxigenic Escherichia coli (Enterotoxigenic E.coli, ETEC), the intestinal infection of livestock is very dangerous for its potential outbreak [Danbara H, Komase K, Arita H, et al. Infect Immun, 1988, 56 (6): 1513-1517.], a method that can quickly and accurately detect Escherichia coli O150 is urgently needed in agriculture.

O-antigen is the O-specific polysaccharide component of Gram-negative bacterial lipopolysaccharide, which consists of many repeating oligosaccharide units. The synthesis process of O-antigen has been studied clearly: first, the nucleoside diphosphate monosaccharide is transferred to a lipid molecule fixed on the inner membrane of the cell by glycosyltransferase, and then the oligosaccharide unit is synthesized inside the inner membrane, O- The oligosaccharide unit of the antigen is transferred to the outside of the inner membrane by a transport enzyme, and then polymerized into a polysaccharide by a polymerase, and then connected to a glycolipid molecule to form a lipopolysaccharide molecule [Whitfield, C. (1995) "Biosynthesis of lipopolysaccharide O Antigens". Trends in Microbiology. 3: 178-185; Schnaitman, C.A. and J.D. Klena. (1993) "Genetics oflipopolysaccharide biosynthesis in entericbacteria". Microbiological Reviews, 57(3): 655-682]. The genes encoding all enzyme molecules responsible for O-antigen synthesis are generally arranged adjacently on the chromosome to form a gene cluster [Reeves, P.R., et al. (1996) "Bacterial polysaccharide synthesis and gene nomenclature" Trends in Microbiology, 4: 495- 503]. In Shigella, Escherichia coli and Salmonella, the O-antigen gene cluster is located between the galF and gnd genes [Lei Wang. et al (2001) "Sequence analysis of four Shigella boydii O-antigenloci: implication for Escherichia coli and Shigella relationships". Infection and Immunity, 11: 6923-6930; Lei Wang and Peter Reeves (2000) "The Escherichia coliO111 and Salmonella enterica O35 gene clusters: gene clusters encoding the samecolitose-containing act O antigen are highly of 5B 1 of our log 2 conserved: 2 -5261]. The O-antigen gene cluster contains three types of genes: sugar synthesis pathway genes, glycosyltransferase genes, and oligosaccharide unit processing genes. The enzymes encoded by the sugar synthesis pathway genes synthesize nucleoside diphosphate monosaccharides required for O-antigen; The enzyme encoded by the base transferase gene transfers nucleoside diphosphate monosaccharides and other molecules to monosaccharides so that monosaccharides can be polymerized into oligosaccharide units; oligosaccharide unit processing genes include transferase genes and polymerase genes, which convert oligosaccharides Units are transferred to the outside of the bacterial inner membrane, where they polymerize into polysaccharides. Glycosyltransferase genes and oligosaccharide unit processing genes were only present in gene clusters carrying these genes. The difference of monosaccharides in O-antigens, the difference of linkages between monosaccharides and the linkages between oligosaccharide units constitute the diversity of O-antigens, and the composition of monosaccharides, linkages between monosaccharides and oligosaccharides The connection between units is controlled by the genes in the O-antigen gene cluster, so the O-antigen gene cluster determines the synthesis of O-antigen and also determines the diversity of O-antigen.

Because the O-antigen is a very strong antigen, it is one of the important pathogenic factors of Escherichia coli, and at the same time it has a strong diversity, which enlightens us to study a rapid and accurate detection of Escherichia coli and its O- A method with good antigen specificity and high sensitivity. Serological immune responses targeting surface polysaccharides have been used to type and identify bacteria since the 1930s and are the only means of identifying pathogenic bacteria. This diagnostic method requires a large amount of antiserum, but the types of antiserum are generally not complete and the quantity is insufficient. There are also some difficulties in the preparation and storage of a large amount of antiserum. On the other hand, this method takes a long time, has low sensitivity, high missed detection rate, and poor accuracy. Therefore, it is generally believed that this traditional serological detection method will be replaced by modern molecular biological methods. In 1993, Luk, J.M.C et.al identified the O-antigen of Salmonella by PCR method [Luk, J.M.C.et.al. (1993) "Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of S.enterica major serogroups (A, B, C2, and D)", J.Clin.Microbiol.31:2118-2123]. The method of Luk, et.al is to align the nucleotide sequences of the synthetic genes corresponding to CDP-abicosose and CDP-tyvelose within the O-antigen of Salmonella serotypes E1, D1, A, B and C2 Afterwards, oligonucleotides specific to different serotypes of Salmonella were obtained. In 1996, Paton, A.W et.al identified a toxin-producing serotype of E.coli O111 with an oligonucleotide specific to the O-antigen of E.coli O111 derived from the wbdI gene ["Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli". The method of identifying the serotype of E.coli O111 with the oligonucleotide of wbdI gene has false positive results. Bastin D.A.and Reeves, P.R. believe that this is because the wbdI gene is a putative sugar synthesis pathway gene [Bastin D.A.andReeves, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111. Gene 164: 17 -23], and there may also be this sugar in the structure of the O-antigen of other bacteria, so the sugar synthesis pathway gene is not highly specific for the O-antigen Shigella has 46 serotypes, but only 33 different Escherichia coli has 166 different O-antigens [Reeves, P.R (1992) "Variation in O antigens, niche specific selection and bacterial populations". FEMS Microbiol. Lett, 100:509-516], the two relatives The relationship is very close, and 12 species are shared by Escherichia coli and Shigella [Ewing, W.H. (1986) "Edwards and Ewing's identification of the Enterobacteriaceae". Elsevier Science Publishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983 ) "Antigenic relationships between the enteroinvasive Escherichia coli antigens O28ac, O112ac, O124, O136, O143, O144, O152and and Shigella O antigens" J.clinMicrobiol, 17(4):681-684]

Contents of the invention

The object of the present invention is to provide a nucleotide specific for the O-antigen of Escherichia coli O150 type. It is a nucleotide in the O-antigen gene cluster of Escherichia coli O150 type, and is a specific nucleotide derived from a glycosyltransferase gene, a transportase gene and a polymerase gene.

The second object of the present invention is to provide the full-length nucleotide sequence of the O-antigen gene cluster of Escherichia coli O150.

Another object of the present invention is to provide the gene that constitutes the O-antigen gene cluster of Escherichia coli O150 type: the gene of transferase is wzx gene or the gene that has similar function with wzx; The polymerase gene is wzy gene or has similar function with wzy Functional genes; glycosyltransferase genes, including orf8, orf9, orf10 genes.

Another object of the present invention is to provide oligonucleotides, which are respectively derived from the genes encoding glycosyltransferases in the O-antigen gene cluster of Escherichia coli O150 type, including orf8, orf9, orf10 genes; Genes are wzx genes or genes with similar functions to wzx, derived from genes encoding polymerases, namely wzy genes or genes with similar functions to wzy; they are oligonucleotides in the above genes, with a length of 10-20nt; they Specific for the O-antigen of E. coli O150; especially the oligonucleotides listed in Table 1, which are highly specific for the O-antigen of E. coli O150, and these oligonucleotides can be regenerated In combination, the combined oligonucleotides are also highly specific to the O-antigen of Escherichia coli O150.

Another object of the present invention is that the above-mentioned oligonucleotides provided can be used as primers for nucleic acid amplification reactions, or as probes for hybridization reactions, or for making gene chips or microarrays, thereby detecting and Identification of O-antigen of Escherichia coli O150 and detection and identification of Escherichia coli O150.

Another object of the present invention is to provide a method for isolating the complete sequence of the O-antigen gene cluster of Escherichia coli O150 type. According to this method, the complete sequence of O-antigen gene clusters of other bacteria can be obtained, and the complete sequence of bacterial gene clusters encoding other polysaccharide antigens can also be obtained.

The purpose of the present invention is achieved by the following technical solutions.

The nucleotides specific to the O-antigen of Escherichia coli O150 type according to the present invention are isolated nucleotides as shown in SEQ ID NO: 1, with a total length of 13551 bases; or have one or more insertions and deletions or substituted bases while maintaining the nucleotides of SEQ ID NO: 1 that are functional as said isolated nucleotides.

The aforementioned nucleotides specific to the O-antigen of Escherichia coli O150 type consist of 11 genes, all of which are located between the galF gene and the gnd gene.

The aforementioned nucleotides specific to the O-antigen of Escherichia coli O150 type, wherein said gene is: a gene of a transport enzyme, including a wzx gene or a gene with a similar function to wzx; a polymerase gene wzy gene or a gene that has a similar function to wzy Genes with similar functions; glycosyltransferase genes, including orf8, orf9, orf10 genes; the genes described therein: wzx is the nucleotide of 5632 to 7134 bases in SEQ ID NO: 1; orf8 is SEQ ID NO: The nucleotide of 8307 to 9197 bases in 1; orf9 is the nucleotide of 9184 to 10098 bases in SEQ ID NO: 1; orf10 is the nucleotide of 10098 to 10994 bases in SEQ ID NO: 1; wzy is the nucleotide of bases 11012 to 12109 in SEQ ID NO:1.

The aforementioned nucleotides specific to the O-antigen of Escherichia coli O150 type, wherein it is derived from said wzx gene, wzy gene or glycosyltransferase gene orf8, orf9, orf10 gene; and their mixture or their reorganize.

The aforementioned nucleotides specific to the O-antigen of Escherichia coli O150 type, wherein the oligonucleotide pair derived from the wzx gene is: nucleotides from 5706 to 5722 bases in SEQ ID NO: 1 and Nucleotides of 6458 to 6475 bases; nucleotides of 5719 to 5736 bases and 6681 to 6698 bases of SEQ ID NO: 1; 5978 to 5995 bases of SEQ ID NO: 1 Nucleotides and nucleotides of 6789 to 6806 bases; the oligonucleotide pair derived from the orf8 gene is: the nucleotides of 8681 to 8699 bases and the core of 9169 to 9185 bases in SEQ ID NO: 1 Nucleotide; SEQ ID NO: the nucleotide of 8418 to 8435 bases and the nucleotide of 8995 to 9012 bases in SEQ ID NO: 1; The nucleotide of 8333 to 8350 bases and 9050 to 9067 in SEQ ID NO: 1 The nucleotide of base; The oligonucleotide pair derived from orf9 gene is: the nucleotide of 9397 to 9413 bases and the nucleotide of 9882 to 9899 bases in SEQ ID NO: 1; SEQ ID NO: The nucleotides of 9337 to 9354 bases and the nucleotides of 9899 to 9916 bases in 1; the nucleotides of 9489 to 9506 bases and the nucleotides of 10035 to 10051 bases in SEQ ID NO: 1; The oligonucleotide pairs derived from the orf10 gene are: 10302 to 10319 base nucleotides and 10852 to 10869 base nucleotides in SEQ ID NO: 1; 10293 to 10310 bases in SEQ ID NO: 1 The nucleotides of bases and 10954 to 10971 bases; the nucleotides of 10377 to 10394 bases and the nucleotides of 10973 to 10989 bases in SEQ ID NO: 1; the oligonucleotide derived from wzy gene The nucleotide pair is: 11217 to 11234 base nucleotides and 11985 to 12002 base nucleotides in SEQ ID NO: 1; 11225 to 11241 base nucleotides and 12035 to 12002 base nucleotides in SEQ ID NO: 1 12052 base nucleotides; 11172 to 11189 base nucleotides and 11960 to 11977 base nucleotides in SEQ ID NO: 1.

Application of the aforementioned nucleotides specific to the O-antigen of Escherichia coli O150 type in detecting bacteria expressing O-antigen, identifying bacterial O-antigen and other polysaccharide antigens of bacteria in diagnosis.

The aforementioned recombinant molecule of nucleotides specific to the O-antigen of Escherichia coli O150 can provide and express the O-antigen of Escherichia coli O150 through insertion and expression, and become a bacterial vaccine.

The application of the aforementioned O-antigen-specific nucleotides of Escherichia coli O150 type, wherein it is used as a primer for PCR, as a probe for hybridization reaction and fluorescence detection, or for making gene chips or microarrays to detect livestock and bacteria in the environment.

The aforementioned method for isolating nucleotides specific to the O-antigen of Escherichia coli O150 type is characterized in that it comprises the following steps:

(1) Genome extraction: Escherichia coli O150 was cultured overnight at 37° C. in 5 mL of LB medium, and the cells were collected by centrifugation. Resuspend the cells with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubate at 37°C for 20 minutes, then add 10ul 10mg/ml lysozyme and continue to incubate for 20 minutes, then add 3ul 20mg/ml proteinase K , 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg/ml RNase, incubate at 65°C for 30 minutes, add an equal volume of phenol extraction mixture, take the supernatant and use an equal volume of phenol: chloroform: iso Pentanol (25:24:1) mixed solution was extracted twice, the supernatant was extracted with an equal volume of ether to remove residual phenol, the supernatant was used to precipitate DNA with 2 times the volume of ethanol, the DNA was rolled up with glass wool and washed with 70 Wash the DNA with % ethanol, resuspend the DNA in 30ul TE, and detect the genomic DNA by 0.4% agarose gel electrophoresis;

(2) Amplify the O-antigen gene cluster in Escherichia coli O150 by PCR: use the genome of Escherichia coli O150 as a template to amplify its O-antigen gene cluster by Long PCR. Design an upstream primer (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence in the promoter region, and then design a downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C); use the Expand Long Template PCR method of Boehringer Mannheim to amplify the O-antigen gene cluster. The PCR reaction procedure is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 68°C 15 minutes, 30 cycles in this way, and finally, continue to extend at 68°C for 7 minutes to obtain PCR products, use 0.8% agarose gel electrophoresis to detect the size and specificity of PCR products, combine 6 tubes of long PCR products, and use The Wizard PCR Preps purification kit of Promega Company purified the PCR product;

(3) Construction of the O-antigen gene cluster library: use the modified Novagen DNaseI shot gun method to construct the O-antigen gene cluster library. The reaction system is 300ng of PCR purified product, 0.9ul of 0.1M MnCl ₂ , 1ul of 1mg diluted at 1:2000 /ml of DNaseI, the reaction was carried out at room temperature, digested for 10 minutes to concentrate the size of DNA fragments between 1kb-3kb, then added 2ul 0.1M EDTA to terminate the reaction, combined 4 tubes of the same reaction system, and extracted with an equal volume of phenol Extract once, use an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixed solution to extract once, then use an equal volume of ether to extract once, use 2.5 times the volume of absolute ethanol to precipitate DNA, and use Wash the pellet with 70% ethanol, and finally resuspend it in 18ul water, then add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul 100mM DTT and 5 units of T4 DNA polymerase to the mixture, 11°C for 30 minutes, The digested product was made into a blunt end. After the reaction was terminated at 75°C, 5 units of Tth DNA polymerase and its corresponding buffer were added to expand the system to 80ul, and the reaction was carried out at 70°C for 20 minutes to add a dA tail to the 3′ end of the DNA. The mixture was extracted with an equal volume of chloroform: isoamyl alcohol (24:1) mixed solution and an equal volume of ether, and then ligated with the 3×10 ^-3 pGEM-T-Easy carrier of Promega Company at 16°C for 24 hours. The volume is 90ul, which contains 9ul of 10×buffer and 25 units of T4 DNA ligase. Finally, the connection mixture was precipitated with 1/10 volume of 3M NaAc (pH5.2) and 2 times the volume of absolute ethanol, then washed with 70% ethanol, dried and dissolved in 30ul water to obtain the connection product; Preparation method of electroporation competent cells Prepare competent E. coli DH5a cells, mix 2-3ul of the ligation product with 50ul of competent E. coli DH5a, transfer to Bi0-Rad’s 0.2cm electric shock cup for electric shock, the voltage is 2.5 KV, the time is 5.0 milliseconds to 6.0 milliseconds, immediately after the electric shock, add 1ml of SOC medium to the cup to revive the bacteria, and then spread the bacteria on the LB solid medium containing ampicillin, X-Gal and IPTG, at 37 Cultivate overnight at ℃, get blue-white colonies the next day, transfer the obtained white colonies, that is, white clones, to LB solid medium containing ampicillin for culture, and extract plasmids from each clone, and use EcoRI digestion to identify the insertion The size of the fragments, the obtained white clonal group constitutes the O-antigen gene cluster library of Escherichia coli O150 type;

(4) Sequencing the clones in the library: select 120 clones with inserts of more than 700 bp from the library, and use the ABI377 type DNA automatic sequencer to sequence the inserts in the clones in one direction by Shanghai Bioengineering Co., Ltd., so that the sequence reaches 90% coverage, and the remaining 10% of the sequence is reverse-sequenced to obtain all the sequences of the O-antigen gene cluster;

(5) Splicing and analysis of nucleotide sequences: use the Pregap4 and Gap4 software of the Staden package software package published by the Cambridge MRC (Medical Research Council) Molecular Biology Laboratory to splice and edit all sequences to obtain Escherichia coli O150 The full-length nucleotide sequence of the O-antigen gene cluster; the quality of the sequence is mainly guaranteed by two aspects: 1) do 6 Long PCR reactions to the genome of Escherichia coli O150 type, and then mix these products to generate the library, 2 ) for each base, to ensure more than 3 high-quality coverages, after obtaining the nucleotide sequence of the Escherichia coli O150 type O-antigen gene cluster, use the National Center for Biotechnology Information (The National Center for Biotechnology Information, NCBI's orffinder found genes, found 11 open reading frames, and compared them with the genes in GenBank with blast series software to discover the functions of these open reading frames and determine what genes they are, and then use the Artemis software of the British Sanger Center to complete Gene annotation, precise alignment between DNA and protein sequences using Clustral W software, and finally the structure of the O-antigen gene cluster of Escherichia coli O150;

(6) Screening of specific genes: design primers for wzx, orf8, orf9, orf10, wzy genes in the O-antigen gene cluster of E. distributed in different places within the corresponding genes to ensure their specificity; PCR with these primers using the genomes of 166 strains of Escherichia coli and 43 strains of Shigella as a template did not amplify any of the correct size Bands, that is, no PCR product bands were obtained in most groups, although PDR product bands were obtained in a small number of groups, but its size did not meet the expected size, so wzx, orf8, orf9, orf10, wzy genes have no effect on Escherichia coli O150 type O-antigens are highly specific.

That is, the first aspect of the present invention provides the full-length nucleotide sequence of the O-antigen gene cluster of Escherichia coli O150 type, its full sequence is shown in SEQ ID NO: 1, with a full length of 9227 bases or have one or more inserted, deleted or substituted bases while maintaining the nucleotide of SEQ ID NO: 1 of the isolated nucleotide function. The structure of the O-antigen gene cluster of Escherichia coli O150 was obtained by the method of the present invention. As shown in Table 3, it consists of 11 genes in total, all of which are located between the galF gene and the gnd gene.

The second aspect of the present invention provides the gene in the O-antigen gene cluster of Escherichia coli O150 type, i.e. translocator gene (wzx gene or the gene that has similar function with wzx); Polymerase gene (wzy gene or with wzy Genes with similar functions); Glycosyltransferase genes, including orf8, orf9, orf10 genes; Special sugar synthesis pathway genes in bacterial polysaccharide antigens, including gne genes. Their starting position and termination position and nucleotide sequence in O-antigen gene cluster are all listed in table 4; The O-antigen is highly specific.

The third aspect of the present invention provides the wzx gene or the gene with similar function to wzx, the wzy gene or the gene and glycosyltransferase that are derived from the O-antigen gene cluster of Escherichia coli O150 type Genes, including oligonucleotides of orf8, orf9, orf10 genes, which are oligonucleotides of any of these genes. However, it is preferred to use the oligonucleotide pairs listed in Table 1, which also lists the position of these oligonucleotide pairs in the O-antigen gene cluster and the For the size of the products of the PCR reactions performed for the primers, these PCR reactions can be performed with the annealing temperatures in the table. These primers only obtained products of the expected size in the PCR amplification using Escherichia coli O150 as a template, but did not obtain products of the expected size in the PCR amplification using other bacteria listed in Table 2 as templates. In more detail, PCR reactions using these oligonucleotide pairs as primers did not yield any products in most bacteria. Therefore, it was confirmed that these primers, oligonucleotides listed in Table 1, are highly specific to the O-antigen of Escherichia coli O150 type.

The method for isolating the O-antigen-specific nucleotides of Escherichia coli O150 type comprises the following steps: 1) extraction of the genome; 2) PCR amplification of the O-antigen gene cluster in Escherichia coli O150 type; 3) Construction of O-antigen gene cluster library; 4) sequencing of clones in the library; 5) splicing and analysis of nucleotide sequences; 6) screening of specific genes.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

As used in the present invention, "oligonucleotide" mainly refers to a nucleotide molecule derived from the gene encoding glycosyltransferase, gene encoding transportase and gene encoding polymerase in the O-antigen gene cluster, They may vary in length, generally ranging from 10 to 20 nucleotides. More precisely, these oligonucleotides are derived from wzx gene (nucleotide position is from 5632 to 7134 bases of SEQ ID NO: 1), orf8 gene (nucleotide position is from 8307 bases of SEQ ID NO: 1 to 9197 bases), orf9 gene (nucleotide position is from 9184 to 10098 bases of SEQ ID NO: 1), orf10 gene (nucleotide position is from 10098 to 10994 bases of SEQ ID NO: 1), wzy gene (nucleotide position is from 11012 to 12109 bases of SEQ ID NO: 1). Oligonucleotides derived from within the above genes are highly specific for E. coli type O150.

In addition, sometimes two genetically similar gene clusters encoding different O-antigens produce new O-antigens through gene recombination or mutation, thereby producing new bacterial types, new mutant strains. In this environment, it is necessary to select multiple pairs of oligonucleotides to hybridize with the recombinant gene to improve the specificity of detection. Accordingly, the present invention provides a set of mixtures of pairs of oligonucleotides derived from glycosyltransferase genes; from transporter and polymerase genes, including wzx genes or genes with similar functions to wzx, wzy genes or Genes with similar functions to wzy. The mixture of these genes is specific for a particular bacterial polysaccharide antigen, making the set of oligonucleotides specific for that bacterial polysaccharide antigen. More specifically, the mixture of oligonucleotides is a combination of oligonucleotides derived from a glycosyltransferase gene, a wzx gene or a gene with a similar function to wzx, a wzy gene or a gene with a similar function to wzy .

In another aspect, the invention relates to the identification of oligonucleotides which can be used to detect bacteria expressing O-antigen and to identify bacterial O-antigen in diagnostics.

The present invention relates to a method for the detection of one or more bacterial polysaccharide antigens in foodstuffs which allow a sample to specifically hybridize to an oligonucleotide of at least one of the following genes: (i) encoding a glycosyl Transferase gene (ii) gene encoding transferase and polymerase, including wzx gene or gene with similar function to wzx, wzy gene or gene with similar function to wzy. Where conditions permit, at least one oligonucleotide is capable of specifically hybridizing to more than one such gene of at least one bacterium expressing a particular O-antigen, which is E. coli type O150. It can be detected by PCR method, and the nucleotides in the method of the present invention can be labeled as probes through hybridization reactions such as southern-blot or fluorescence detection, or through gene chips or microarrays to detect antigens and bacteria in samples.

The designers of the present invention took into account the fact that a mixture of oligonucleotides can specifically hybridize to a target region to detect a sample when a single specific oligonucleotide is ineffective for detection. Therefore the present invention provides a set of oligonucleotides for use in the detection method of the present invention. The oligonucleotides mentioned here refer to genes derived from genes encoding glycosyltransferases, genes encoding transferases, and polymerases, including wzx genes or genes with similar functions to wzx, wzy genes or genes with similar functions to wzy Functional gene oligonucleotides. This set of oligonucleotides is specific for a particular bacterial O-antigen expressed by E. coli type O150.

In another aspect, the present invention relates to a method for detecting in excreta one or more bacterial polysaccharide antigens that allow a sample to specifically hybridize to oligonucleotides of at least one of the following genes: ( i) genes encoding glycosyltransferases (ii) genes encoding transportases and polymerases, including wzx genes or genes with similar functions to wzx, wzy genes or genes with similar functions to wzy. At least one oligonucleotide is capable of specifically hybridizing to at least one such gene of at least one bacterium expressing a particular O-antigen if conditions permit. These bacteria are E. coli type O150. The oligonucleotides in the present invention can be used as primers to detect samples by PCR, and the oligonucleotide molecules in the present invention can also be labeled as probes through hybridization reactions such as southern-blot or fluorescence detection, or through gene chips Or microarray detection of antigens and bacteria in samples.

Generally, a pair of oligonucleotides may hybridize to the same gene or to different genes, but one of them must be able to specifically hybridize to a specific sequence of a specific antigenic type, and the other oligonucleotide Can hybridize to non-specific regions. Therefore, when the oligonucleotides in a specific polysaccharide antigen gene cluster are recombined, at least one pair of oligonucleotides can be selected to hybridize with the specific gene mixture in the polysaccharide antigen gene cluster, or multiple pairs of oligonucleotides can be selected Crosses with a mixture of specific genes. Even when all genes in a particular gene cluster are unique, the method can be applied to identify nucleotide molecules of gene mixtures within this gene cluster. The present invention therefore provides a complete set of pairs of oligonucleotides for testing the method of the invention, where the pairs of oligonucleotides are derived from genes encoding glycosyltransferases, genes encoding transportases and polymerases including wzx Genes or genes with similar functions to wzx, wzy genes or genes with similar functions to wzy, this set of oligonucleotides is specific to a particular bacterial polysaccharide, and this set of oligonucleotides may be involved in sugar synthesis Nucleotides of essential genes.

In another aspect, the present invention also relates to a method for detecting one or more bacterial polysaccharide antigens in a sample derived from a diseased animal. One or more bacterial polysaccharide antigens in the sample can allow the sample to specifically hybridize to one of a pair of oligonucleotides in at least one of the following genes: (i) a gene encoding a glycosyltransferase (ii) ) genes encoding transportases and polymerases, including wzx genes or genes with similar functions to wzx, wzy genes or genes with similar functions to wzy. Where conditions permit, at least one oligonucleotide is capable of specifically hybridizing to one or more such genes of at least one bacteria expressing a particular O-antigen in the sample, these bacteria being E. coli type O150. The oligonucleotides of the present invention can be used as primers to detect samples by PCR, and the oligonucleotides of the present invention can also be labeled as probes through hybridization reactions, or antigens in samples can be detected by gene chips or microarrays and bacteria.

In more detail, the method described above can be understood as that when oligonucleotide pairs are used, one of the oligonucleotide molecules can hybridize to a gene that is not derived from the glycosyltransferase gene, the wzx gene or has On the sequence of genes with similar functions, wzy genes or genes with similar functions to wzy. Furthermore, when both oligonucleotides hybridize, they may hybridize to the same gene or to different genes. That is, when cross-reactivity is an issue, mixtures of oligonucleotides can be selected to detect mixed genes to provide specificity of detection.

The present inventors believe that the present invention is not necessarily limited to the specific O-antigen encoded by the above-mentioned nucleotide sequence, but is broadly applicable to the detection of all O-antigen expressing and identification of O-antigen bacteria. Furthermore, due to the similarities between O-antigen synthesis and the synthesis of other polysaccharide antigens, such as bacterial extracellular antigens, the methods and molecules of the present invention are also applicable to these other polysaccharide antigens.

The present invention discloses the full-length sequence of the O-antigen gene cluster of Escherichia coli O150 type for the first time, and can produce recombinant molecules from the sequence of this uncloned full-length gene cluster, and can produce the expression Escherichia coli O150 type by inserting expression O-antigen, and become a useful vaccine.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (NewYork: Cold Spring Harbor Laboratory Press, 1989) are generally followed.

Embodiment 1: the extraction of genome:

Escherichia coli O150 was cultured overnight at 37°C in 5 mL of LB medium, and the cells were collected by centrifugation. Resuspend the cells with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubate at 37°C for 20 minutes, then add 10ul 10mg/ml lysozyme and continue to incubate for 20 minutes. Then add 3ul 20mg/ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg/ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant and extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixed solution, take the supernatant and extract with an equal volume of ether to remove Residual phenol; the supernatant used 2 times the volume of ethanol to precipitate DNA, rolled out the DNA with glass wool and washed the DNA with 70% ethanol, and finally resuspended the DNA in 30ul TE. Genomic DNA was detected by 0.4% agarose gel electrophoresis.

Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O150 by PCR:

The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O150 as a template. First, design the upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C); the O-antigen gene cluster was amplified by the Expand Long Template PCR method of Boehringer Mannheim, and the PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, 30 cycles of annealing at 60°C for 30 seconds and extension at 68°C for 15 minutes. Finally, continue extending at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to detect the size and specificity of the PCR products. Combine 6 tubes of long PCR products and purify the PCR products with the Wizard PCR Preps purification kit from Promega.

Embodiment 3: construct O-antigen gene cluster library:

The first is the acquisition of the ligation product: use the modified Novagen DNaseI shot gun method to construct the O-antigen gene cluster library. The reaction system was 300ng PCR purified product, 0.9ul 0.1M MnCl ₂ , 1ul 1mg/ml DNaseI diluted 1:2000, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the DNA fragment size between 1kb-3kb, then add 2ul 0.1M EDTA to terminate the reaction. Combine 4 tubes of the same reaction system, extract once with an equal volume of phenol, once with an equal volume of a mixed solution of phenol:chloroform:isoamyl alcohol (25:24:1), and once again with an equal volume of diethyl ether Finally, the DNA was precipitated with 2.5 times the volume of absolute ethanol, washed with 70% ethanol, and finally resuspended in 18ul of water. Then add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul100mM DTT and 5 units of T4 DNA polymerase to this mixture, 11°C for 30 minutes, make the end of the digested product blunt, stop the reaction at 75°C, add 5 units of Tth DNA polymerase and its corresponding buffer solution and expand the system to 80ul, react at 70°C for 20 minutes, add dA tail to the 3′ end of DNA; ) mixed solution extraction and equal volume ether extraction, and then ligated with 3×10 ⁻³ pGEM-T-Easy carrier from Promega Company at 16° C. for 24 hours, with a total volume of 90 ul. There are 9ul of 10×buffer and 25 units of T4DNA ligase. Finally, the ligation mixture was precipitated with 1/10 volume of 3M NaAc (pH 5.2) and 2 times the volume of absolute ethanol, washed with 70% ethanol, dried and dissolved in 30ul of water to obtain the ligation product.

The second is the preparation of competent cells: prepare competent cells Escherichia coli DH5□ according to the method provided by Bio-Rad Company. Take a single colony of Escherichia coli DH5□ in 5ml of LB medium, culture it at 180rpm for 10 hours, take 2ml of the culture and transfer it to 200ml of LB medium, shake vigorously at 37°C and 250rpm to about OD6000.5, then Cool in an ice bath for 20 minutes, and centrifuge at 4000 rpm for 15 minutes at 4°C. Drain the supernatant, blow off the bacteria with 200ml of cold ice-precooled deionized sterilized water, and centrifuge at 4000rpm at 4°C for 15 minutes. Blow off the bacterial cells with 100 ml of cold ice-precooled deionized sterilized water, and centrifuge at 4000 rpm for 15 minutes at 4°C. Suspend the cells with cold ice-precooled 10% glycerol, centrifuge at 6000 rpm at 4°C for 10 minutes, discard the supernatant, and finally precipitate the cells with 1ml ice-precooled 10% glycerol, which are competent cells. The prepared competent cells were divided into 50ul tubes and stored at -70°C.

Finally, electroporation competent cells: take 2-3ul of ligation products and mix them with 50ul of competent Escherichia coli DH5□, then transfer to Bio-Rad’s 0.2cm electric shock cup for electric shock, the voltage is 2.5 kV, and the time is 5.0 milliseconds -6.0 milliseconds. Immediately after electric shock, add 1ml of SOC medium to the cup to revive the bacteria. Then immediately smear the bacteria on LB solid medium containing ampicillin, X-Gal and IPTG and culture them upside down overnight at 37°C, and blue-white colonies are obtained the next day. The obtained white colonies, that is, white clones, were transferred to LB solid medium containing ampicillin for culture, and at the same time, plasmids were extracted from each clone and digested with EcoRI to identify the size of the inserted fragment, and the white clone group constituted Escherichia coli O150 Type O-antigen gene cluster library.

Example 4: Sequencing the clones in the library:

From the library, 120 clones with inserts above 700 bp were selected, and the inserts in the clones were sequenced unidirectionally by Shanghai Bioengineering Co., Ltd. with an ABI377 DNA automatic sequencer, so that the sequence coverage reached 90%. The remaining 10% of the sequences were reverse-sequenced to obtain all the sequences of the O-antigen gene cluster.

Embodiment 5: splicing and analysis of nucleotide sequences:

Use the Pregap4 and Gap4 software of the Staden package software package published by Cambridge MRC (Medical Research Council) Molecular Biology Laboratory to splice and edit all the sequences to obtain the full-length nucleotides of the O-antigen gene cluster of Escherichia coli O150 sequence (see list of sequences). The quality of the sequence is mainly guaranteed by two aspects: 1) do 6 Long PCR reactions to the genome of Escherichia coli O150 type, and then mix these products to generate a library. 2) For each base, ensure more than 3 high-quality coverages. After obtaining the nucleotide sequence of the Escherichia coli O150 type O-antigen gene cluster, the orffinder of the National Center for Biotechnology Information (NCBI) was used to discover genes, and 11 open reading frames were found, and blast A series of software is compared with the genes in GenBank to discover the functions of these open reading frames and determine what genes they are, and then use the Artemis software of the Sanger Center in the UK to complete the gene annotation, and use the ClustralW software to make precise alignments between DNA and protein sequences. Finally, the structure of the O-antigen gene cluster of Escherichia coli O150 was obtained, as shown in Table 3.

Through retrieval and comparison, it was found that the protein encoded by orf6 contained 14 potential transmembrane segments, through the algorithm of Eisenberg et al [Eisenberg, D, Schwarz, E.etal (1984) "Analysis of membrane and surface protein sequences with the hydrophobic moment plot.J .Mol.Biol.179:125-142] also found that Orf6 has 14 potential transmembrane regions, and is similar to many Wzx proteins, for example, it has 21% identity with Wzx of pyrococcus in 419 amino acids, 41 % similarity, so it can be determined that orf6 is a wzx gene, named wzx. Orf8 is similar to glycosyltransferase family 2 (pfam00535, 3e-07), and compared with blast, it has 27% identity, 51% similarity, indicating a high degree of homology between them, it can be speculated that orf8 is also a glycosyltransferase gene, tentatively named orf8. orf9 and rfbF of Shigella flexneri 2a The genes have 47% identity and 63% similarity in 285 amino acids. The rfbF gene encodes the glycosyltransferase gene of dTDP-rhamnose, so it is speculated that orf9 is a glycosyltransferase gene, which is tentatively named orf9. orf10 has 48% identity and 67% similarity with the rfbG gene of Shigella flexneri 2a in 289 amino acids. The rfbG gene encodes the glycosyltransferase gene of dTDP-rhamnose, so it is speculated that orf10 is a Glycosyltransferase gene, tentatively named orf10. The protein encoded by orf11 is found to contain 10 potential transmembrane segments through analysis, and its inner membrane topology has the typical characteristics of the well-known O-antigen polymerase (Wzy). In addition, It has 25% identity and 47% identity with Wzy of Shigella baumannii encoding O-antigen polymerase in 317 amino acids, indicating that there is a high degree of homology between them, so orf11 is named wzy Gene.

Example 6: Screening of specific genes: Primers were designed for the wzx, orf8, orf9, orf10, and wzy genes in the O-antigen gene cluster of Escherichia coli O150. The positions of these genes in the nucleotide sequence are shown in Table 1.

Table 1 lists the glycosyltransferase gene and oligosaccharide unit processing gene in the O antigen gene cluster of Escherichia coli O150 and the primers and PCR data in the gene. The glycosyltransferase genes, transportase genes and polymerase genes of the O antigen gene cluster of Escherichia coli O150 and their corresponding functions and sizes are listed in the table. In each gene, we designed three pairs of primers, and each pair of primers was distributed in different places in the corresponding gene to ensure its specificity. The position and size of each primer in SEQ ID NO: 1 are also listed in the table. The genomes of all bacteria in Table 2 were used as templates for PCR with each pair of primers at the corresponding annealing temperature listed in the table, and corresponding PCR products were obtained, and their sizes were also listed in the table.

The mdh (malate dehydrogenase) gene is a highly conserved gene present in all Escherichia coli genomes, so we designed primers #101 (-TTC ATC CTAAAC TCC TTA TT) and #102 (-TAA TCG CAG GGG AAA GCA GG), and then the genome was extracted from 166 strains of Escherichia coli, and the method was as described above. Use this pair of primers to PCR from the genomes of 166 strains of Escherichia coli to identify Escherichia coli and test the quality of their genomes.

Table 2 is 166 strains of Escherichia coli and 43 strains of Shigella and their sources for screening specific genes. For the convenience of detection, we divide them into a group of 8-10 bacteria, a total of 27 groups, their Sources are listed in the table.

Genomic DNA of Escherichia coli O150 was contained in group 24 as a positive control. Use each group of bacteria as a template, and use each pair of primers in Table 1 to perform PCR under the following conditions: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 15 seconds, and the annealing temperature varies with different primers (refer to Table 1). The annealing time was 50 seconds and the extension was 2 minutes at 72°C for 30 cycles. Finally, continue to extend at 72°C for 10 minutes, and the reaction system is 25ul. After the reaction was completed, 10 ul of the PCR product was taken to detect the amplified fragment by 0.8% agarose gel electrophoresis.

For wzx, orf8, orf9, orf10, and wzy genes, each gene has three pairs of primers to be detected, and each pair of primers has a correct band of the expected size after PCR in the 24th group. No bands of the correct size were amplified, that is, no PCR product bands were obtained in most groups, so the wzx, orf8, orf9, orf10, wzy genes are highly specific to E. coli O150 type and its O-antigen of.

Finally, genes highly specific to the O-antigen of E. coli O150 were screened from E. coli O150 by PCR: wzx, wzy and three glycosyltransferase genes. Any 10-20nt oligonucleotide in these genes is specific to the O-antigen of Escherichia coli O150 type, especially the primers in each of the above-mentioned genes, that is, the oligonucleotide pair, is confirmed to be effective for large intestine after PCR detection. Bacillus type O150 is highly specific. All of these oligonucleotides can be used to quickly and accurately detect E. coli O150 in livestock and the environment, and can identify their O-antigens.

Table 3 is the structural table of the O-antigen gene cluster of Escherichia coli O150 type, lists the structure of the O-antigen gene cluster of Escherichia coli O150 type in the table, is made up of 11 genes altogether, and each gene is represented with box , and write the name of the gene in the box, and the number indicates the order of the open reading frame (orf) in the O-antigen gene cluster. At both ends of the O-antigen gene cluster are the galF gene and the gnd gene, which do not belong to the O-antigen gene cluster, and we only use a part of their sequence to design primers to amplify the full-length sequence of the O-antigen gene cluster.

Table 4 is the position figure of the gene in the O-antigen gene cluster of Escherichia coli O150 type, has listed the accurate position of all open reading frames in the O-antigen gene cluster of Escherichia coli O150 type in the complete sequence in the figure, The start and stop codons of each open reading frame are underlined. There are two start codons for the open reading frame in Escherichia coli: ATG and GTG.

sequence list

<110> Nankai University

<120> Nucleotides specific to the O-antigen of Escherichia coli O150

<130> Nucleotide specific for the O-antigen of Escherichia coli O150

<160>1

<170>PatentIn version 3.1

<210>1

<211>13552

<212>DNA

<213>Escherichia coli

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attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaactgcg cgtgaagcgt 120

caactactgg cggaagttca gtccatctgc ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttgtgtg cgcgacccgc cattggtgac 240

aatccatttg tcgtggtgct gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgttg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccactg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtaggt cgctatgtgc tttctgccga tatttggccg 540

gaactggaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgctattgcc 600

gagctggcga aaaaacaatc cgttgatgtc atgctgatga ccggtgacag ttacgactgc 660

ggcaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gcctacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg tattgagaag ttgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt taacaagaaa attataacgg cagtgaagat ttgcagcaaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa accatcagaa tagcaacgag ttagcagtag 900

ggtttttc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc ataggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

tcaaactgag agccgcttat ttcacagcat gctctgaagt aatatggaat aaattaagtg 1140

aaaatacttg ttactggtgg cgcaggattt attggttctg ctgtagttcg tcacattta 1200

aataatacgc aggatagtgt tgttaatgtc gataaattaa cgtacgccgg aaacctggaa 1260

tcacttgctg atgtttctga ttccgaacgc tatgtttttg aacatgcgga tatttgcgat 1320

gcagctgtaa tggcacggat ttttgctcag catcagccgg atgcagtgat gcacctggct 1380

gctgaaagcc atgttgaccg ttcaattaca ggtcctgcgg catttattga aaccaatatt 1440

gttggtacat atgtcctttt ggaagccgct cgcaattact ggtctgctct tgatagcgac 1500

aagaaaaata gcttccgttt tcatcatatt tctactgacg aagtctatgg tgatttgcct 1560

catcctgacg aggtaaataa tacagaagaa ttacccttat ttactgagac aacagcttac 1620

gcgccaagca gcccttattc cgcatccaaa gcatccagcg atcatttagt ccgcgcgtgg 1680

aaacgtacct atggtttacc gaccattgtg actaattgct ctaacaatta tggtccttat 1740

catttcccgg aaaaattgat tccattggtt attctcaatg ctctggaagg taaagcatta 1800

cctatttatg gtaaagggga tcaaattcgc gactggctgt atgttgaaga tcatgcgcgt 1860

gcgttatata ccgtcgtaac cgaaggtaaa gcgggtgaaa cttataacat tggtgggcac 1920

aacgaaaaga aaaacatcga tgtagtgctc actatttgtg atttgttgga tgagattgta 1980

ccgaaagaga aatcttaccg cgagcaaatt acttatgttg ccgatcgtcc gggacacgat 2040

cgccgttatg ccattgatgc tgagaagatt ggtcgcgaat tgggatggaa accacaggaa 2100

acgtttgaga gcgggattcg gaagacagtg gaatggtacc tgtccaatac aaaatgggtt 2160

gataatgtga aaagtggtgc ctatcaatcg tggattgaac agaactatga gggccgccag 2220

taatgaatat cctccttttt ggcaaaacag ggcaggtagg ttgggaacta cagcgtgctc 2280

tggtaccttt gggtaatttg attgctcttg atgttcactc cactgattac tgtggtgatt 2340

ttagtaatcc tgaaggtgta gctgaaaccg taagaagcat tcggccggat attattgtca 2400

acgcagccgc tcacaccgca gtagacaaag cagaatcaga accgaagttt gcacaattac 2460

tgaacgcgac gagtgtcgaa gcgatcgcga aagcagccaa tgaagtcggc gcctgggtta 2520

ttcactactc tactgactac gtatttccgg ggaccggtga aataccatgg caggaggagg 2580

atgcaaccgc accgctaaat gtttacggtg aaaccaagtt agcgggagaa aaagcattac 2640

aagagcattg tgtgaagcac cttattttcc ggaccagctg ggtctatgca ggtaaaggaa 2700

ataacttcgc caaaacaatg ttacgtctgg caaaagagcg tgaagaattg gccgttatta 2760

acgatcagtt tggtgcgcca acaggtgctg agctgctggc tgattgtaca gcacatgcca 2820

ttcgtgtcgc attgaataaa ccggaagtcg caggcttgta ccatctggta gccagtggta 2880

ccacaacctg gtacgattat gctgcgctgg tttttgaaga ggcgcgcaaa gcagatattc 2940

cccttgcact caacaaactc aatgcggtac caacaacagc ctatcctaca tcagctcgtc 3000

gtccacataa ctctcgcctt aatacagaaa aatttcagca gaattttgcg cttgttttgc 3060

ctgactggca ggttggcgtg aaacgaatgc ttaacgaatt atttacgact acagcaattt 3120

aatagttttt gcatcttgtt cgtgatgatg gagcaagatg aattaaaagg aatgatgaaa 3180

tgaaaacgcg taaaggtatt attttagcgg gtggctctgg tactcgtctt tatcctgtga 3240

ctatggctgt cagtaaacag ctattaccta tttatgataa gccgatgatc tattacccgc 3300

tctctacact gatgttggcg ggtattcgcg atattttgat tatcagcacg ccacaggata 3360

ctcctcgttt tcaacaactg ctgggtgatg ggagccagtg ggggctaaat cttcactaca 3420

aagtgcaacc gagtccggat ggtcttgcgc aggcgtttat tatcggtgaa gagtttattg 3480

gtggtgatga ttgtgctttg gtacttggtg ataatatctt ctacggtcac gacctgccta 3540

agttaatgga tgccgctgtt aacaaagaaa gtggtgcaac ggtatttgcc tatcacgtta 3600

atgatcctga acgctacggt gtcgttgagt ttgataaaaa cggtacggca ataagcctgg 3660

aagaaaaacc gttacaacca aaaagtaatt atgcggtaac tgggctttat ttctatgata 3720

acgacgttgt cgaaatggcg aaaaacctta agccttctgc ccgtggtgaa ctagaaatta 3780

ccgatattaa ccgtatttat atggaacagg ggcgtttatc tgttgccatg atgggacgtg 3840

gttatgcatg gttggacacg gggacacatc aaagtcttat tgaggcaagc aatttcatcg 3900

cgacaataga agaacgacag gggctgaaag tttcctgccc ggaagaaatt gcttaccgta 3960

aggggtttat cgatgctgag caggtgaaag tattagctga accgttgaag aaaaatgctt 4020

atggccagta cctgctaaaa atgattaaag gttattaata aaatgaacgt aattaaaaca 4080

gagattccag acgtattaat tttcgaaccg aaagtttttg gtgatgagcg tggtttcttt 4140

atggaaagct ttaatcagaa agttttcgaa gaagctgtag gacgtaaggt tgaatttgtt 4200

caggataacc attcgaagtc ttgtaaaggt gttttacgcg ggctgcatta tcagttagaa 4260

cccttatgcgc aagggaaatt ggtacgttgc gttgttggtg aggtttttga tgttgcggtt 4320

gatattcgta aatggtcacc tacctttggc aaatgggttg gggtgaattt atctgctgag 4380

aataagcgcc agttgtggat ccctgaagga tttgcacatg gttttttggt gctaagtgag 4440

acggcagaat ttgtttataa gacgacgaat tattatgcac ctgggtatga aggtagcatc 4500

atttggaatg ataaaatact atctattgat tggcctgagt taacactgga gtatattctt 4560

tccgataaag atctaaaggc cccaattcta agtgaatcta tagagaatat ttaaaatgtt 4620

tattagtaga gtaaaacgta aaattcaatc tttgataagc cttgagttga attttttaca 4680

cgattattgg aggtttaaaa aatattacac gaagggatct caagcttcga aagataagca 4740

gaaacttgaa tcttggatat tgcaagataa acatcgaata gaaaaagctt tctctttacc 4800

taagccaaaa cttggatttg gtaaagatgt aatacctcgt ctcattgaca atcttgttaa 4860

ttatagcagt aaatttggaa atgatcaggt atattatatt ggcttaggtg cattacaatc 4920

gtataagaga ttccacgacg aacaaaaatt tttatgcct gaattttat caaagaatat 4980

ttgtaaaata cggaatgatg attttctcga tccccgatgc aatgtcgctg gctattttaa 5040

aaaagatgat tcattagtta caggtaagat aacagtagaa tcattgatga acgaacggcg 5100

tagctgtcgg cattttgatg ttgatgaaag tcttaatatc ggagataaac ttttaaagga 5160

tgttacaaaa ctatctatta cagccccttc tgtttgtaat agacaacatt ggcgcattca 5220

ctatttttca ggtgaattaa agaataaaat tcttagttat caaaatggaa attcaggatt 5280

tacagaaaac ataccttatg tagcagttat aacaagtgat cttagagcat tttattctac 5340

tgacgaaaga aaccagccct atacagacgg tgggatcttt gccatgaatt ttatgtatgc 5400

tctacaacac tatggtcttg cttcttgtcc attgaattgg tgtaattcat ctcacattga 5460

gaatgagttt agagaaacta agttgattcc tgattacgaa gtagtagtac ttgtggttgc 5520

ttttggctat ccaaataaag atgccttata tgctaaatca ccacgattgt ctctaaataa 5580

cctttacaca attaattagg cgaaggccac ctttaaaggt ggctgacaat aatgaagaaa 5640

caaatcatta aaaatatttc agccaatttt actaatttga ttgttagtat tttattgga 5700

ctattattga cgcctttact cgtcaaaagt ttaggcattg aagtatatgg agttctaccg 5760

atagctttat ttttaacatt ttatatggga gtcattgctc aagcattaac tgcatctgta 5820

aatagatttt taattgagga ttttattaaa ggagcatata ctgaggcaag tgtcgtattt 5880

agtacatcgt ttattcttat tctaggatat ctatctgtta ttagtattgg atttatttat 5940

ccaatattgc atataagtga tttttttaac ataccaccgg gatatgaaca tgatgcagtt 6000

cttctattct cgtgtgtaat attttcgttt gttattgcta tgatcagttc atcattgtca 6060

gtttctatgt atgcaaaaaa taggattgac ttgatgcaat atggaaacat tttaaggaat 6120

attackaaag tcttaatcat tataatgttt gtttatttat ctgaaataaa tttaacaaaa 6180

ataggaatat caattgtttt atctgagctg ttttatctaa tctatgtcat tataacttgg 6240

aaaatattga caccacaact ttctctgcag ataggatttt tcagaaagga taaggtaaag 6300

agattaacat ctttcagtgg ctggatattg tttgatcaaa ttgggtcaat tctttttctt 6360

aaaacagatc tgattttagt taattctcta agtggtagta aagctaatgg tgaatattca 6420

atagctacac aatttagtga tcttttgcgt tcacttgctg gtttggtagc tggtagtta 6480

ggtcctatga tggtaatttt atatgaagaa aagaaattag aagaacttac ttcattgacc 6540

attacatttg ttaaattcct aagcttgacg atagctattc ctattattat tctttgtgta 6600

tactctaaag aaattcttga gctttggtta ggttcggggt ttgtatttct ttcaccatta 6660

atatgtatcg taacgcttcc actgattatc aatttgggca cactgccaat attatcgatc 6720

aatattgcag tcaaaaaaat tcaagtgcct gcactcataa atttctttct tgcaatcgtt 6780

ggaattatag gctctgtagt attgttcaaa accactacat taggtttatta tgctgtagct 6840

ataagttatg tgattacatt aactattaag aactccattt tcataccatg gtatgcaaca 6900

attatattaa ataaaccaag gtatcttttt tttaaaactc atctaattac tatcatcttc 6960

tcttctctat tttttatttt tatatatata cttaaaagta taatgcagat tactaattta 7020

aatttgcttt ttgaattgat attggttggt tgctttggac ttctatctac atttcctttc 7080

tatagtaaag aggaaaggcg atcatttatg ttaatgctca agaggaaagt gtaataatgg 7140

attcgactca gaaaataggc gttctgaatt tccagtattc ggatcataat tatggtgctg 7200

ttttacaagc cgcagcaata gaacaatttc tgaaaagcaa tggatataat gctgaacata 7260

tagactatat atctagtcca gagataaagg gtaatagggt aattatacaa ctaaaatgta 7320

tattgaaaaa attgggacta tctgattctg taagaaaaat gcttggaaaa caagtccact 7380

tgacacctgt ggtcacaaac gaaaaagtat ttgaggaatt tagaaaaaat tggttggtga 7440

gaacgaagtg tttcagaaac tttgaagaat taaaaaaaac tgattttaat tttaaagcgg 7500

ttatagttgg gagtgatcag gtttggcgac catctcaatt tactcggttt tccgattata 7560

aggtatattt cttaagcttt ttaccttcgc aggttaagaa aatatcttt gcagcaagtt 7620

ttggtattga taagtgggaa gttagtgatg ctaacgtcac gaacgaaata aaaaaatata 7680

ttcaagattt caatgctgtt tcagtcagag aagatagtgg cgttaatatt tgtgcaacca 7740

tttttaatgt cgacgctgaa catgttcttt atcctacttt actggttggt agtgactttt 7800

ttaacagtat aatagctaag gaaatacgtg ctgatgctta ttacgatgct atagtttt 7860

acaaacttga tattgatcaa cattttatag attcagttac tatactaggt agaaagaaat 7920

gcaagccagt aaaaaatatt tattacaacc aagtagatgg ccgaaatgag tattattctg 7980

ttcctgaatg gttaagcaat attaagaatg ctaattttgt tatcacagat tcttttcatt 8040

gtgtatgttt ttgtcttctt tttcaaaaag agtttttatg ctgtgtgaat gaaagtagag 8100

gattatctag attgcaaagt ttacttggaa tgcttgagct tactgatcgt atttgctcgt 8160

caaaagatga ttttgataat aagttaaatt cgttaaagcc aatagactat tctaaagtgg 8220

gcgatatatt gaataagcat agagatattt ctaaggcgtt tttattaagt aatttatctt 8280

gagtttgagt gtgggttaaa tattatga atgcaaataa tagaaatgca tttccgatca 8340

ttgttatgac tcgcaacgaa ggcgccgact tacaacgttg cgttgattct atattacata 8400

ctgtatctat agatgtacat atatatatca tagacaataa ttctgatgat ttatcacata 8460

ttcagatcct tgctaacctt gaagacctga atcgtaaaaa tttaacagtt ataagaaata 8520

caaggaattt atggatttta ggtgtaaata acacactatt gaaaatcaag aaaaatcatg 8580

atactaaata ttttattctc actgatggtg atatagactt tcatggtttt aaagaagaaa 8640

gttgttggtt aacatattta atcaataaaa tggaatccaa cgtgataatc ggtaagctcg 8700

gaatttctct ggattggtca tttctggcct caaaaacgga gatgaaagat atttaccggc 8760

aagaacaaaa tctctataat gaaagaagaa aaatcggtga tctttatata tctgccgttg 8820

acacgacagt agccattatt cgttgggatt ggtctataga gcgtaaagct ttaattttatc 8880

ctgaccatat gaggtattta cgtccagaac tgtactcttg ccgtactaaa cgtggtttaa 8940

cagtagagca tttaggatgg tatagatacc ttgataactc cctgtctacg caaagtataa 9000

actcgaaagt tttttgtttt acaattgttg gtggtactgt taaacctgag gttttaaaac 9060

tggctagtaa accctatcag tatttccata ttttgttttc aaaacctttc gctatgcttt 9120

ggtatttcag acgatatttt aaacttttta tatattttat tactaaaggg agacgtggat 9180

ttgatggaca gtcataataa taagttatca aaacgtattc cgtcacctgc actacgttgt 9240

tatgcagtaa tagttacatt taaccctgat attaataagg ttagaagttt aatagatatt 9300

ctacaaaaga attttatcac cgcagttgta gtagataata cgattggagc aataaattat 9360

ccattctcat gtcatgttat taatttaggt gataatttag gtattgccac agcacaaaat 9420

aaaggcatag aatattgttt gactaaacaa gctgaagcta tctggttttt tgatcaggac 9480

agcgtaatca cttcttcatt tgtcgaaaga tttctatcta ctgtccatga aaatcctcat 9540

gataagatct ttgctcctgt gttctgggat gagaagaaag ggtttgaata tgcaattaca 9600

aatatagatt caaagggcaa cagacaaaag ttgttatcat catcttttcc atcggatttt 9660

tatagttcga ttgttatttc atcagggtgc ttaataaaaa gtgatttgct gatgaaaatt 9720

ggtccaatgc tagatttttt atttatagat tacgttgata cggaatggtg tttacgtgct 9780

ttctattatg gctacaaagt tcacataatc aaaaatgcaa ggatgaaaca ttctataggt 9840

gataacacta tttctttaat cggttttaat gtaccaattc actctcctct tcgacgttat 9900

tatcgaataa gaaatgcatt tttattgatt aggtttaagc atatcccaaa aaaactagca 9960

tttagagaag tgatattttg ttgctgccat caattaataa ttatcctgac ccaagaaaaa 10020

aaactggcat atgttaagta ttttttacgg gctgtatatg atggccttag aaataaaact 10080

gggaagtttg taaactaatg agttttgtca tgaagccaaa ggtattagtg ctagttgcag 10140

tttataatgg tgaaaagtgg ttaatagagc aacttacatc tataataaat caaaaaaatg 10200

ttgatattga tgtgattatt aatatcgata aatcaactga ttcttcatac aaattgtgtg 10260

agaattttat tgcagataat gtcaagatac taccgtacgg agctgtattt ggcggtgcag 10320

gtgccaattt ttatcatcta atccatgaat ctgaattttc tggttacgat tacattgcat 10380

tttccgatca agacgatatt tggtttgaaa ctaaaatata tgatgccatc aataggatgc 10440

atgattatga tgcatattca agtaatgtaa tagcattctg ggaaaatggc aaacaatgtc 10500

taattgagaa atctcaacca cagacagaat ttgattattt ttttgaggca gcaggacccg 10560

ggtgcactta tatagtgaga caggaactag cactcgcatt taaatatttc gtttgcaaga 10620

attatgtggc tgtaaataaa gtttgtttac atgattggct gttatatgcg tttgccagag 10680

aaagaaaata taaatggttt attgatcctc aacctggtat gttgtataga cagcattcga 10740

ataatcaagt tggggcaaat gcttcattat taggtgctat caagcgtata aaaaaaatta 10800

aagattcttg gtatagaaat gaggtaaaaa aaatagtcaa tttaacttcg attgataata 10860

atttaataag tgaatgcctt atgagagata gatacttatc atctttaaaa ttggctttta 10920

tagttagaaa attaagacgt cgtcctcgag atcaatttgc tatgtttata gccttagttc 10980

tgggctggtt ttaacatcga tcgggagcac tatgctcatt ttaacaaaca atattatatt 11040

gctgattttt ataatctata cttttttggt atttataagt gcacttggag taaaattaaa 11100

tagcagaggg cttgataatt tttcgtgcct cattacagtc ctgacatttg ttatactcat 11160

tttcgctcgt acaggattag gtgttgacga aagtacatat ttagaatctt atcatttatta 11220

tgttcaagga ggtgaagcag attttgaata cggatttaat atcctttttt ggggagtcag 11280

actgttcggg gtaacagaat cttcattcaa taccatattc ccgttattga tattaacagc 11340

attatacttc gctgttactg tatcagttaa aaatcctttt agatcttttg tattaatatt 11400

tacaatattt tcttctttct ttctggattt ttcatttaat gcatataggc aggggctttc 11460

attctggttt gttctacttg ccatagaatc acatctgtct gggaataggt ggaaatttat 11520

agtttattgc ttaattggac ttggtttcca ctggtcagca gcagttgttt tattcatgct 11580

tttcctaaca agatttttat ctacaaggtt agcgatattc tgtaatattt ttctattttt 11640

cttaacgcta gtggcagcaa tgatgccatt acatgtactt tcaatgcttg taacgatgat 11700

acaatacttg ccattaaact catcttactt gcaaaaagtt attttttatc ttactacaat 11760

taagtcttcg ttttacgatt taaatttctt tggacgtgct ccattaataa tttatgcgct 11820

tattcttatt tctctcttgg ctatatatag aaaactatta ccaattgtgc aatttaaatt 11880

gttaattctt ttattagtct acagtgtgtt gttacttgag atgtcataca gttttagaaa 11940

ttactactgg gtgctaccat ttactccatt tattatggcg aaaattttga gttgttactc 12000

taataagaaa aaggtaatgg taatgtatac tatgttttct acagggttgt ggtctctatc 12060

aatagctagt ttctattcat ttcctatact atctatgata tttcagtgaa atcatataat 12120

aatatggttc attataaata cttttttggt taataatatt ttatataaac tgtgcattaa 12180

cgcacggtga ccacccatga caggagtaaa caatgtcaaa gcaacagatc ggcgtcgtcg 12240

gtatggcagt gatggggcgc aacctagcgc tcaacatcga aagccgtggt tataccgtct 12300

ctattttcaa ccgctcccgt gagaagacgg aagaagtgat tgccgaaaat ccaggcaaga 12360

aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg cctcgtcgca 12420

tcctgttaat ggtgaaagca ggcgcaggca cggatgctgc tactgattct cccaaaccat 12480

acctcgataa aggcgacatc areattgatg gtggtaacac cttcttccag gacaccattc 12540

gtcgtaaccg tgagctttct gctgaaggct ttaacttcat tggtaccggt gtctctggag 12600

gcgaagaagg tgcgctgaaa ggtccttcca ttatgcctgg tgggcagaaa gaagcctatg 12660

aactggttgc gccgatcctg accaaaatcg ccgcagcggc tgaagacggt gagccatgcg l2720

ttacctatat cggtgccgat ggcgcaggcc actatgtgaa gatggttcac aatggtattg 12780

aatacggaga tatgcaactg attgctgaag cccactctct gcttaaaggt ggcccgaacc 12840

tcaccaacga agaactggct cagacctcta ccgagtggaa caacggtgaa ctgagcagtt 12900

acctgatcga catcaccaaa gatatcttca ccaaaaaaga tgaagacggc aactacctgg 12960

ttgatgtgat tctggatgaa gcggctaaca aagglaccgg taaatggacc agccagagcg 13020

cgctggatct cggcgaaccg ctgtcgctaa ttacegagtc tgtgtttgct cgttatatat 13080

cttctctgaa agatcagcgc gttgccgcgt ctaaagttct ctctggtccg caagcgcagc 13140

cagcaggcga caaagctgag tttatcgaga aagttcgtcg tgcgctgtat ctgggcaaaa 13200

tcgtttccta cgctcagggc ttctctcagc tgcgcgcggc gtctgaagag tacaactggg 13260

atctgaacfa cggcgaaatc gcgaagattt tccgtgctgg ctgcatcatc cgtgcgcagt 13320

tcctacagaa aalcaccgat gcttatgccg aaaatccgca gatcgctaac ctgctgctgg 13380

ctccgtactt taaacaaatt gccgatgatt accagcaggc gctgcgtgat gtcgttgctt 13440

atgcggtaca gaacggtatc ccggttccga ccttcgccgc tgcggttgca tattatgaca 13500

gctaccgttc cgctgttctg cctgcgaacc tgatccaggc acagcgtgac ta 13552

Table 1 Escherichia coli O15O type O antigen gene cluster in the erythrotransferase gene and oligosaccharide unit processing gene and primers and PCR data therein Gene Function base position of gene forward primer reverse primer length of PCR product The number of groups that produced the correct size bands PCR annealing temperature (°C) wxya transferase 5632-7134 5706-5721 6458-6475 770bp 0 ^* 50 5719-5736 6681-6698 980bp 0 ^* 52 5978-5995 6789-6806 829bp 0 ^* 54 orf8 Glycosyltransferase 8307-9197 8681-8699 9169-9185 505bp 0 ^* 48 8418-8435 8995-9012 595bp 0 ^* 48 8333-8350 9050-9067 735bp 0 ^* 48 orf9 Glycotransferase 9184-10098 9397-9413 9882-9899 503bp 0 ^* 50 9337-9354 9899-9916 580bp 0 ^* 54 9489-9506 10035-10051 563bp 0 ^* 54 orf10 before transglycosylation 10098-10994 10302-40319 10852-10869 568bp 0 ^* 50 10193-10310 10954-10971 679bp 0 ^* 44 10377-10394 10973-10989 613bp 0 ^* 48 Wzy polymerase 11012-12109 112l7-11234 11985-12002 786bp 0 ^* 52 11225-11241 12035-12052 828bp 0 ^* 53 11172-11189 11960-11977 806bp 0 ^* 54

^* A correct band was obtained in E. coli Ol50 type

Table 2 166 strains of Escherichia coli and 43 strains of Shigella and their sources

Group number Strains contained in this group Source

1 Wild-type E. coli O1, O2, O3, O4, O10, O16, O18, O39 IMVS ^a

2 Wild-type Escherichia coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS

3 Wild-type Escherichia coli O102, O109, O119, O120, O121, O125, O126, O137 IMVS

4 Wild-type Escherichia coli O138, O139, O149, O7, O5, O6, O11, O12 IMVS

5 Wild-type Escherichia coli O13, O14, O15, O17, O19ab, O20, O21, O22 IMVS

6 Wild-type Escherichia coli O23, O24, O25, O26, O27, O28, O29, O30 IMVS

7 Wild-type Escherichia coli O32, O33, O34, O35, O36, O37, O38, O42 IMVS

8 Wild-type Escherichia coli O43, O44, O45, O46, O50, O51, O52, O53 IMVS

9 Wild-type Escherichia coli O54, O55, O56, O57, O58, O59, O60, O61 IMVS

10 Wild-type Escherichia coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS

11 Wild-type Escherichia coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS

12 Wild-type Escherichia coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS

13 Wild-type Escherichia coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS

14 Wild-type Escherichia coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS

15 Wild-type Escherichia coli O123, O165, O166, O167, O168, O169, O170, O171 See b

16 Wild-type Escherichia coli O172, O173, O127, O128, O129, O130, O131, O132, See c

17 Wild-type Escherichia coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS

18 Wild-type Escherichia coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS

19 Wild-type Escherichia coli O153, 0154, O155, O156, O157, O158, O159, IMVS

20 Wild-type Escherichia coli O160, O161, O163, O8, O9, O124, O111 IMVS

21 Wild-type Escherichia coli O103, O104, O105, O106, O107, O108, O110 IMVS

22 Shigella baumannii serotypes B4, B5, B6, B8, B9, B11, B12, B14 See d

23 Shigella baumannii serotypes B1, B3, B7, B8, B10, B13, B15, B16, B17, B18 See d

24 Shigella dysenteriae serotypes D1, D2, D3, D4, D5, D6, D7, D8 See d

25 Shigella dysenteriae serum D9, D10, D11, D12, D13 See d

26 Shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5(v:7)F5(v:4) See d

27 Shigella terminalis D5, DR See d

a. Institute of Medical and Veterinary Science, Anelaide, Australia

b. O123 from IMVS; the rest from Statens Serum Institut, Copenhagen, Denmark

c. 172 and 173 from Statens Serum Institut, Copenhagen, Denmark, the rest from IMVS

d. Institute of Epidemiology, Chinese Academy of Preventive Medicine

Table 3 Escherichia coli O150 type O antigen gene structure diagram

Table 4 Escherichia coli O150 type O antigen gene cluster gene position

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgaactgcg cgtgaagcgt 120

caactactgg cggaagttca gtccatctgc ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttgtgtg cgcgacccgc cattggtgac 240

aatccatttg tcgtggtgct gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgttg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccactg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtaggt cgctatgtgc tttctgccga tatttggccg 540

gaactggaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgctattgcc 600

gagctggcga aaaaacaatc cgttgatgtc atgctgatga ccggtgacag ttacgactgc 660

ggcaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gcctacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg tattgagaag ttgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt taacaagaaa attataacgg cagtgaagat ttgcagcaaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa accatcagaa tagcaacgag ttagcagtag 900

ggtttttc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc ataggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

or orf1 start

tcaaactgag agccgcttat ttcacagcat gctctgaagt aatatggaat aaattaa gtg 1140

aaaatacttg ttactggtgg cgcaggattt attggttctg ctgtagttcg tcacattta 1200

aataatacgc aggatagtgt tgttaatgtc gataaattaa cgtacgccgg aaacctggaa 1260

tcacttgctg atgtttctga ttccgaacgc tatgtttttg aacatgcgga tatttgcgat 1320

gcagctgtaa tggcacggat ttttgctcag catcagccgg atgcagtgat gcacctggct 1380

gctgaaagcc atgttgaccg ttcaattaca ggtcctgcgg catttattga aaccaatatt 1440

gttggtacat atgtcctttt ggaagccgct cgcaattact ggtctgctct tgatagcgac 1500

aagaaaaata gcttccgttt tcatcatatt tctactgacg aagtctatgg tgatttgcct 1560

catcctgacg aggtaaataa tacagaagaa ttacccttat ttactgagac aacagcttac 1620

gcgccaagca gcccttattc cgcatccaaa gcatccagcg atcatttagt ccgcgcgtgg 1680

aaacgtacct atggtttacc gaccattgtg actaattgct ctaacaatta tggtccttat 1740

catttcccgg aaaaattgat tccattggtt attctcaatg ctctggaagg taaagcatta 1800

cctatttatg gtaaagggga tcaaattcgc gactggctgt atgttgaaga tcatgcgcgt 1860

gcgttatata ccgtcgtaac cgaaggtaaa gcgggtgaaa cttataacat tggtgggcac 1920

aacgaaaaga aaaacatcga tgtagtgctc actatttgtg atttgttgga tgagattgta 1980

ccgaaagaga aatcttaccg cgagcaaatt acttatgttg ccgatcgtcc gggacacgat 2040

cgccgttatg ccattgatgc tgagaagatt ggtcgcgaat tgggatggaa accacaggaa 2100

acgtttgaga gcgggattcg gaagacagtg gaatggtacc tgtccaatac aaaatgggtt 2160

orf1 ends, orf2 starts

ga taatg tga aaagtggtgc ctatcaatcg tggattgaac agaactatga gggccgccag 2220

taatgaatat cctccttttt ggcaaaacag ggcaggtagg ttgggaacta cagcgtgctc 2280

tggtaccttt gggtaatttg attgctcttg atgttcactc cactgattac tgtggtgatt 2340

ttagtaatcc tgaaggtgta gctgaaaccg taagaagcat tcggccggat attattgtca 2400

acgcagccgc tcacaccgca gtagacaaag cagaatcaga accgaagttt gcacaattac 2460

tgaacgcgac gagtgtcgaa gcgatcgcga aagcagccaa tgaagtcggc gcctgggtta 2520

ttcactactc tactgactac gtatttccgg ggaccggtga aataccatgg caggaggagg 2580

atgcaaccgc accgctaaat gtttacggtg aaaccaagtt agcgggagaa aaagcattac 2640

aagagcattg tgtgaagcac cttattttcc ggaccagctg ggtctatgca ggtaaaggaa 2700

ataacttcgc caaaacaatg ttacgtctgg caaaagagcg tgaagaattg gccgttatta 2760

acgatcagtt tggtgcgcca acaggtgctg agctgctggc tgattgtaca gcacatgcca 2820

ttcgtgtcgc attgaataaa ccggaagtcg caggcttgta ccatctggta gccagtggta 2880

ccacaacctg gtacgattat gctgcgctgg tttttgaaga ggcgcgcaaa gcagatattc 2940

cccttgcact caacaaactc aatgcggtac caacaacagc ctatcctaca tcagctcgtc 3000

gtccacataa ctctcgcctt aatacagaaa aatttcagca gaattttgcg cttgttttgc 3060

ctgactggca ggttggcgtg aaacgaatgc ttaacgaatt atttacgact acagcaattt 3120

orf2 terminates orf3 starts

aatagttttt gcatcttgtt cgtgatgatg gagcaagatg aattaaaagg aatgatgaaa 3180

tgaaaacgcg taaaggtatt attttagcgg tgggctctgg tactcgtctt tatcctgtga 3240

ctatggctgt cagtaaacag ctattaccta tttatgataa gccgatgatc tattacccgc 3300

tctctacact gatgttggcg ggtattcgcg atattttgat tatcagcacg ccacaggata 3360

ctcctcgttt tcaacaactg ctgggtgatg ggagccagtg ggggctaaat cttcactaca 3420

aagtgcaacc gagtccggat ggtcttgcgc aggcgtttat tatcggtgaa gagtttattg 3480

gtggtgatga ttgtgctttg gtacttggtg aataatctt ctacggtcac gacctgccta 3540

agttaatgga tgccgctgtt aacaaagaaa gtggtgcaac ggtatttgcc tatcacgtta 3600

atgatcctga acgctacggt gtcgttgagt ttgataaaaa cggtacggca ataagcctgg 3660

aagaaaaacc gttacaacca aaaagtaatt atgcggtaac tgggctttat ttctatgata 3720

acgacgttgt cgaaatggcg aaaaacctta agccttctgc ccgtggtgaa ctagaaatta 3780

ccgatattaa ccgtatttat atggaacagg ggcgtttatc tgttgccatg atgggacgtg 3840

gttatgcatg gttggacacg gggacacatc aaagtcttat tgaggcaagc aatttcatcg 3900

cgacaataga agaacgacag gggctgaaag tttcctgccc ggaagaaatt gcttaccgta 3960

aggggtttat cgatgctgag caggtgaaag tattagctga accgttgaag aaaaatgctt 4020

orf3 terminates orf4 starts

atggccagta cctgctaaaa atgattaaag gttattaata aaatgaacgt aattaaaaca 4080

gagattccag acgtattaat tttcgaaccg aaagtttttg gtgatgagcg tggtttcttt 4140

atggaaagct ttaatcagaa agttttcgaa gaagctgtag gacgtaaggt tgaatttgtt 4200

caggataacc attcgaagtc ttgtaaaggt gttttacgcg ggctgcatta tcagttagaa 4260

cccttatgcgc aagggaaatt ggtacgttgc gttgttggtg aggtttttga tgttgcggtt 4320

gatattcgta aatggtcacc tacctttggc aaatgggttg gggtgaattt atctgctgag 4380

aataagcgcc agttgtggat ccctgaagga tttgcacatg gttttttggt gctaagtgag 4440

acggcagaat ttgtttataa gacgacgaat tattatgcac ctgggtatga aggtagcatc 4500

atttggaatg ataaaatact atctattgat tggcctgagt taacactgga gtatattctt 4560

Orf4 ends, orf5 starts

tccgataaag atctaaaggc cccaattcta agtgaatcta tagagaatat t taa a atg tt 4620

tattagtaga gtaaaacgta aaattcaatc tttgataagc cttgagttga attttttaca 4680

cgattattgg aggtttaaaa aatattacac gaagggatct caagcttcga aagataagca 4740

gaaacttgaa tcttggatat tgcaagataa acatcgaata gaaaaagctt tctctttacc 4800

taagccaaaa cttggatttg gtaaagatgt aatacctcgt ctcattgaca atcttgttaa 4860

ttatagcagt aaatttggaa atgatcaggt atattatatt ggcttaggtg cattacaatc 4920

gtataagaga ttccacgacg aacaaaaatt tttatgcct gaattttat caaagaatat 4980

ttgtaaaata cggaatgatg attttctcga tccccgatgc aatgtcgctg gctattttaa 5040

aaaagatgat tcattagtta caggtaagat aacagtagaa tcattgatga acgaacggcg 5100

tagctgtcgg cattttgatg ttgatgaaag tcttaatatc ggagataaac ttttaaagga 5160

tgttacaaaa ctatctatta cagccccttc tgtttgtaat aagacaacatt ggcgcattca 5220

ctatttttca ggtgaattaa agaataaaat tcttagttat caaaatggaa attcaggatt 5280

tacagaaaac ataccttatg tagcagttat aacaagtgat cttagagcat tttattctac 5340

tgacgaaaga aaccagccct atacagacgg tgggatcttt gccatgaatt ttatgtatgc 5400

tctacaacac tatggtcttg cttcttgtcc attgaattgg tgtaattcat ctcacattga 5460

gaatgagttt agagaaacta agttgattcc tgattacgaa gtagtagtac ttgtggttgc 5520

ttttggctat ccaaataaag atgccttata tgctaaatca ccacgattgt ctctaaataa 5580

orf5 terminates orf6 starts

cctttacaca attaat tag g cgaaggccac ctttaaaggt ggctgacaat a atg aagaaa 5640

caaatcatta aaaatatttc agccaatttt actaatttga ttgttagtat tttattgga 5700

ctattattga cgcctttact cgtcaaaagt ttaggcattg aagtatatgg agttctaccg 5760

atagctttat ttttaacatt ttatatggga gtcattgctc aagcattaac tgcatctgta 5820

aatagatttt taattgagga ttttattaaa ggagcatata ctgaggcaag tgtcgtattt 5880

agtacatcgt ttattcttat tctagatat ctatctgtta ttagtattgg atttatttat 5940

ccaatattgc atataagtga tttttttaac ataccaccgg gatatgaaca tgatgcagtt 6000

cttctattct cgtgtgtaat attttcgttt gttattgcta tgatcagttc atcattgtca 6060

gtttctatgt atgcaaaaaa taggattgac ttgatgcaat atggaaacat tttaaggaat 6120

attackaaag tcttaatcat tataatgttt gtttatttat ctgaaataaa tttaacaaaa 6180

ataggaatat caattgtttt atctgagctg ttttatctaa tctatgtcat tataacttgg 6240

aaaatattga caccacaact ttctctgcag ataggatttt tcagaaagga taaggtaaag 6300

agattaacat ctttcagtgg ctggatattg tttgatcaaa ttgggtcaat tctttttctt 6360

aaaacagatc tgattttagt taattctcta agtggtagta aagctaatgg tgaatattca 6420

atagctacac aatttagtga tcttttgcgt tcacttgctg gtttggtagc tggtagtta 6480

ggtcctatga tggtaatttt atatgaagaa aagaaattag aagaacttac ttcattgacc 6540

attacatttg ttaaattcct aagcttgacg atagctattc ctattattat tctttgtgta 6600

tactctaaag aaattcttga gctttggtta ggttcggggt ttgtatttct ttcaccatta 6660

atatgtatcg taacgcttcc actgattatc aatttgggca cactgccaat attatcgatc 6720

aatattgcag tcaaaaaaat tcaagtgcct gcactcataa atttctttct tgcaatcgtt 6780

ggaattatag gctctgtagt attgttcaaa accactacat taggtttatta tgctgtagct 6840

ataagttatg tgattacatt aactattaag aactccatt tcataccatg gtatgcaaca 6900

attatattaa ataaaccaag gtatcttttt tttaaaactc atctaattac tatcatcttc 6960

tcttctctat tttttatttt tatatatata cttaaaagta taatgcagat tactaattta 7020

aatttgcttt ttgaattgat attggttggt tgctttggac ttctatctac atttcctttc 7080

orf6 ends, orf7 starts

tatagtaaag aggaaaggcg atcatttatg ttaatgctca agaggaaagt gtaataatgg 7140

attcgactca gaaaataggc gttctgaatt tccagtattc ggatcataat tatggtgctg 7200

ttttacaagc cgcagcaata gaacaatttc tgaaaagcaa tggatataat gctgaacata 7260

tagactatat atctagtcca gagataaagg gtaatagggt aattatacaa ctaaaatgta 7320

tattgaaaaa attgggacta tctgattctg taagaaaaat gcttggaaaa caagtccact 7380

tgacacctgt ggtcacaaac gaaaaagtat ttgaggaatt tagaaaaaat tggttggtga 7440

gaacgaagtg tttcagaaac tttgaagaat taaaaaaaac tgattttaat tttaaagcgg 7500

ttatagttgg gagtgatcag gtttggcgac catctcaatt tactcggttt tccgattata 7560

aggtatattt cttaagcttt ttaccttcgc aggttaagaa aatatcttt gcagcaagtt 7620

ttggtattga taagtgggaa gttagtgatg ctaacgtcac gaacgaaata aaaaaatata 7680

ttcaagattt caatgctgtt tcagtcagag aagatagtgg cgttaatatt tgtgcaacca 7740

tttttaatgt cgacgctgaa catgttcttt atcctacttt actggttggt agtgactttt 7800

ttaacagtat aatagctaag gaaatacgtg ctgatgctta ttacgatgct atagtttt 7860

acaaacttga tattgatcaa cattttatag attcagttac tatactaggt agaaagaaat 7920

gcaagccagt aaaaaatatt tattacaacc aagtagatgg ccgaaatgag tattattctg 7980

ttcctgaatg gttaagcaat attaagaatg ctaattttgt tatcacagat tcttttcatt 8040

gtgtatgttt ttgtcttctt tttcaaaaag agtttttatg ctgtgtgaat gaaagtagag 8100

gattatctag attgcaaagt ttacttggaa tgcttgagct tactgatcgt atttgctcgt 8160

caaaagatga ttttgataat aagttaaatt cgttaaagcc aatagactat tctaaagtgg 8220

orf7 termination

gcgatatatt gaataagcat agagatattt ctaaggcgtt tttattaagt aatttatctt 8280

orf8 start

gagtttgagt gtgggttaaa tattatga atgcaaataa tagaaatgca tttccgatca 8340

ttgttatgac tcgcaacgaa ggcgccgact tacaacgttg cgttgattct atattacata 8400

ctgtatctat agatgtacat atatatatca tagacaataa ttctgatgat ttatcacata 8460

ttcagatcct tgctaacctt gaagacctga atcgtaaaaa tttaacagtt ataagaaata 8520

caaggaattt atggatttta ggtgtaaata acacactatt gaaaatcaag aaaaatcatg 8580

atactaaata ttttattctc actgatggtg atatagactt tcatggtttt aaagaagaaa 8640

gttgttggtt aacatattta atcaataaaa tggaatccaa cgtgataatc ggtaagctcg 8700

gaatttctct ggattggtca tttctggcct caaaaacgga gatgaaagat atttaccggc 8760

aagaacaaaa tctctataat gaaagaagaa aaatcggtga tctttatata tctgccgttg 8820

acacgacagt agccattatt cgttgggatt ggtctataga gcgtaaagct ttaattttatc 8880

ctgaccatat gaggtattta cgtccagaac tgtactcttg ccgtactaaa cgtggtttaa 8940

cagtagagca tttaggatgg tatagatacc ttgataactc cctgtctacg caaagtataa 9000

actcgaaagt tttttgtttt acaattgttg gtggtactgt taaacctgag gttttaaaac 9060

tggctagtaa accctatcag tatttccata ttttgttttc aaaacctttc gctatgcttt 9120

ggtatttcag acgatatttt aaacttttta tatattttat tactaaaggg agacgtggat 9180

orf9 start orf8 end

ttgatggaca gtcataataa taagttatca aaacgtattc cgtcacctgc actacgttgt 9240

tatgcagtaa tagttacatt taaccctgat attaataagg ttagaagttt aatagatatt 9300

ctacaaaaga attttatcac cgcagttgta gtagataata cgattggagc aataaattat 9360

ccattctcat gtcatgttat taatttaggt gataatttag gtattgccac agcacaaaat 9420

aaaggcatag aatattgttt gactaaacaa gctgaagcta tctggttttt tgatcaggac 9480

agcgtaatca cttcttcatt tgtcgaaaga tttctatcta ctgtccatga aaatcctcat 9540

gataagatct ttgctcctgt gttctgggat gagaagaaag ggtttgaata tgcaattaca 9600

aatatagatt caaagggcaa cagacaaaag ttgttatcat catcttttcc atcggatttt 9660

tatagttcga ttgttatttc atcagggtgc ttaataaaaa gtgatttgct gatgaaaatt 9720

ggtccaatgc tagatttttt atttatagat tacgttgata cggaatggtg tttacgtgct 9780

ttctattatg gctacaaagt tcacataatc aaaaatgcaa ggatgaaaca ttctataggt 9840

gataacacta tttctttaat cggttttaat gtaccaattc actctcctct tcgacgttat 9900

tatcgaataa gaaatgcatt tttattgatt aggtttaagc atatcccaaa aaaactagca 9960

tttagagaag tgatattttg ttgctgccat caattaataa ttatcctgac ccaagaaaaa 10020

aaactggcat atgttaagta ttttttacgg gctgtatatg atggccttag aaataaaact 10080

  orf9 ends, orf10 starts

gggaagtttg taaactaatg agttttgtca tgaagccaaa ggtattagtg ctagttgcag 10140

tttataatgg tgaaaagtgg ttaatagagc aacttacatc tataataaat caaaaaaatg 10200

ttgatattga tgtgattatt aatatcgata aatcaactga ttcttcatac aaattgtgtg 10260

agaattttat tgcagataat gtcaagatac taccgtacgg agctgtattt ggcggtgcag 10320

gtgccaattt ttatcatcta atccatgaat ctgaattttc tggttacgat tacattgcat 10380

tttccgatca agacgatatt tggtttgaaa ctaaaatata tgatgccatc aataggatgc 10440

atgattatga tgcatattca agtaatgtaa tagcattctg ggaaaatggc aaacaatgtc 10500

taattgagaa atctcaacca cagacagaat ttgattattt ttttgaggca gcaggacccg 10560

ggtgcactta tatagtgaga caggaactag cactcgcatt taaatatttc gtttgcaaga 10620

attatgtggc tgtaaataaa gtttgtttac atgattggct gttatatgcg tttgccagag 10680

aaagaaaata taaatggttt attgatcctc aacctggtat gttgtataga cagcattcga 10740

ataatcaagt tggggcaaat gcttcattat taggtgctat caagcgtata aaaaaaatta 10800

aagattcttg gtatagaaat gaggtaaaaa aaatagtcaa tttaacttcg attgataata 10860

atttaataag tgaatgcctt atgagagata gatacttatc atctttaaaa ttggctttta 10920

tagttagaaa attaagacgt cgtcctcgag atcaatttgc tatgtttata gccttagttc 10980

orf10 ends orf11 starts

tgggctggtt ttaacatcga tcgggagcac tatgctcatt ttaacaaaca atattatatt 11040

gctgattttt ataatctata cttttttggt atttataagt gcacttggag taaaattaaa 11100

tagcagagggg cttgataatt tttcgtgcct cattacagtc ctgacatttg ttatactcat 11160

tttcgctcgt acaggattag gtgttgacga aagtacatat ttagaatctt atcatttatta 11220

tgttcaagga ggtgaagcag attttgaata cggatttaat atcctttttt ggggagtcag 11280

actgttcggg gtaacagaat cttcattcaa taccatattc ccgttattga tattaacagc 11340

attatacttc gctgttactg tatcagttaa aaatcctttt agatcttttg tattaatatt 11400

tacaatattt tcttctttct ttctggattt ttcatttaat gcatataggc aggggctttc 11460

attctggttt gttctacttg ccatagaatc acatctgtct gggaataggt ggaaatttat 11520

agtttattgc ttaattggac ttggtttcca ctggtcagca gcagttgttt tattcatgct 11580

tttcctaaca agatttttat ctacaaggtt agcgatattc tgtaatattt ttctattttt 11640

cttaacgcta gtggcagcaa tgatgccatt acatgtactt tcaatgcttg taacgatgat 11700

acaatacttg ccattaaact catcttactt gcaaaaagtt attttttatc ttactacaat 11760

taagtcttcg ttttacgatt taaatttctt tggacgtgct ccattaataa tttatgcgct 11820

tattcttatt tctctcttgg ctatatatag aaaactatta ccaattgtgc aatttaaatt 11880

gttaattctt ttattagtct acagtgtgtt gttacttgag atgtcataca gttttagaaa 11940

ttactactgg gtgctaccat ttactccatt tattatggcg aaaattttga gttgttactc 12000

taataagaaa aaggtaatgg taatgtatac tatgttttct acagggttgt ggtctctatc 12060

orf11 termination

aatagctagt ttctattcat ttcctatact atctatgata tttcagtgaa atcatataat 12120

aatatggttc attataaata cttttttggt taataatatt ttatataaac tgtgcattaa 12180

cgcacggtga ccacccatga caggagtaaa caatgtcaaa gcaacagatc ggcgtcgtcg 12240

gtatggcagt gatggggcgc aacctagcgc tcaacatcga aagccgtggt tataccgtct 12300

ctattttcaa ccgctcccgt gagaagacgg aagaagtgat tgccgaaaat ccaggcaaga 12360

aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg cctcgtcgca 12420

tcctgttaat ggtgaaagca ggtgcaggca cggatgctgc tattgattct ctcaaaccat 12480

acctcgataa aggcgacatc atcattgatg gtggtaacac cttcttccag gacaccattc 12540

gtcgtaaccg tgagctttct gctgaaggct ttaacttcat tggtaccggt gtctctggag 12600

gcgaagaagg tgcgctgaaa ggtccttcca ttatgcctgg tgggcagaaa gaagcctatg 12660

aactggttgc gccgatcctg accaaaatcg ccgcagtggc tgaagacggt gagccatgcg 12720

ttacctatat cggtgccgat ggcgcaggcc attatgtgaa gatggttcac aatggtattg 12780

aatacggaga tatgcaactg attgctgaag cctattctct gcttaaaggt ggcctgaacc 12840

tcaccaacga agaactggct cagaccttta ccgagtggaa taacggtgaa ctgagcagtt 12900

acctgatcga catcaccaaa gatatcttca ccaaaaaaga tgaagacggt aactacctgg 12960

ttgatgtgat tctggatgaa gcggctaaca aaggtaccgg taaatggacc agccagagcg 13020

cgctggatct cggcgaaccg ctgtcgctaa ttaccgagtc tgtgtttgct cgttatat 13080

cttctctgaa agatcagcgc gttgccgcgt ctaaagttct ctctggtccg caagcgcagc 13140

cagcaggcga caaagctgag tttatcgaga aagttcgtcg tgcgctgtat ctgggcaaaa 13200

tcgtttctta tgctcagggc ttctctcagc tgcgtgcggc gtctgaagag tacaactggg 13260

atctgaacta cggcgaaatc gcgaagattt tccgtgctgg ctgcatcatc cgtgcgcagt 13320

tcctacagaa aatcaccgat gcttatgccg aaaatccgca gatcgctaac ctgctgctgg 13380

ctccgtactt taaacaaatt gccgatgatt accagcaggc gctgcgtgat gtcgttgctt 13440

atgcggtaca gaacggtatc ccggttccga ccttcgccgc tgcggttgca tattatgaca 13500

gctaccgttc cgctgttctg cctgcgaacc tgatccaggc acagcgtgac ta 13552

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still belong to within the scope of the technical solutions of the present invention.

Claims (2)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O150 type is characterized in that described oligonucleotide is to being: the Nucleotide of 5706 to 5722 bases among the SEQ ID NO:1 and the Nucleotide of 6458 to 6475 bases; The Nucleotide of 5719 to 5736 bases among the SEQ ID NO:1 and the Nucleotide of 6681 to 6698 bases; The Nucleotide of 5978 to 5995 bases among the SEQ ID NO:1 and the Nucleotide of 6789 to 6806 bases; The Nucleotide of 8681 to 8699 bases among the SEQ ID NO:1 and the Nucleotide of 9169 to 9185 bases; The Nucleotide of 8418 to 8435 bases among the SEQ ID NO:1 and the Nucleotide of 8995 to 9012 bases; The Nucleotide of 8333 to 8350 bases among the SEQ ID NO:1 and the Nucleotide of 9050 to 9067 bases; The Nucleotide of 9397 to 9413 bases among the SEQ ID NO:1 and the Nucleotide of 9882 to 9899 bases; The Nucleotide of 9337 to 9354 bases among the SEQ ID NO:1 and the Nucleotide of 9899 to 9916 bases; The Nucleotide of 9489 to 9506 bases among the SEQ ID NO:1 and the Nucleotide of 10035 to 10051 bases; The Nucleotide of 10302 to 10319 bases among the SEQ ID NO:1 and the Nucleotide of 10852 to 10869 bases; The Nucleotide of 10293 to 10310 bases among the SEQ ID NO:1 and the Nucleotide of 10954 to 10971 bases; The Nucleotide of 10377 to 10394 bases among the SEQ ID NO:1 and the Nucleotide of 10973 to 10989 bases; The Nucleotide of 11217 to 11234 bases among the SEQ ID NO:1 and the Nucleotide of 11985 to 12002 bases; The Nucleotide of 11225 to 11241 bases among the SEQ ID NO:1 and the Nucleotide of 12035 to 12052 bases; The Nucleotide of 11172 to 11189 bases among the SEQ ID NO:1 and the Nucleotide of 11960 to 11977 bases.

2, the application of the described Nucleotide of claim 1 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray confession detection intestinal bacteria O150 type as probe.