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Nucleotide specific for escherichia coli 0141 O-antigen

A technology of Escherichia coli and nucleotides, applied in antibacterial drugs, antibody medical components, determination/inspection of microorganisms, etc., can solve problems such as false positives

Inactive Publication Date: 2004-11-17
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1935, Paton, A.W et.al identified a toxin-producing serotype of E.coli O111 using oligonucleotides 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".J.Clin.Microbiol.34:1622-1627], but later studies showed that Paton, A.W et. There are false positive results in the method of identifying the serotype of E.coli O111 by oligonucleotide of wbdI gene

Method used

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  • Nucleotide specific for escherichia coli 0141 O-antigen

Examples

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Embodiment 1

[0044] Example 1: Genome extraction.

[0045] E. coli type O141 was cultured overnight at 37°C in 5 mL of LB medium, and the cells were collected by centrifugation. Cells were resuspended with 500 ul of 50 mM Tris-HCl (pH 8.0) and 10 ul of 0.4 M EDTA, incubated at 37°C for 20 minutes, and then 10 ul of 10 mg / ml lysozyme was added for a further 20 minutes. Then, 3ul of 20mg / ml proteinase K and 15ul of 10% SDS were added and incubated at 50°C for 2 hours, and then 3ul of 10mg / ml RNase was added and incubated at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant and extract it 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, supernatant DNA was precipitated with 2 volumes of ethanol, DNA was rolled out with glass silk and DNA was washed with 70% ethanol, and finally DNA was resuspended in 30ul TE. Genomic...

Embodiment 2

[0046] Example 2: Amplification of the O-antigen gene cluster in E. coli type O141 by PCR.

[0047] The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O141 as a template. First design the upstream primers (#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 primers (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the gnd gene downstream of the O-antigen gene cluster 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 program 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 15 seconds and elongation at 68°C for 15 minutes. Finally, the extension was continued at 68°C for 7 minutes to obtain the PCR product, and the size a...

Embodiment 3

[0048] Example 3: Construction of O-antigen gene cluster library.

[0049] The first is the acquisition of ligation products: a modified Novagen DNaseI shot gun method is used to construct the O-antigen gene cluster library. The reaction system is 300ng PCR purified product, 0.9ul 0.1M MnCl 2 , 1ul1:2000 dilution of 1mg / ml DNaseI, the reaction was carried out at room temperature. The DNA fragment size was concentrated between 1.5kb-3kb after digestion for 10 minutes, and then 2ul of 0.1M EDTA was added to stop the reaction. Combine 4 tubes of the same reaction system, extract once with an equal volume of phenol, extract once with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixed solution, and extract once with an equal volume of ether After that, the DNA was precipitated with 2.5 volumes of absolute ethanol, washed with 70% ethanol, and finally resuspended in 18 ul water. Then add 2.5ul dNTP (1mM dCTP, 1mM dGTP, 1mM dTTP, 10mM dATP), 1.25ul 100mM DTT and 5 ...

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Abstract

The invention provides a nucleotide specific for Escherichia coli 0141 O-antigen, which is the total nucleotide sequence of the gene cluster for controlling the synthesis of O-antigens in Escherichia 0141, e.g. isolated nucleotide represented by SEQ ID No:1, with overall length of 15601 bases, or nucleotide of SEQ ID No:1 including one or more inserted, deleted or substituted bases and sustains the isolated nucleotide functions, it also includes the oligonucleotides of glycosyl transferase genes and oligosaccharide unit treatment genes in the O-antigen gene cluster originated from Escherichia coli 0141. The invention proves the high specifity of oligonucleotides for Escherichia coli 0141 O-antigen through PCR. A method for detecting and identifying Escherichia coli 0141 in human body and the environment by means of the oligonucleotide according to the invention is also disclosed.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of a gene cluster controlling O-antigen synthesis in Escherichia coli O141 type (Escherichic ccli O141), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O141 type , these oligonucleotides specific for O-antigen can be used to rapidly and accurately detect Escherichia coli O141 type in human body and environment and identify O-antigen in these pathogenic bacteria. Background technique [0002] The 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 is well studied: the nucleoside diphosphate monosaccharide is firstly transferred to a lipid molecule fixed on the inner membrane by glycosyltransferase, and then the oligosaccharide unit is synthesized on the inner side of the inner membrane, O- The...

Claims

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Application Information

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IPC IPC(8): A61K39/108A61P31/04C07H21/00C12N15/11C12N15/31C12Q1/68
CPCY02A50/30
Inventor 冯露孔庆科郭宏杰王磊
Owner NANKAI UNIV
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