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Nucleotide specific for escherichia coli 0156 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

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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 0156 O-antigen

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Experimental program
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Embodiment 1

[0043] Embodiment 1: the extraction of genome:

[0044]Escherichia coli O156 was cultured overnight at 37°C in 5 mL of LB medium, and the cells were collected by centrifugation. The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation 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 For residual phenol, the supernatant was used to precipitate DNA with 2 times the volume of ethanol, and the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended in 30ul T...

Embodiment 2

[0045] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O156 by PCR:

[0046] Using the genome of Escherichia coli O156 as a template, its O-antigen gene cluster was amplified by Long PCR. 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 15 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 5 tubes of long ...

Embodiment 3

[0047] Embodiment 3: construct O-antigen gene cluster library:

[0048] 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 is 300ng PCR purified product, 0.9ul 0.1M MnCl 2 , 1 ul of 1 mg / 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 1.5kb-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), 1.25ul 100mM DTT and 5 units ...

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Abstract

The invention provides a nucleotide specific for Escherichia coli 0156 O-antigen, which is the total nucleotide sequence of the gene cluster for controlling the synthesis of O-antigens in Escherichia coli 0156, e.g. isolated nucleotide represented by SEQ ID No:1, with overall length of 15390 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 0156. The invention proves the high specifity of oligonucleotides for Escherichia coli 0156 O-antigen through PCR. A method for detecting and identifying Escherichia coli 0156 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 the gene cluster controlling O-antigen synthesis in Escherichia coli O156 type (Escherichia coli O156), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O156 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O156 in the human body and the environment and identify the O-antigens in these pathogenic bacteria. Background technique [0002] 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,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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