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Nucleotide specific for escherichia coli 054 O-antigen

A technology of Escherichia coli and nucleotides, applied in the direction of antibacterial drugs, antibody medical components, and microorganism determination/inspection, which can solve the problems of false positives and so on.

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

AI Technical Summary

Problems solved by technology

In 1996, 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 ["Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli". J.Clin.Microbiol.34:1622-1627], but later studies showed that Paton, A.W et.al's use was derived from wbdI gene The oligonucleotide method for identifying the serotype of E.coli O111 has false positive results

Method used

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  • Nucleotide specific for escherichia coli 054 O-antigen

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Experimental program
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Embodiment 1

[0044] Escherichia coli O54 was cultured overnight at 37°C in 5 mL of LB medium, and the cells were collected by centrifugation. The cells were resuspended with 500 ul of 50 mM Tris-HCl (pH 8.0) and 10 ul of 0.4M EDTA, incubated at 37° C. for 20 minutes, and then 10 ul of 10 mg / ml lysozyme was added for further 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 then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. The supernatant was used to precipitate DNA with 2 times 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 TE. Genomic DNA was detected by 0.4% aga...

Embodiment 2

[0045] Example 2: Amplification of the O-antigen gene cluster in E. coli O54 by PCR

[0046] The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O54 as a template. First, design the upstream primer (5'-ATT GTG GCT GCA GGG ATC AAA GAA ATC-3') based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primers based on the gnd gene downstream of the O-antigen gene cluster Primer (5'-TAG TCG CGC TGN GCC TGG ATT AAG TTCGC-3'). The O-antigen gene cluster was amplified using the Expand Long Template PCR method of Boehringer Mannheim. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 61°C for 30 seconds, and extension at 68°C for 15 minutes. Carry out 30 cycles in this way; finally, continue to extend at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to...

Embodiment 3

[0047] Example 3: Construction of O-antigen gene cluster library.

[0048] The first is the acquisition of the connection product:

[0049] A modified Novagen DNaseI shot gun method was used 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 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), 1.25ul 100mM ...

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Abstract

The invention provides a nucleotide specific for Escherichia coli 054 O-antigen, which is the total nucleotide sequence of the gene cluster for controlling the synthesis of O-antigens in Escherichia 054, e.g. isolated nucleotide represented by SEQ ID No:1, with overall length of 14062 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 wzx gines in the O-antigen gene cluster originating from the Escherichia 054, and genes and wzy genes having similar functions with wzx, and genes having similar functions with wzy genes or wzy genes. The invention proves the high specifity of oligonucleotides for Escherichia 054 O-antigen through PCR. A method for detecting and identifying Escherichia 054 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 O54 type (Escherichia coli O54), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O54 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O54 in the human body and the environment and identify the O-antigens in these pathogenic bacteria. Background technique [0002] Escherichia coli O54 type is a pathogenic bacterium, which was first isolated from young pigs suffering from diarrhea and edema in 1986 [Garabal JI.et.al. (1993) "Serogroups of Escherichia coliisolated from piglets in Spain". Vet Microbiol .48(1-2):113-23]. It can produce cytotoxic necrosis factor (cytotoxic necrotising factor, CNF), which belongs to the necrotic Escherichia coli (NTEC), and is also potentially pathogenic to humans, and ca...

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 冯露杨静华郭宏杰王磊
Owner NANKAI UNIV
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