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Nucleotide specific against 0-antigen of colibacillus 0107

A technology of Escherichia coli and nucleotides, which is applied in the determination/testing of microorganisms, sugar derivatives, biochemical equipment and methods, etc., and can solve problems such as false positives

Inactive Publication Date: 2003-09-17
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 ["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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Embodiment 1

[0045] Escherichia coli O107 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 residual phenol. The DNA was precipitated with 2 times the volume of ethanol in the supernatant, 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% agarose gel elect...

Embodiment 2

[0046] Using the genome of Escherichia coli O107 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 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 use Promega's Wizard PCR Preps purification kit to purify the PCR products. Emb...

Embodiment 3

[0046] Using the genome of Escherichia coli O107 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 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 use Promega's Wizard PCR Preps purification kit to purify the PCR products. Emb...

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PUM

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Abstract

A nucleotide specific to the O-antigen of Escherichia coli O107 is a complete nucleotide sequence of the genom for controlling the synthesis of O-antigen in Escherichia coli O107 such as the separated nucleotide shown by SEQ ID No.1. It has 13188 bases, or has one or more inserted, deletional or substituted base but keeps the function of said separated nucleotide. It also includes the oligonucleotide of glycosyltransferase gene and oligose unit treating gene in O-antigen genom. A method for using said oligonucleotide to detect Escherichia coli O107 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 O107 (Escherichia coli O107), particularly the oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O107 , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O107 in the human body and the environment and identify the O-antigens in these pathogenic bacteria. Background technique [0002] Escherichia coli O107 was first discovered in Finland in 1997 and can cause diarrhea in infants and tourists. It belongs to enterotoxigenic Escherichia coli, and its potential explosive epidemic is very dangerous [Keskimaki M, Saari M, Heiskanen T. Shiga toxin-producing Escherichia coli in finland from 1990 through 1997: prevalence and characteristics of isolates [J]. J Clin Microbiol, 1998, 36(12): 3641-3646], there is an urgent need for a metho...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12N15/11C12Q1/04C12Q1/68G01N33/569
Inventor 王磊郭宏杰冯露
Owner NANKAI UNIV
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