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Nucleotide specific against o-antigen of colibacillus 0150

A technology of Escherichia coli and nucleotides, which can be used in the determination/inspection 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

AI Technical Summary

Problems solved by technology

In 1996, Paton, A.W et.al identified a toxin-producing serotype of E.coli O11l with an oligonucleotide specific to the O-antigen of E.coli O111 derived from the wbdI gene ["Molecular microbiological investigation ofan 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.al's use originated from wbdI There are false positive results in the method of gene oligonucleotide identification of E.coli O111 serotype

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0045]Escherichia coli O150 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; 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 electrophor...

Embodiment 2

[0046] The O-antigen gene cluster of Escherichia coli O150 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. Embodiment 3: construct O-antigen g...

Embodiment 3

[0046] The O-antigen gene cluster of Escherichia coli O150 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. Embodiment 3: construct O-antigen g...

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PUM

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Abstract

A nucleotide specific to the O-antigen of Escherichia coli O150 is a complete nucleotide sequence of the genom for controlling the synthesis of O-antigen in Escherichia coli O150, such as the separated nucleotide shown by SEQ ID No:1. It has 13552 bases, or has one or more inserted, deletional, or substituted bases, 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. Said oligonucleotide can be used to detect Escherichia coli O150.

Description

technical field [0001] 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 [0002] Escherichia coli O150 was first detected in sick 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 cattle and other livestock Enterotoxigenic Escherichia coli (Enterotoxigenic E.coli, ETEC), the risk of its potential explosive epidemic is very high [Danbara H, Komase K, Arita H, et al.Infect Immun, 1988, 56( 6): 1513-1517.], a method that can q...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C12Q1/04C12Q1/68G01N33/569
Inventor 王磊郭宏杰冯露
Owner NANKAI UNIV
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