Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Nucleotide specific for escherichia coli 0149 O-antigen

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: 2005-01-26
TIANJIN BIOCHIP TECH CO LTD
View PDF0 Cites 1 Cited by
  • 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 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the extraction of genome:

[0054] Escherichia coli O145 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 ...

Embodiment 2

[0055] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O145 by PCR:

[0056] Using the genome of Escherichia coli O145 as a template, its O-antigen gene cluster was amplified by Long PCR. First, the upstream primer w1-1098 (5'-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T-3') was designed according to the JUMPStart sequence often found upstream of the O-antigen gene cluster, and then according to the gnd downstream of the O-antigen gene cluster Gene design downstream primer w1-913 (5'-TAG TCG CGT GNCCCT GGA TTA AGT TCG C-3'); use Boehringer Mannheim's Expand LongTemplate PCR method to amplify the O-antigen gene cluster, and the PCR reaction procedure is as follows: at 94°C Pre-denatured for 2 minutes; then denatured at 94°C for 10 seconds, annealed at 60°C for 15 seconds, and extended at 68°C for 15 minutes, thus performing 30 cycles. Finally, continue extending at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to ...

Embodiment 3

[0057] Embodiment 3: construct O-antigen gene cluster library:

[0058] 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 100mMDTT and 5 units o...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a nucleotide specific for Escherichia coli 0145 O-antigen, which is the total nucleotide sequence of the gene cluster for controlling the synthesis of O-antigens in Escherichia, e.g. isolated nucleotide represented by SEQ ID No:1, with overall length of 16932 bases, or nucleotide of SEQ ID No:1 including one or more inserted, deleted or substituted bases and sustaining the functions of the isolated nucleotides, it also includes the oligonucleotides of glycosyl transferase genes and oligosaccharide unit treatment genes in the O-antigen gene cluster originated from Escherichia 0145. The invention proves the high specifity of oligonucleotides for Escherichia coli 0145 O-antigen through PCR. A method for detecting and identifying Escherichia 0145 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 O145 type (Escherichia coli O145), in particular to the oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O145 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O145 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 membr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C12N15/11C12N15/31C12P19/34C12Q1/68
Inventor 王磊冯露
Owner TIANJIN BIOCHIP TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com