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Nucleotide specific for escherichia coli 0123 O-antigen

A technology of Escherichia coli and nucleotides, applied in the direction of sugar derivatives, biochemical equipment and methods, applications, etc., can solve problems such as false positives

Inactive Publication Date: 2006-04-05
TIANJIN BIOCHIP TECH CO LTD
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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 O111 using oligonucleotides specific to the O-antigen of E.coli O11l derived from the wbdI gene ["Molecularmicrobiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dryfermented sausage contaminated with Shiga-like toxin producing Escherichiacoli".J.Clin.Microbiol.34:1622-1627], but later studies showed that Paton, A.W et.al's use originated from the wbdI gene The oligonucleotide method for identifying the serotype of E.coli O111 has false positive results

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: the extraction of genome:

[0053] Escherichia coli O123 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 μl of 50 mM Tris-HCl (pH 8.0) and 10 μl of 0.4M EDTA, incubated at 37° C. for 20 minutes, and then 10 μl of 10 mg / mL lysozyme was added for further incubation for 20 minutes. Then add 3 μl 20 mg / mL proteinase K, 15 μl 10% SDS, incubate at 50° C. for 2 hours, then add 3 μl 10 mg / 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 precipitated with 2 times the volume of ethanol, the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended i...

Embodiment 2

[0054] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O123 by PCR:

[0055] The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O123 as a template. First, design the upstream primer (#wl-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T) based on the JUMPStart sequence often found in the promoter region of the O-antigen gene cluster, and then design according to the gnd gene downstream of the O-antigen gene cluster Downstream primer (#1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C); use Boehringer Mannheim’s ExpandLong Template PCR method to amplify the O-antigen gene cluster. The PCR reaction procedure is as follows: pre-denaturation at 94°C for 2 minutes; then 94°C Denaturation for 10 seconds, annealing at 55°C for 15 seconds, and extension at 68°C for 15 minutes, for 30 cycles. 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 ...

Embodiment 3

[0056] Embodiment 3: construct O-antigen gene cluster library:

[0057] 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, 0.9μl 0.1M MnCl 2 , 1 μl of 1 mg / mL DNaseI diluted 1:2000, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the size of DNA fragments between 1.5kb-3kb, then add 2μl 0.1M EDTA to stop 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 18 μl of water. Then add 2.5 μl dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25μl 100mM DTT and 5 units...

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Abstract

The invention provides a nucleotide specific for Escherichia coli 0123 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 17084 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 0123.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O123 type (Escherichia coli O123), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O123 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O123 in human body and 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, O- The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12P19/34C07H21/00C12Q1/68
Inventor 王磊冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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