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Nucleotide specific for escherichia coli 0134 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: 2007-05-16
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 O111 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 O134 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 100 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 ...

Embodiment 2

[0054] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O134 by PCR:

[0055] Using the genome of Escherichia coli O134 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 galF gene often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCT GGA TTAAGT TCG C); use Boehringer Mannheim’s Expand Long Template PCR method to amplify the O-antigen gene cluster, and the PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds , 55 annealing for 15 seconds, 68 ° C extension for 15 minutes, so that 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 specificity of the PCR products. Combine 5 tubes of longPCR products and purif...

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 2.5μl dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25μl 100mMDTT and 5 units o...

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PUM

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Abstract

The invention provides a nucleotide specific for escherichia coli 0134 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 7530 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 SEQ ID No:1, it also includes the oligosaccharide unit treatment genes in the O-antigen gene cluster originated from Escherichia coli 0134. The invention proves the high specifity of oligonucleotides for Escherichia coli 0134 O-antigen through PCR, a method for detecting and identifying Escherichia 0134 by means of the oligonucleotide according to The invention discloses 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 O134 type (Escherichia coli O134), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O134 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O134 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 membrane,...

Claims

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Application Information

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