Nucleotide specific to O antigen of 078 type bacillus coli

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

Inactive Publication Date: 2007-12-19
TIANJIN BIOCHIP TECH CO LTD
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  • 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

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  • Nucleotide specific to O antigen of 078 type bacillus coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: the extraction of genome:

[0052] Escherichia coli O78 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 in...

Embodiment 2

[0053] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O78 by PCR:

[0054] Using the genome of Escherichia coli O78 as a template, its O-antigen gene cluster was amplified by Long PCR. First, design upstream primers (#w1-1098-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 Mannheirn’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 spe...

Embodiment 3

[0055] Embodiment 3: construct O-antigen gene cluster library:

[0056] 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 0.1 M 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 100mMDTT and 5 units...

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Abstract

This invention provides a specific nucleotide O-antigen for Escherichia coli 078, a complete sequence of nucleotide which controls the synthesis of O-antigen, e.g. as show in the SEQ ID NO: 1 the nucleic acid for separation has 12655 alkali bases, or compromising one or several insert, lost, or substitutes alkali bases, while keeping the function of the nucleic acid; It also compromises oligonucleotide which processing gene in the single-surge unit of O-antigen gene group form Escherichia coli; this invention proves that the oligonucleotide has a high specific function on Escherichia coli 078 by PCR, and discloses a method of identifying and detecting Escherichia coli by the oligonucleotide.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O78 type (Escherichia coli O78), in particular to the oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O78 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O78 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): C12N15/11C12Q1/68C12N15/10C12N15/31
Inventor 王磊韩巍青冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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