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Nucleotide specific to O-antigen of 025 type b acillus coli

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: 2005-08-31
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 ["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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  • Nucleotide specific to O-antigen of 025 type b acillus coli

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Embodiment 1

[0052] Embodiment 1: the extraction of genome:

[0053] Escherichia coli O25 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

[0054] Embodiment 2: Amplify the O-antigen gene cluster in Escherichia coli O25 type by PCR:

[0055] Using the genome of Escherichia coli O25 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 long PCR products and purify ...

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

A nuclleoside specific to O-antigen of escherichia coli O25 is disclosed. It is the nucleotide shown by SEQ ID No.1 for controlling the synthesis of O-antigen in escherichia coli O25 or the nucleotide shown by SEQ ID No.1, which has one or more inserted, deleted or substituted bases, and contains also the oligonucleoside coming from oligose unit treating gene. A method for using said oligonucleoside to detect the verify escherichia coli O25 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 O25 type (Escherichia coli O25), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O25 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O25 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, O- ...

Claims

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Application Information

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