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Nucleotide peculiar to 0-antigen of 110 type bacillus coli

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: 2005-01-12
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 ["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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the extraction of genome:

[0054] Escherichia coli O110 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 O110 by PCR:

[0056] Using the genome of Escherichia coli O110 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 sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCT GGATTA AGT TCG C); use Boehringer Mannheim’s Expand Long Template PCR method to amplify the O-antigen gene cluster, and the PCR reaction procedure is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds , 60 ° C 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 product...

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...

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PUM

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Abstract

The invention provides a nucletoide which is specific to O-antigen of Escherichia coli type O110, it is a nucleotide complete sequence of gene cluster for controlling synthesis of O-antigen in escherichia coli type, as the separated nucleotide shown by SEQ ID No:1, its total length has 9800 bases; or the nucleotide of SEQ ID No:1, which has one or several inserted, deleted or substituted bases, and retains the function of the described separated nucleotide at the same time; it also includes the oligonucleotide of the glycosyltransferase gene and oligosaccharide unit treatment gene originated from O-antigen gene cluster of Escherichia coli type O110. Said invention utilizes PCR to verify that the oligonucleotide has high specificity to O-antigen of escherichia coli type O110, and said invention also discloses the method for detecting and identifying Escherichia coli type O110 in human body.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O110 type (Escherichia coli O110), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O110 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O110 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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IPC IPC(8): C07H21/00C12N15/31C12P19/34C12Q1/68
Inventor 王磊李雅玥冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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