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Nucleotide peculiar to 0-antigen of 041 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 ["Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome 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.al's use was derived from wbdI gene The oligonucleotide method for identifying the serotype of E.coli O111 has false positive results

Method used

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  • Nucleotide peculiar to 0-antigen of 041 type bacillus coli

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1: Genome extraction.

[0054] Escherichia coli O41 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 ul of 50 mM Tris-HCl (pH 8.0) and 10 ul of 0.4M EDTA, incubated at 37° C. for 20 minutes, and then 10 ul of 10 mg / ml lysozyme was added for further 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 then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. The supernatant was used to precipitate DNA with 2 times 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 T...

Embodiment 2

[0055] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O41 by PCR

[0056] Using the genome of Escherichia coli O41 as a template, its O-antigen gene cluster was amplified by Long PCR. First, the upstream primers (5'-ATTGTG GCT GCA GGG ATC AAA GAA ATC-3') were designed according to the galF gene often found upstream of the O-antigen gene cluster, and then the downstream primers were designed according to the gnd gene downstream of the O-antigen gene cluster (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3'). The O-antigen gene cluster was amplified using the Expand Long Template PCR method of Boehringer Mannheim. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 61°C for 30 seconds, and extension at 68°C for 15 minutes. Carry out 30 cycles in this way; finally, continue to extend at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresi...

Embodiment 3

[0057] Example 3: Construction of 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 1kb-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 100mM DTT and 5 units...

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Abstract

The invention provides a nucleotide which is specific to O-antigen of Escherichia coli O41, it is a nucleotide complete sequence of gene cluster for controlling the synthesis of O-antigen in Escherichia coli O41, as the separated nucleotide shown by SEQ ID No:1, its total length has 15377 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 aligosacharide unit treatment gene (including wzx gene or gene whose function is similar to that of WZX and WZY gene or gene whose function is similar to that of wzy) originated from O-antigen gene cluster of Escherichia coli O41. Said invention utilizes PCR to verify that the oligonucleotide has high specificity to O-antigen of Escherichia coli O41, and said invention also discloses the method for detecting and identifying Escherichia coli O41 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 O41 (Escherichia coli O41), particularly the oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O41, which can be These O-antigen-specific oligonucleotides are used to quickly and accurately detect Escherichia coli O41 in the human body and the environment and identify the O-antigens in these pathogenic bacteria. Background technique [0002] The lipopolysaccharide located on the surface of Escherichia coli is the cause of E. coli pathogenicity, and the O-antigen is the outermost structure of the lipopolysaccharide, which is the target recognized by the immune system and the site of phage adsorption. The lack of O-antigen will cause the serum sensitivity of many pathogens, or severely weaken the virulence of pathogens [Frank et al (1987) "The function of antibody and complement i...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12N15/31C12P19/34C12Q1/68
Inventor 王磊杨静华冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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