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Nucleotide peculiar to 0-antigen of 051 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 051 type bacillus coli

Examples

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Embodiment 1

[0054] Example 1: Genome extraction.

[0055] Escherichia coli O51 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

[0056] Example 2: Amplification of the O-antigen gene cluster in E. coli O51 by PCR

[0057] Using the genome of Escherichia coli O51 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 frequently 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 electrophoresis to ...

Embodiment 3

[0058] Example 3: Construction of O-antigen gene cluster library.

[0059] 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 O51, it is a nucleotide complete sequence of gene cluster for controlling synthesis of O-antigen in Escherichia coli O51, as the separated nucleotide shown by SEQ ID No:1, its total length has 13343 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 oligosaccharide unit treatment gene (including wzx gene or gene whose function is similar to that a wzx and wzy gene or gene whose function is simlar to that of wzy) originated from O-antigen gene cluster of Escherichia coli O51. Said invention utilizes PCR to verify that the oligonucleotide has high specificity to O-antigen of Escherichia coli O51, and said invention also discloses the method for detecting and identifying Escherichia coli O51 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 O51 (Escherichia coli O51), particularly the oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O51, which can be These O-antigen-specific oligonucleotides are used to quickly and accurately detect Escherichia coli O51 in the human body and the environment and identify the O-antigens in these pathogenic bacteria. Background technique [0002] Escherichia coli O51 is a pathogenic bacterium that has been isolated from the urine of patients with cystitis [Hebelka M et al (1993) "Sexual acquisition of acutepyelonephritis in a man". Scand J Infect Dis, 25 (1):141-3]. It is also reported that it is a facultative EPEC (Enteropathogenic Escherichia coli), which can cause enteritis in children under two years old [Czirok E et al (1976) "The role in sporadicenteritis of facultatively ente...

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 王磊于浩冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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