Nucleotide to 0-antigen specificity of Escherichia coli 0140 type

Abstract

The invention provides a nucleotide specific to O-antigens of Escherichia coli O140, a full sequence of gene cluster in Escherichia coli O140, controlling O-antigen synthesis, such as the separated nucleotide shown in SEQ IN No.1, with a overall length of 14009 basic groups; or a nucleotide in SEQ IN No.1 having one or many inserted, deleted or substituted basic groups and simultaneously maintaining functions of the separated nucleotide; also including an oligonucleotide coming from glycosyltransferase gene and oligosaccharide unit-processing gene in the O-antigen gene cluster of Escherichia coli O140; by PCR, the invention verifies that oligonucleotide has specificity to all the O-antigens of Escherichia coli O140; the invention also discloses a method of using the oligonucleotide to detect and identify Escherichia coli O140 in the human body and environment.

Description

technical field

[0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O140 type (Escherichia coli O140), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O140 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O140 in human body and 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- The...

Examples

Embodiment 1

[0044] Embodiment 1: the extraction of genome:

[0045]Escherichia coli O140 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 In order to remove 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 3...

Embodiment 2

[0046] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O140 by PCR:

[0047] The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O140 as a template. First, design the upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C); the O-antigen gene cluster was amplified by the Expand Long Template PCR method of Boehringer Mannheim, and the PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, 30 cycles of annealing at 60°C for 15 seconds and extension at 68°C for 15 minutes. 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 P...

Embodiment 3

[0048] Embodiment 3: construct O-antigen gene cluster library:

[0049] 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 100mM DTT and 5 units ...

technical field

[0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O140 type (Escherichia coli O140), in particular to oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O140 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O140 in human body and 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- The...

Examples

Embodiment 1

[0044] Embodiment 1: the extraction of genome:

[0045]Escherichia coli O140 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 In order to remove 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 3...

Embodiment 2

[0046] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O140 by PCR:

[0047] The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O140 as a template. First, design the upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C); the O-antigen gene cluster was amplified by the Expand Long Template PCR method of Boehringer Mannheim, and the PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, 30 cycles of annealing at 60°C for 15 seconds and extension at 68°C for 15 minutes. 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 P...

Embodiment 3

[0048] Embodiment 3: construct O-antigen gene cluster library:

[0049] 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 100mM DTT and 5 units ...