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Nucleotide specific for bacillus coli O167 type and Sh.boydii 3 type O-antigen

A technology of Shigella baumannii and Escherichia coli, which is applied in the field of nucleotides specific to the O-antigen of Escherichia coli O167 and Shigella baumannii type 3, which can solve problems such as false positives

Inactive Publication Date: 2005-01-26
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-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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Extraction of genome:

[0053] Culture E. coli O167 or Shigella baumannii type 3 overnight at 37°C in 5 mL of LB medium, and collect the cells by centrifugation. The cells were resuspended with 500 μl of 50 mM Tris-HCl (pH 8.0) and 10 μl of 0.4 M 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 3μl of 20mg / mL proteinase K and 15μl of 10% SDS were added and incubated at 50°C for 2 hours, and then 3μl of 10mg / mL RNase was added and incubated at 65°C for 30 minutes. Add an equal volume of phenol extraction mixture, take the supernatant and extract twice with an equal volume of phenol: chloroform: isoamyl alcohol extraction (25:24:1) mixed solution, take the supernatant and extract with an equal volume of ether to remove For the remaining phenol, the supernatant is used to precipitate the DNA with 2 times the volume of ethanol, the DNA is rolled out with glass wool and the DNA is washe...

Embodiment 2

[0054] Example 2: Amplification of O-antigen gene clusters in Escherichia coli O167 or Shigella baumannii type 3 by PCR:

[0055] Using the genome of Escherichia coli O167 or Shigella baumannii type 3 as a template, the O-antigen gene cluster was amplified by Long PCR. First, design the upstream primers (#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 primers based on the gnd gene downstream of the O-antigen gene cluster (# 1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C); Use Boehringer Mannheim's ExpandLong Template PCR method to amplify O-antigen gene clusters. The PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, Annealing is performed for 15 seconds, and extension is performed at 68°C for 15 minutes, so that 30 cycles are performed. Finally, the extension was continued for 7 minutes at 68°C to obta...

Embodiment 3

[0056] Example 3: Construction of O-antigen gene cluster library:

[0057] The first is the acquisition of ligation products: the modified Novagen DNaseI shot gun method is used to construct an O-antigen gene cluster library. The reaction system is 300ng PCR purified product, 0.9μl 0.1M MnCl 2 , 1μl 1:2000 diluted 1mg / mL DNaseI, the reaction was carried out at room temperature. Enzyme digestion was performed for 10 minutes to concentrate the DNA fragment size between 1.5kb-3kb, and then 2μl 0.1M EDTA was added to stop the reaction. Combine 4 tubes of the same reaction system, extract once with an equal volume of phenol, extract once with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixed solution, and then extract once with an equal volume of ether Then, the DNA was precipitated with 2.5 times the volume of absolute ethanol, and the precipitate was washed with 70% ethanol, and finally resuspended in 18 μl of water. Then add 2.5μl dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP...

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Abstract

The invention provides an O-antigen specific nucleotide to Escherichia coli O167 and Shigella boydii3, which is the whole sequence of gene cluster controlling O-antigen synthesis in Escherichia coli O167 and Shigella boydii3, such as separating nucleotide sequence of SEQ ID NO:1 , Escherichia coli O167 being 12864 base, Shigella boydii3 being 12199 base, or one or several base substituted, defected or inserted SEQ ID NO:1 and at same time have same function with SEQ ID NO:1, also comprise oligonucleotide of glycosyltransferase gene and oligosaccharide disposing gene derived from O-antigen gene cluster of Escherichia coli O167 and Shigella boydii3. It is validated that oligonucleotide is highly specific to Escherichia coli O167 and Shigella boydii3 O-antigen. The invention also discloses Escherichia coli O167 and Shigella boydii3 detection and identification method using inventive oligonucleotide.

Description

Technical field [0001] The present invention relates to the complete nucleotide sequence of the gene clusters controlling O-antigen synthesis in Escherichia coli O167 and Shigella boydii 3, and in particular to Escherichia coli O167 and Oligonucleotides in the gene cluster that controls O-antigen synthesis in Shigella baumannii type 3 can be used to quickly and accurately detect E. coli O167 in humans and the environment by using these O-antigen-specific oligonucleotides And Shigella baumannii type 3 and identify O-antigens in these pathogenic bacteria. Background technique [0002] O-antigen is the O-specific polysaccharide component in the Gram-negative bacterial lipopolysaccharide, which is composed of many repeated oligosaccharide units. The synthesis process of O-antigen has been studied more clearly: firstly, glycosyltransferase transfers the nucleoside diphosphate monosaccharide to a lipid molecule fixed on the inner membrane of the cell, and then synthesizes oligosacchari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61K39/108A61K39/112C12N15/31C12Q1/02C12Q1/68G01N33/53
CPCY02A50/30
Inventor 王磊冯露韩巍青
Owner TIANJIN BIOCHIP TECH CO LTD
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