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Nucleotide specific to O antigen of 078 type bacillus coli

A technology of Escherichia coli and nucleotides, which is applied in the direction of antibody medical components, microbial measurement/testing, medical preparations containing active ingredients, etc., and can solve problems such as false positives

Inactive Publication Date: 2005-10-26
TIANJIN BIOCHIP TECH CO LTD
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  • 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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  • Nucleotide specific to O antigen of 078 type bacillus coli

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Embodiment 1

[0051] Embodiment 1: the extraction of genome:

[0052] Escherichia coli O78 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 μl of 50 mM Tris-HCl (pH 8.0) and 10 μl of 0.4M 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 add 3 μl 20 mg / mL proteinase K, 15 μl 10% SDS, incubate at 50° C. for 2 hours, then add 3 μl 10 mg / 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 precipitated with 2 times the volume of ethanol, the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended in...

Embodiment 2

[0053] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O78 by PCR:

[0054] Using the genome of Escherichia coli O78 as a template, its O-antigen gene cluster was amplified by Long PCR. First, design the upstream primer (#wl-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T) based on the JUMPStart sequence often found in the promoter region of the O-antigen gene cluster, and then design according to the gnd gene downstream of the O-antigen gene cluster Downstream primer (#1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C); use Boehringer Mannheim’s ExpandLong Template PCR method to amplify the O-antigen gene cluster. The PCR reaction procedure is as follows: pre-denaturation at 94°C for 2 minutes; then 94°C Denaturation for 10 seconds, annealing at 55□ for 15 seconds, and extension at 68°C for 15 minutes were performed for 30 cycles. Finally, continue extending at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to detect t...

Embodiment 3

[0055] Embodiment 3: construct O-antigen gene cluster library:

[0056] 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, 0.9μl 0.1M MnCl 2 , 1 μl of 1 mg / mL DNaseI diluted 1:2000, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the size of DNA fragments between 1.5kb-3kb, then add 2μl 0.1M EDTA to stop 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 18 μl of water. Then add 2.5μl dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25μl 100mMDTT and 5 units o...

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Abstract

This invention provides a specific nucleotide O-antigen for Escherichia coli 078, a complete sequence of nucleotide which controls the synthesis of O-antigen, e.g. as show in the SEQ ID NO: 1 the nucleic acid for separation has 12655 alkali bases, or compromising one or several insert, lost, or substitutes alkali bases, while keeping the function of the nucleic acid; It also compromises oligonucleotide which processing gene in the single-surge unit of O-antigen gene group form Escherichia coli; this invention proves that the oligonucleotide has a high specific function on Escherichia coli 078 by PCR, and discloses a method of identifying and detecting Escherichia coli by the oligonucleotide.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Escherichia coli O78 type (Escherichia coli O78), in particular to the oligonucleotides in the gene cluster controlling O-antigen synthesis in Escherichia coli O78 type , these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Escherichia coli O78 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): C12N15/11C12N15/31
Inventor 王磊韩巍青冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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