Nucleotide and its use

A technology of Shigella baumannii and nucleotides, which can be used in the fields of application, microbiological determination/testing, sugar derivatives, etc., and can solve the problems of indistinguishability

Inactive Publication Date: 2003-08-27
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Shigella has 46 serotypes, Escherichia coli has 166 different O-antigens, the relationship between the two is very close, and there are 12 O-antigens shared by Escherichia coli and Shigella [Ewing, W.H. (1986 ) "Edwards and Ewing's identification of the Enterobacteriaceae". Elsevier Science Publishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) "Antigenic relationships between the enteroinvasive Escherichia coli antigens O28ac, O112ac and O154, O114, O136, O14 and Shigella Oantigens” J.clin Microbiol, 17(4):681-684], traditional serological methods cannot distinguish them

Method used

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  • Nucleotide and its use

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Embodiment 1

[0045] Shigella baumannii type 13 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), and extract the supernatant with an equal volume of ether to remove 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, the DNA was resuspended in 30ul TE, and the genomic DNA was detected by 0.4% agarose gel electrophoresis. Example 2: Amplificati...

Embodiment 2

[0046] The O-antigen gene cluster of Shigella baumannii type 13 was amplified by Long PCR. First design the upstream primer (5'-ATT GTGGCT GCA GGG ATC AAA GAA AT-3') based on the JumpStart 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 (5'-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3'). Use the Expand Long Template PCR method of BoehringerMannheim to amplify the O-antigen gene cluster. The PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 15 minutes. Do 30 cycles. 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 6 tubes of long PCR products, and use Promega's Wizard PCR Preps purification kit to p...

Embodiment 3

[0046] The O-antigen gene cluster of Shigella baumannii type 13 was amplified by Long PCR. First design the upstream primer (5'-ATT GTGGCT GCA GGG ATC AAA GAA AT-3') based on the JumpStart 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 (5'-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3'). Use the Expand Long Template PCR method of BoehringerMannheim to amplify the O-antigen gene cluster. The PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 15 minutes. Do 30 cycles. 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 6 tubes of long PCR products, and use Promega's Wizard PCR Preps purification kit to p...

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Abstract

The invention provides a nucleotide special to the O-antigen of Shigella dysenteriae 13, namely the nucleotide total sequence of gene group controlling the composition of the O-antigen in Shigella dysenteriae 13. It also includes the O-antigen gene group's construction and the oligonucleotide deriving from the glycosyl-transfer ase gene and the oligose unit processing gene (including wzx gene or the gene with the function similar to wzx gene and wzy gene or the gene with the function similar to wzx gene) in the O-antigen group of Shigella dysenteriae 13, and includes the method to get the O-antigen gene group. It verifies high speciality of the oligonucleotide. It also discloses the method of testing the oligonucleotide and determining Shigella dysenteriae 13 in human body and the environment.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling the synthesis of O-antigen in Shigella bodyii 13 (Shigella bodyii 13), in particular to the gene cluster controlling the synthesis of O-antigen in Shigella bodyii 13 Oligonucleotides in the gene cluster, these O-antigen-specific oligonucleotides can be used to quickly and accurately detect Shigella baumannii type 13 in humans and the environment and identify O- antigen. Background technique [0002] Shigella is a pathogenic bacterium developed along with human evolution, which can invade colonic epithelial cells, cause self-limited purulent infection lesions, and cause bacillary dysentery in humans. Humans have a high sensitivity to Shigella, and only need less than ten bacteria to cause human infection. Children and adults are susceptible to infection, especially children, who are prone to cause acute toxic dysentery, and Shigella O-antigen is one of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C12N15/10C12N15/31C12Q1/68G01N33/569
Inventor 王磊杨静华冯露
Owner NANKAI UNIV
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