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Nucleotide specific against 0-antigen of shigella dysenteria 3, colibacillus 0124 and 0164

A technology for Shigella dysenteriae and Escherichia coli, which can be used in the determination/inspection of microorganisms, resistance to vector-borne diseases, sugar derivatives, etc., and can solve problems such as indistinguishability

Inactive Publication Date: 2003-09-17
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen from the figure that their O-antigen structures are very similar, the difference is that there is one more group on the last sugar of the branched chain in the O-antigen structure of Shigella dysenteriae type 3 than that of E. coli O164; There is a β-D-Galp on the main chain in the O-antigen structure of Shigella dysenteriae type 3, and there is an α-D-Galp in the main chain in the O-antigen structure of Escherichia coli O164, traditional serological method cannot distinguish them

Method used

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Experimental program
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Effect test

Embodiment 1

[0057] Shigella dysenteriae type 3 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 then use an equal volume of ether Extract 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, and finally the DNA was resuspended in 30ul TE. Genomic DNA was detected by 0.4% agarose gel electrop...

Embodiment 2

[0058] Using the genome of Shigella dysenteriae type 3 as template, its O-antigen gene cluster was amplified by Long PCR. First, design the upstream primer (5'-ATT GTG GCT 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 Primer (5'-TAG TCG CGT GNG CCT GGATTA AGT TCG C-3'). Use the Expand Long Template PCR method of Boehringer Mannheim 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'...

Embodiment 3

[0058] Using the genome of Shigella dysenteriae type 3 as template, its O-antigen gene cluster was amplified by Long PCR. First, design the upstream primer (5'-ATT GTG GCT 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 Primer (5'-TAG TCG CGT GNG CCT GGATTA AGT TCG C-3'). Use the Expand Long Template PCR method of Boehringer Mannheim 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'...

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Abstract

A nucleotide specific to the O-antigen of Shigella dysenteriae 3, Escherichia coli O124 and Escherichia coil O164 is a complete nucleotide sequence of the genom for controlling the synthesis of O-antigen is shigella dysenteriae 3, such as the separated nucleotide shown by SEQ ID No.1. It has 12629 bases, or has one or more inserted, deletional or substituted bases but keeps the function of said separated neucleotide. It also includes the oligonucleotide of glycosyltransferase gene and oligose unit treating gene in O-antigen genom. A method for using said oligonucleotide to detect said three bacteria is also disclosed.

Description

technical field [0001] The present invention relates to the complete nucleotide sequence of the gene cluster controlling O-antigen synthesis in Shigella dysenteriae type 3 (Shigella dysenteriae 3), in particular to the gene cluster controlling O-antigen synthesis in Shigella dysenteriae type 3 The oligonucleotides in the O-antigen can be used to quickly and accurately detect Shigella dysenteriae type 3, Escherichia coli O124 (Escherichia coliO124) and Escherichia coli O164 ( Escherichia coli O164) and identify the O-antigen in these pathogenic bacteria. 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...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12N15/11C12Q1/04C12Q1/68G01N33/569
CPCY02A50/30
Inventor 王磊杨静华冯露
Owner NANKAI UNIV
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