Nucleotide with specificity to o-antigen of type-12 shigella shigae and colibacillus 0152

A technology of Shigella dysenteriae and Escherichia coli, applied in the field of nucleotides specific to the O-antigen of Shigella dysenteriae type 12 and Escherichia coli O152, which can solve the problems of indistinguishability

Inactive Publication Date: 2003-07-30
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 but only 33 different O-antigens and E. coli has 166 different O-antigens [Reeves, P.R (1992) "Variation in O antigens, niche specific selection and bacterial populations". FEMS Microbiol.Lett, 100:509-516], the relationship between the two is very close, and there are 12 O-antigens shared by Escherichia coli and Shigella, among which Shigella dysenteriae type 12 and Escherichia coli O152 have The same O-antigen [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 O128ac, O , O124, O136, O143, O144, O152 and O164 and Shigella O antigens” J.clinMicrobiol, 17(4):681-684], traditional serotyping methods cannot distinguish them

Method used

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  • Nucleotide with specificity to o-antigen of type-12 shigella shigae and colibacillus 0152
  • Nucleotide with specificity to o-antigen of type-12 shigella shigae and colibacillus 0152
  • Nucleotide with specificity to o-antigen of type-12 shigella shigae and colibacillus 0152

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Experimental program
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Embodiment 1

[0030] Shigella 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, 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, and extract the supernatant with an equal volume of ether to remove residual phenol. The DNA was precipitated with 2 times the volume of ethanol in the supernatant, and 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 electrophoresis. Example 2: Amplification of the O-antigen gene ...

Embodiment 2

[0031] The O-antigen gene cluster of Shigella dysenteriae type 12 was amplified by Long PCR. First, design the upstream primer (#1523-ATT GTG GCT GCA GGGATC 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 -TAGTCG CGT GNG CCT GGA TTA AGT TCG C). Use the ExpandLong 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's Wizard PCR Preps purification kit to purify the PCR products. Embodiment 3: Construction of O-anti...

Embodiment 3

[0032] A modified Novagen DNaseI shot gun method was used 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 1kb-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 phenol:chloroform:isoamyl alcohol (25:24:1), and then once with an equal volume of diethyl ether. Precipitate DNA with 2.5 times the volume of absolute ethanol, wash the precipitate with 70% ethanol, and finally resuspend in 18ul of water. Then add 2.5uldNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul 100mM DTT and 5 units of T4 DNA polymerase to this mixture, 11°C for 30 minutes, make the end of the digested product blunt, stop the reaction at 75°C, add 5 units of ...

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Abstract

The present invention includes nucleotide with specificity to O-antigen of Shigella dysenteriae 12 and Escharichia coli O152; the whole nucleotide sequence of gene cluster of Shigella dysenteriae 12 to control the synthesis of O-antigen; the structure of O-antigen gene cluster, oligoneotide of glycosyltransferase gene and oligosaccharide unit processing gene original from O-antigen of Shigella dysenteriae 12; and method of obtaining bacterial O-antigen gene cluster. PCR proves the specificity to O-antigen of shigella dysenteriae 12 and Escherichia coli O152 of the present invention. The method of detecting and identifying Shigella dysenteriae 12 and Escherichia coli O152 in body and in environment with the oligoneotide of the present invention 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 12, in particular to the gene cluster controlling O-antigen synthesis in Shigella dysenteriae 12 Oligonucleotides in O-antigen can be used to quickly and accurately detect Shigella dysenteriae type 12 and Escherichia coli O152 (Escherichia coli O152) in humans and the environment and identify these pathogenic O-antigen in 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 cause acute toxic dysentery, and Shigella O-antigen is one ...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12P19/34C12Q1/68
Inventor 王磊杨静华
Owner NANKAI UNIV
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