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Specific nucleotides for citrobacter 017 and 039 and application of special nucleotides

A Citrobacter, nucleotide technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc., can solve the difficulty of sugar synthesis gene identification, complex polysaccharide antigen synthesis gene cluster composition, and genetic Unpredictable functions, etc.

Active Publication Date: 2016-01-20
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reasons for the above phenomenon are: polysaccharide antigen synthesis gene cluster composition is complex, gene function is difficult to predict, sugar synthesis gene identification is difficult, etc.
In addition, most of the units that have carried out some relevant research lack the foundation of previous research

Method used

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  • Specific nucleotides for citrobacter 017 and 039 and application of special nucleotides
  • Specific nucleotides for citrobacter 017 and 039 and application of special nucleotides
  • Specific nucleotides for citrobacter 017 and 039 and application of special nucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 : Genome Extraction

[0035] Citrobacter was cultured at 37°C in brain-heart infusion broth medium, the bacteria were collected, and the genome was extracted with a bacterial genome extraction kit (Kangwei Century CW0552). The specific steps are as follows:

[0036] 1. Take 1-5ml of bacterial culture (106-108 cells, no more than 2×109 cells), centrifuge at 10000rpm for 1 minute, and discard the supernatant.

[0037] 2. Add 180μl BufferGTL to the pellet and shake to resuspend the bacteria.

[0038] 3. Add 20 μl ProteinaseK, vortex to mix, and incubate at 56°C until the cells are completely lysed. During the incubation, invert or shake the centrifuge tube at regular intervals to disperse the sample.

[0039] 4. Add 4 μl of RNaseA solution (Product No. CW0601) with a concentration of 100 mg / ml, vortex to mix, incubate at room temperature for 5 minutes, and then proceed to step 5.

[0040] 5. Add 200μl BufferGL, vortex and mix well; add 200μl absolute ethano...

Embodiment 2

[0052] Example 2: sequence deciphering

[0053] The genomes of the standard strains of Citrobacter 017 and O39 serotypes were extracted, and the genomes of each serotype of Citrobacter were sequenced by Solexapair-end sequencing technology to obtain the sequence of the serotype, and the sequences were compared using Blast and PSI-Blast. The TMHMM2.0 program was used to predict the transmembrane structure, and the ClustalW program was used to perform sequence alignment and screen conservative and specific gene fragments. Finally, the O antigen gene cluster sequences and decipher results of each serotype of Citrobacter were obtained.

Embodiment 3

[0054] Example 3 : Primer design

[0055] According to the deciphering of gene clusters, we found that wzm and wzy The gene is indeed a serotype-specific gene, so a specific segment of the gene is selected to design specific primers.

[0056] Primer design is a core part of this invention. Primers were designed according to the specific genes described in the literature. wzm and wzy The gene is a relatively specific gene in the Citrobacter O antigen gene cluster, which can be used as the target gene for serotype identification. Import the above genes into PrimerPremier5 for primer design. The length of the primers should preferably be between 18 and 24 bp, and the Tm value should be around 55°C. A pair of primers are designed for each gene, with a single target band.

[0057] After the primers are designed, BLAST is performed in Genbank. The designed primers should not have too high sequence similarity with other related bacteria, so as to ensure that the primers ca...

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Abstract

The invention relates to specific nucleotides for 017 and 039 serotypes of citrobacter and an application of the specific nucleotides. The nucleotides include at least one of nucleotides represented as SEQ ID NO: 1-4, the nucleotides can be used for preparing a PCR (polymerase chain reaction) kit for detecting the citrobacter. The citrobacter can be sampled from crude extract of culture of the digestive tract, urine, blood and wounds and can also be obtained through separation in feces environment of humans and animals. According to the specific nucleotides for the 017 and 039 serotypes of the citrobacter and the PCR kit containing the nucleotides, a preparation method of the PCR kit is simple, the detection period is short, the speed is high, the operability is high, industrial production is facilitated, and the detection cost is relatively lower; the accuracy is high; the sensitivity is high.

Description

technical field [0001] The invention relates to nucleotides specific to Citrobacter 017 and O39 serotypes, in particular to nucleotides specific to a single gene in the Citrobacter 017 and O39 serotype O antigen gene cluster and application thereof. Background technique [0002] Citrobacter is a common Gram-negative bacterium in the Enterobacteriaceae family, which can usually use citric acid as the only carbon source and belongs to facultative anaerobic bacteria. Citrobacter, the closest relative to Salmonella and Escherichia coli, is an opportunistic pathogen. [0003] Citrobacter can be divided into 11 gene species by DNA hybridization method, namely Citrobacter freundii (C.freundii), Citrobacter koseri (C. koseri), Citrobacter nonmalonate (C. .amalonatIzcus), Citrobacter fageri (C.farmeri), Citrobacter youngae (C.youngae), Citrobacter braakii (C.braakii), Citrobacter walkmanii (C.werkmanii), plug C. sedlakif, C. rodentium, C. gillenii, C. murliniae, which have obvious ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 王磊钱澄倩郭玺刘斌
Owner NANKAI UNIV
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