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Nucleotides specific to Aeromonas hydrophila o29, o30, o33 and o35 and their application

A technology of Aeromonas hydrophila and nucleotides, which is applied to Aeromonas hydrophila O29, can solve the problems of insufficient quantity, high missed detection rate, and low sensitivity, and achieve strong practicability, high accuracy, The effect of detecting low cost

Active Publication Date: 2018-10-26
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the development of molecular biology, there are certain problems in the traditional serotyping and identification methods. For example, the diagnostic method of serotyping requires a large amount of antiserum, and the antiserum is generally incomplete and insufficient. Antiserum also presents some difficulties in preparation and storage
On the other hand, the serotyping method takes a long time, has low sensitivity, high missed detection rate, and poor accuracy, and there are often cross-reactions between antisera produced by different O antigens

Method used

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  • Nucleotides specific to Aeromonas hydrophila o29, o30, o33 and o35 and their application
  • Nucleotides specific to Aeromonas hydrophila o29, o30, o33 and o35 and their application
  • Nucleotides specific to Aeromonas hydrophila o29, o30, o33 and o35 and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 : Genome Extraction

[0037] Aeromonas hydrophila was cultivated in nutrient broth medium at 37°C, the bacteria were collected, and the genome was extracted. The specific steps are as follows:

[0038] 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, 15ul10% 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 then extract twice with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. Precipitate DNA with 2 times the volume of ethanol in the supernatant, roll out the DNA with glass wool and wash the DNA with 70% ethanol, and finally r...

Embodiment 2

[0039] Example 2: sequence deciphering

[0040] Extract the genomes of the standard strains of each serotype of Aeromonas hydrophila, and use Solexa pair-end sequencing technology to perform whole-genome sequencing on the genomes of each serotype of Aeromonas hydrophila to obtain the sequence of the serotype, using Blast and PSI-Blast Sequence alignment was carried out, TMHMM 2.0 program was used for transmembrane structure prediction, and ClustalW program was used for sequence alignment and screening of conserved and specific gene fragments, and finally the O antigen gene cluster sequence and deciphering results of each serotype of Aeromonas hydrophila were obtained.

Embodiment 3

[0041] Example 3 : Primer design

[0042] The O antigen gene cluster sequences of each serotype of Aeromonas hydrophila were self-tested by our laboratory. Through comparative analysis, we selected gene-specific segments with relatively low identity and similarity values ​​in the Blast comparison results to design primers. Among them, the identity value and similarity value of the wzx gene comparison result of O29 serotype were 56% and 74%; the identity value and similarity value of the wzz gene comparison result of O30 serotype were 60% and 74%; the wzm gene ratio of O33 serotype The result identity value and similarity value are 73% and 83%; the wzy gene comparison result identity value and similarity value of O35 serotype are 51% and 70%; For specific target genes, specific primers are designed for the specific segments of the genes of each serotype.

[0043] Primer design is a core part of this invention. Import the above genes into Primer Premier 5 for primer design, ...

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Abstract

The invention relates to a nucleotide specific to Aeromonas hydrophila O antigens and an application thereof. The nucleotide comprises: (1) at least one of nucleotides shown as SEQ ID NO:1 to 8; and (2) at least one of nucleotides being complementary with the nucleotides shown as SEQ ID NO:1 to 8. The nucleotides can be used for preparing a PCR (Polymerase Chain Reaction) kit for detecting Aeromonas hydrophila and a gene chip. The nucleotide specific to the Aeromonas hydrophila O antigens and the PCR kit and the gene chip including the nucleotide have high practicability. The PCR kit has the advantages of easiness and convenience in preparation, short detection period, high speed, high operability, easiness for industrial production, relatively low detection cost, high accuracy and high sensitivity.

Description

technical field [0001] The present invention relates to nucleotides specific to Aeromonas hydrophile O29, O30, O33 and O35 serotypes, in particular to individual genes in the O antigen gene cluster of Aeromonas hydrophilia O29, O30, O33 and O35 serotypes Specific nucleotides and their applications. Background technique [0002] Aeromonas hydrophila (Aeromonas hydrophila) belongs to the Vibriaceae Aeromonas genus, is a Gram-negative short bacillus, without spores and capsules, also known as Aeromonas hydrophila. Aeromonas hydrophila widely exists in many water bodies in nature and is the primary pathogenic bacteria of many aquatic animals. It is an opportunistic pathogenic bacterium and a typical human-animal-fish comorbid pathogen. It enters the body through the intestinal tract and can produce highly toxic exotoxins, causing fulminant hemorrhagic disease. In general, the stronger the adhesion of the bacteria to the intestinal tissue, the stronger the toxicity of the exot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 王磊许广楠王敏许玲玲张新杰
Owner NANKAI UNIV
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