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Nucleotides specific to Aeromonas hydrophila o44, o24, o25 and o28 and their application

A hydrophilic Aeromonas, nucleotide technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high missed detection rate, low sensitivity, insufficient quantity, etc. , to achieve the effect of high accuracy, low detection cost and strong practicability

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 o44, o24, o25 and o28 and their application
  • Nucleotides specific to Aeromonas hydrophila o44, o24, o25 and o28 and their application
  • Nucleotides specific to Aeromonas hydrophila o44, o24, o25 and o28 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. Of which O44 serotype wxya The identity value and similarity value of the gene comparison result are 67% and 78%; the identity value and similarity value of the wzz gene comparison result of O24 serotype are 87% and 93%; the identity value and similarity value of the wzy gene comparison result of O25 serotype Values ​​of 79% and 88%; O28 serotype GT gene comparison results identity value and similarity value of 65% and 79%; so each serotype selects the above-mentioned corresponding gene as the specific target gene of the serotype, for each serotype Specific primers were designed for the specific segments of the genes of the serotypes.

[0043] Primer de...

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Abstract

The invention relates to a specific nucleotide for aeromonas hydrophila O antigens and application thereof. The nucleotide comprises: (1) at least one of nucleotides shown as SEQ ID NO:1 to SEQ ID NO:8; (2) at least one of nucleotides which are complementary with the nucleotides shown as SEQ ID NO:1 to SEQ ID NO:8. The nucleotides can be used for preparing a PCR (Polymerase Chain Reaction) kit and a gene chip, which are used for detecting aeromonas hydrophila. The specific nucleotide for theaeromonas hydrophila O antigens and the PCR kit and the gene chip, which contain the nucleotide, have high practicability; the PCR kit has the advantages of simple and convenient preparation method, short detection period, rapid speed and high operability; the industrial production is facilitated and the detection cost is relatively lower; the accuracy is high and the sensitivity is high.

Description

technical field [0001] The present invention relates to nucleotides specific to Aeromonas hydrophila O44, O24, O25 and O28 serotypes, in particular to individual genes in the O antigen gene cluster of Aeromonas hydrophilia O44, O24, O25 and O28 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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