Nucleotide specific to Aeromonas hydrophila O29, O30, O33 and O35 and application

A hydrophilic Aeromonas, nucleotide technology, applied to the hydrophilic Aeromonas O29, can solve the problems of high missed detection rate, insufficient quantity, low sensitivity, etc., to achieve high accuracy, strong practicability, Detect the effect of low cost

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

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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  • Nucleotide specific to Aeromonas hydrophila O29, O30, O33 and O35 and application
  • Nucleotide specific to Aeromonas hydrophila O29, O30, O33 and O35 and application
  • Nucleotide specific to Aeromonas hydrophila O29, O30, O33 and O35 and 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 500 ul of 50 mM Tris-HCl (pH 8.0) and 10 ul of 0.4 MEDTA, incubated at 37° C. for 20 minutes, and then 10 ul of 10 mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul of 20mg / ml proteinase K, 15ul of 10% SDS, incubate at 50°C for 2 hours, then add 3ul of 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 wi...

Embodiment 2

[0039] Example 2: sequence deciphering

[0040] Extract the genomes of the standard strains of each serotype of Aeromonas hydrophila, and use Solexapair-end sequencing technology to perform whole-genome sequencing on the genomes of each serotype of Aeromonas hydrophila to obtain the sequence of the serotype, and use Blast and PSI-Blast to perform Sequence comparison, TMHMM2.0program was used for transmembrane structure prediction, and ClustalWprogram was used for sequence alignment and screening of conserved and specific gene fragments, and finally obtained the O antigen gene cluster sequences and deciphered results of each serotype of Aeromonas hydrophila.

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 PrimerPremier5 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 ex...

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 王磊许广楠王敏许玲玲张新杰
Owner NANKAI UNIV
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