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Nucleotides specific to vibrio riverine o11, o14, o16 and o17 and their application

A technology of Vibrio riverina and nucleotides, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of low sensitivity, incomplete antiserum types, and high missed detection rate, and achieve The effect of low detection cost

Active Publication Date: 2018-02-23
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 vibrio riverine o11, o14, o16 and o17 and their application
  • Nucleotides specific to vibrio riverine o11, o14, o16 and o17 and their application
  • Nucleotides specific to vibrio riverine o11, o14, o16 and o17 and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 : Genome Extraction

[0040] 37 ℃ nutrient broth medium to culture Vibrio rivers, collect the bacteria, and extract the genome. The specific steps are as follows:

[0041] 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 resuspend the DNA in 30ul TE. ...

Embodiment 2

[0042] Example 2: sequence deciphering

[0043] Extract the genomes of the standard strains of Vibrio riverinae O11, O14, O16 and O17 serotypes, and use Solexa pair-end sequencing technology to perform whole-genome sequencing on the genomes of each serotype of Vibrio riverinae to obtain the sequence of the serotype, using Blast and PSI-Blast Sequence comparison was carried out, the transmembrane structure prediction was performed by TMHMM 2.0 program, sequence alignment was carried out by ClustalW program and conservative and specific gene fragments were screened, and finally the O antigen gene cluster sequence and deciphering results of each serotype of Vibrio riverina were obtained.

Embodiment 3

[0044] Example 3 : Primer design

[0045] The O antigen gene cluster sequences of each serotype of Vibrio riverine O11, O14, O16 and O17 were self-tested by our laboratory. Through comparison analysis, we selected gene-specific segments with relatively low identity and similarity values ​​in the Blast comparison results Design primers. Of which O11 serotype wzy The identity and similarity values ​​of the gene comparison results were 48% and 70%; the O14 serotype wzy The identity and similarity values ​​of the gene comparison results were 55% and 74%; the O16 serotype wzy The identity and similarity values ​​of the gene comparison results are 44% and 63%, and the O17 serotype wzy The identity and similarity values ​​of the gene comparison results were 41% and 63%. Therefore, for each serotype, the above-mentioned corresponding genes were selected as the specific target genes of the serotype, and specific primers were designed for the specific segments of the genes of ...

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Abstract

The invention relates to nucleotides specific to vibrio fluvialis O11, O14, O16 and O17 as well as application of the nucleotides. The nucleotides comprise one or more of nucleotides shown in SEQ ID NO.1-8. The nucleotides can be used for preparation of a PCR kit and a gene chip which are used for detecting vibrio fluvialis. The inventor has applied for a patent on nucleotides specific to vibrio fluvialis O2, O4, O13, O15 and O18 serotypes, wherein the application number is CN201410158813. The nucleotides specific to vibrio fluvialis O11, O14, O16 and O17 serotypes, as well as the PCR kit and the gene chip comprising the nucleotides have the advantages that the detection sensitivity and efficiency are improved as the length of the product is greatly shortened; the practicability is high; the preparation method of the PCR kit is simple and convenient; the detection period is short; the speed is high; the operability is strong; the industrial production is easy; the detection cost is relatively low; the accuracy as well as the sensitivity is high.

Description

technical field [0001] The present invention relates to nucleotides specific to Vibrio riverine O11, O14, O16 and O17 serotypes, in particular to nucleotides specific to a single gene in the O antigen gene cluster of Vibrio riverines O11, O14, O16 and O17 serotypes and its application. Background technique [0002] Vibrio riverine is a gram-negative bacterium, one of the main habitat bacteria in the marine environment, widely present in the environmental waters of rivers and estuaries, and also one of the main bacterial pathogens of humans and aquatic organisms, it can cause fish, shrimp, shellfish and other diseases of a variety of farmed animals, bringing serious economic losses to the breeding industry. Vibrio riverine can also cause severe epidemic diarrhea in humans through various foods. It is a pathogenic Vibrio in the genus Vibrio, second only to Vibrio cholerae and Vibrio parahaemolyticus, and is considered to be a global zoonosis Comorbid novel pathogens. Its ty...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/689C12Q1/04C12R1/63
Inventor 王磊许玲玲张新杰王敏胡少辉
Owner NANKAI UNIV