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Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof

A fluvibrio vibrio and nucleotide technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of low sensitivity, incomplete types of antiserum, and high missed detection rate. Detecting the effect of low cost

Active Publication Date: 2014-07-02
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 Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof
  • Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof
  • Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 : Genome Extraction

[0045] 37 ℃ nutrient broth medium to culture Vibrio rivers, collect the bacteria, and extract the genome. The specific steps are as follows:

[0046] 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, 15ul 10% 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

[0047] Example 2: sequence deciphering

[0048] The genomes of the standard strains of Vibrio riverinae O2, 04, 013, 015 and 018 serotypes were extracted, and the genomes of each serotype of Vibrio riverinae were sequenced by Solexa pair-end sequencing technology to obtain the sequence of the serotype, using Blast and PSI -Blast for sequence comparison, TMHMM 2.0 program for transmembrane structure prediction, and ClustalW program 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 Vibrio riverine.

Embodiment 3

[0049] Example 3 : Primer design

[0050] The O antigen gene cluster sequences of each serotype of Vibrio riverine O2, O4, O13, O15, and O18 were self-tested by our laboratory. Through comparative analysis, we selected specific genes with relatively low identity and similarity values ​​in the Blast comparison results. Design primers for the segment. Of which the O2 serotype wzy The identity and similarity values ​​of the gene comparison results are 44% and 68%, orf9 The identity and similarity values ​​of the gene comparison results were 44% and 65%; the O4 serotype wxya The identity and similarity values ​​of the gene comparison results were 22% and 42%; the O13 serotype wxya The identity and similarity values ​​of the gene comparison results were 26% and 48%; the O15 serotype orf13 The identity and similarity values ​​of the gene comparison results were 27% and 52%; the O18 serotype wxya The identity value and similarity value of the gene comparison results ar...

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Abstract

The invention relates to a nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 serotypes and an application thereof. The nucleotide comprises 1) at least one of nucleotides shown in SEQIDNO: 1-14; and 2) at least one of nucleotides shown in SEQIDNO: 1-14. The nucleotides can be used for preparing PCR (polymerase chain reaction) kits and gene chips for detecting Vibrio fluvialis. The nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 serotypes disclosed by the invention, as well as a PCR kit and a gene chip which contain the nucleotide, are strong in practicability, and the PCR kit is simple in preparation method, short in detection cycle, rapid in speed, strong in maneuverability, convenient for industrialized production, and low in detection cost; the accuracy is high; and the sensitivity is high.

Description

technical field [0001] The present invention relates to nucleotides specific to O2, O4, O13, O15 and O18 serotypes of Vibrio riverines, in particular to nucleotides specific to individual genes in the O antigen gene cluster of Vibrio riverines O2, O4, O13, O15 and O18 serotypes Nucleotides and their applications. 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 Comor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 王敏王磊胡少辉冯露
Owner NANKAI UNIV
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