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Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide

A technology of Vibrio cholerae and nucleotides, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of low sensitivity, high missed detection rate, insufficient quantity, etc. The effect of low cost, high accuracy and strong practicability

Active Publication Date: 2015-12-23
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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  • Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide
  • Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide
  • Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 : Genome Extraction

[0038] Cultivate Vibrio cholerae in 37°C nutrient broth medium, collect the bacteria, and extract the genome. The specific steps are as follows:

[0039] 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 with 70% ethanol, and finall...

Embodiment 2

[0041] Example 2: sequence deciphering

[0042] The genomes of the standard strains of each serotype of Vibrio cholerae were extracted, and the genomes of each serotype of Vibrio cholerae were sequenced by Solexapair-end sequencing technology to obtain the sequence of the serotype, and the sequences were compared using Blast and PSI-Blast, and TMHMM2 .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 cholerae.

[0043]

Embodiment 3

[0044] Example 3 : Primer design

[0045] The O antigen gene cluster sequences of each serotype of Vibrio cholerae were self-tested by our laboratory. Through comparative analysis, we selected gene-specific segments with relatively low values ​​of identity and similarity in Blast comparison results to design primers. Of which 06 serotype wxya The identity and similarity values ​​of the gene comparison results were 98% and 99%; the 04 serotype wzz The identity and similarity values ​​of the gene comparison results were 98% and 99%; the 07 serotype wzz The identity and similarity values ​​of the gene comparison results were 98% and 99%; the 015 serotype wxya The identity and similarity values ​​of the gene comparison results were 99% and 100%; therefore, each serotype selected the above-mentioned corresponding gene as the specific target gene of the serotype, and designed specific primers for the gene-specific segments of each serotype.

[0046] Primer design is a cor...

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Abstract

The invention relates to a nucleotide specific to vibrio cholerae O antigens and application of the nucleotide, wherein the nucleotide comprises 1) at least one of nucleotides as shown in SEQ ID NO: 1-8; and 2) at least one of nucleotides supplementary with the nucleotides as shown in SEQ ID NO: 1-8. The nucleotides can be used for preparing a PCR kit and a gene chip for detecting vibrio cholerae. The nucleotide specific to vibrio cholerae O antigens disclosed by the invention as well as the PCR kit and the gene chip containing the nucleotide is strong in practicability; the PCR kit is simple and convenient in preparation method, short in detection cycle, rapid in speed, strong in operability and easy in industrial production, while the PCR kit is relatively low in detection cost; and the nucleotide is high in accuracy and high in sensitivity.

Description

technical field [0001] The present invention relates to nucleotides specific to Vibrio cholerae O6, O4, O7 and O15 serotypes, in particular to nucleotides specific to a single gene in the O antigen gene cluster of Vibrio cholerae O6, O4, O7 and O15 serotypes and its application. Background technique [0002] Vibrio cholerae (Vibriocholerae) belongs to the genus Vibrio and is the pathogenic bacterium of cholera. Cholera is a severe intestinal infectious disease. It is a foodborne disease that spreads for a long time and affects a wide range. And the resulting loss of body fluids, dehydration, systemic circulatory failure, electrolyte disturbance, hypokalemia syndrome, abdominal cramps and even death are identified by the World Health Organization as one of the infectious diseases that require international quarantine. "Listed it as a Class A infectious disease that should be subject to "compulsory management". [0003] Bacterial typing and identification methods mainly incl...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/63
CPCC12Q1/689Y02A50/30
Inventor 王磊许广楠许玲玲王敏王来友
Owner NANKAI UNIV
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