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Specific nucleotides for Vibrio cholerae O18, O19, O23 and O12 and applications of specific nucleotides

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

Active Publication Date: 2015-11-25
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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  • Specific nucleotides for Vibrio cholerae O18, O19, O23 and O12 and applications of specific nucleotides
  • Specific nucleotides for Vibrio cholerae O18, O19, O23 and O12 and applications of specific nucleotides
  • Specific nucleotides for Vibrio cholerae O18, O19, O23 and O12 and applications of specific nucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 : Genome Extraction

[0037] Cultivate Vibrio cholerae in 37°C nutrient broth medium, collect the bacteria, and extract the genome. 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 with 70% ethanol, and finally ...

Embodiment 2

[0039] Example 2: sequence deciphering

[0040] 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.

Embodiment 3

[0041] Example 3 : Primer design

[0042] 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 O18 serotype wzz The identity and similarity values ​​of the gene comparison results were 91% and 95%; the O19 serotype wxya The identity and similarity values ​​of the gene comparison results were 84% and 93%; the O23 serotype wzz The identity and similarity values ​​of the gene comparison results were 90% and 94%; the O12 serotype wxya The identity and similarity values ​​of the gene comparison results were 100% and 100%; therefore, the above-mentioned corresponding genes were selected as the specific target genes of the serotypes for each serotype, and specific primers were designed for the gene-specific segments of each serotype.

[0043] Pri...

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Abstract

The invention relates to specific nucleotides for Vibrio cholerae O-antigens and applications of the specific nucleotides. The nucleotides comprise 1), at least one of nucleotides represented in SEQ ID NO: 1-8; 2), at least one of nucleotides complemented with the nucleotides represented in SEQ ID NO:1-8. The nucleotides can be used for preparing a PCR (polymerase chain reaction) kit and a gene chip for detecting Vibrio cholerae. The specific nucleotides for Vibrio cholerae O-antigens as well as the PCR kit and the gene chip containing the nucleotides have high practicability, a preparation method of the PCR kit is simple and convenient, the detection period is short, the speed is high, the operability is high, industrial production is facilitated, and the detection cost is relatively lower; the accuracy and sensitivity are high.

Description

technical field [0001] The present invention relates to nucleotides specific to Vibrio cholerae O18, O19, O23 and O12 serotypes, in particular to nucleotides specific to a single gene in the O antigen gene cluster of Vibrio cholerae O18, O19, O12 and O18 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 mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 王磊许广楠张新杰王敏王来友
Owner NANKAI UNIV