O18, O19, O23, and O12 specific nucleotides and applications

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 (Vibrio cholerae) belongs to the genus Vibrio and is the pathogenic bacterium of cholera. Cholera is a severe intestinal infectious disease. It is a food-borne disease that spreads for a long time and affects a wide range. Vomiting 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. The Law listed it as a Class A infectious disease that should be subject to "compulsory management".

[0003] Bacterial typing and identifica...

Examples

Embodiment 1

[0037] Example 1 : Genome Extraction

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

[0039] 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 3...

Embodiment 2

[0040] Example 2: sequence deciphering

[0041] 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 Solexa pair-end sequencing technology to obtain the sequence of the serotype, and the sequences were compared using Blast and PSI-Blast. The TMHMM 2.0 program was used to predict the transmembrane structure, and the ClustalW program was used to perform sequence alignment and screen conservative and specific gene fragments, and finally obtained the O antigen gene cluster sequences and deciphered results of each serotype of Vibrio cholerae.

Embodiment 3

[0042] Example 3 : Primer design

[0043] 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.

[0044] Pri...

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 (Vibrio cholerae) belongs to the genus Vibrio and is the pathogenic bacterium of cholera. Cholera is a severe intestinal infectious disease. It is a food-borne disease that spreads for a long time and affects a wide range. Vomiting 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. The Law listed it as a Class A infectious disease that should be subject to "compulsory management".

[0003] Bacterial typing and identifica...

Examples

Embodiment 1

[0037] Example 1 : Genome Extraction

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

[0039] 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 3...

Embodiment 2

[0040] Example 2: sequence deciphering

[0041] 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 Solexa pair-end sequencing technology to obtain the sequence of the serotype, and the sequences were compared using Blast and PSI-Blast. The TMHMM 2.0 program was used to predict the transmembrane structure, and the ClustalW program was used to perform sequence alignment and screen conservative and specific gene fragments, and finally obtained the O antigen gene cluster sequences and deciphered results of each serotype of Vibrio cholerae.

Embodiment 3

[0042] Example 3 : Primer design

[0043] 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.

[0044] Pri...

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