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
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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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