Nucleotide sequences specific to Hafnia alvei g5902, g5903, g5904, g5906
A Hafnia bacteria and nucleotide sequence technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The problem of high rate, to achieve the effect of low detection cost
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Embodiment 1
[0037] Example 1 : Genome Extraction
[0038] Hafnia alvei was cultured in nutrient broth medium at 37°C, the bacteria were collected, and the genome was extracted. 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 for extraction, 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 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
[0040] Example 2: sequence deciphering
[0041] The genomes of Hafnia alvei G5902, G5903, G5904, and G5906 standard strains were extracted, and the whole genome sequence of each standard genome of Hafnia alvei was obtained by Solexapair-end sequencing technology to obtain the sequence of the O serotype. Blast and PSI-Blast for sequence comparison, TMHMM Server2.0 program for transmembrane structure prediction, ClustalW program for sequence alignment and screening of conserved and specific gene fragments, and finally the O antigen gene clusters of each serotype of Hafnia alvei Sequence and Deciphering Results.
Embodiment 3
[0042] Example 3 : Primer design
[0043] According to the deciphering of the gene cluster and the results of database comparison, it was found that the wzy and wzx genes are specific genes for the O antigen of Hafnia alvei, so the specific segment of the gene was selected to design specific primers. Since wzy is more specific, the wzy gene is mainly used as the target gene.
[0044] Primer design is a core part of this invention. Primers were designed according to the specific genes described in the literature. The two genes wzx and wzy are relatively specific genes in the O antigen gene cluster of Hafnia alvei, which can be used as target genes for serotype identification. The above genes were introduced into Primer Premier 5 for primer design. The length of the primers should preferably be between 18 and 24 bp, and the Tm value should be between 53 and 58°C. A pair of primers are designed for each gene, with a single target band.
[0045] After the primers are designe...
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