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Nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application

A Hafnia bacterium and nucleotide technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as low sensitivity, insufficient quantity, and incomplete antiserum types

Active Publication Date: 2015-12-02
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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  • Nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application
  • Nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application
  • Nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 : Genome Extraction

[0036] 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:

[0037]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% ethano...

Embodiment 2

[0038] Example 2: sequence deciphering

[0039] The genomes of Hafnia alvei G5897, G5898, and G5900 standard strains were extracted, and the whole genome sequence of the standard genomes of Hafnia alvei was obtained by Solexapair-end sequencing technology to obtain the sequence of the O serotype. Blast and PSI- Blast was used for sequence comparison, TMHMMServer2.0program was used for transmembrane structure prediction, and ClustalWprogram was used 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 Hafnia alvei.

Embodiment 3

[0040] Example 3 : Primer design

[0041] 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.

[0042] 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-mentioned genes were introduced into PrimerPremier5 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.

[0043] After the primers are...

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Abstract

The invention relates to nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application. The nucleotides include one type of nucleotides shown in SEQ ID NO: 1 to 6. These nucleotides are applicable to the preparation of PCR kits and gene chips used for detecting Hafnia alveibifermentans. The invention relates to the nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900, and a PCR kit and a gene chip both including the nucleotides; an operating method is simple, a detection period is short, detection cost is lower, accuracy is high, sensitivity is high, and the nucleotides, the PCR kit and the gene chip are easy to industrially produce.

Description

technical field [0001] The present invention relates to nucleotides specific to Hafnia alvei G5897, G5898, G5900, in particular to nucleotides specific to a single gene in the O antigen gene cluster of Hafnia alvei G5897, G5898, G5900 and its application. Background technique [0002] Hafnia alvei is a kind of Enterobacteriaceae. It is straight rod-shaped, non-capsulated, motile, facultative anaerobic, and has two types of metabolism, respiration and fermentation. It is in the human respiratory tract , intestines, animal intestines and meat dairy products and other foods are more common. Hafnia alveoli is an opportunistic pathogen that can cause bacteremia, respiratory tract infection, urinary tract infection and other infections in people with low immunity. It may be related to gastroenteritis. In addition, It can also cause catarrhal enteritis and hepatosplenomegaly in laying hens, animal diseases such as zoonotic septicemia in Bulgarian rainbow trout. [0003] Bacteria...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 王磊王天威冯露刘斌
Owner NANKAI UNIV
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