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Novel tet34 resistance gene of tetracycline and application of novel tet34 resistance gene

A tetracycline resistance and resistance gene technology, applied in DNA/RNA fragmentation, microbial determination/inspection, instruments, etc., can solve the problems of complicated operation, disappearance, low efficiency, etc., achieve simple and fast operation, shorten the detection cycle, The effect of high sequencing throughput

Pending Publication Date: 2021-10-15
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The emergence of tetracycline-resistant strains and resistance genes has led to a significant decline in the efficacy of drug treatment, reducing or even disappearing the clinical efficacy of tetracycline and even the entire tetracycline antibiotic family, posing a serious threat to human and animal health
The identification methods of new antibiotic resistance genes currently used are mainly based on pure culture and molecular biology methods. This method has a long identification cycle, complicated operation, and low efficiency, which is not conducive to large-scale detection.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Extraction and sequencing of genomic DNA of tetracycline-resistant strains

[0032] Here, the resistant strain Escherichia coli TET with tetracycline resistance is used as an example. Centrifuge 2ml of the overnight cultured bacterial solution at 6000rpm at 4°C for 5 minutes, discard the supernatant and collect the precipitate, use the Bacteria DNAKit kit to complete the extraction of its genomic DNA, through 1% agarose gel electrophoresis and NanoDrop One ultra-micro spectrometry A photometer (Thermo Fisher Scientific, USA) was used to check the integrity and concentration of genomic DNA, and then the obtained genomic DNA was sent to Beijing Novogene Bioinformation Technology Co., Ltd. for sequencing using the Nanopore PromethION and IlluminaNovaSeq 6000 platforms.

Embodiment 2

[0033] Example 2: Assembly and ORF prediction of strain genome

[0034] Perform quality control on the sequencing data obtained in Example 1 to obtain clean data, and then use Unicycler software to mix and assemble the sequencing data of the samples to obtain the complete genome data of the strain. The specific parameters of the Unicycler software are as follows:

[0035] unicycler-1TET_1.fq-2TET_2.fq-l TET.nano.fq-o TET-t 46 --mode normal --min_polish_size 2000

[0036] Among them, TET_1.fq and TET_2.fq are paired-end data sequenced by Illumina, and TET.nano.fq is sequenced data by Nanopore.

[0037] Carry out open reading frame (ORF) prediction to the complete genome data of the bacterial strain that obtains through prodigal software again, the concrete parameter of described prodigal software is as follows:

[0038] prodigal -i TET.fa -m -o TET_temp.txt -a TET_ORF.aa -d TET_ORF.fa

[0039] Among them, TET.fa is the genome obtained by unicycler hybrid assembly, and TET_ORF...

Embodiment 3

[0040] Embodiment 3: Comparison of ORF and SARG databases

[0041] Using DIAMOND software to compare the ORF amino acid sequence obtained in Example 2 with the SARG database, the results showed that the annotation result of ORF516 was a tetracycline resistance gene, and its similarity with the reference gene sequence tet34 in the database was 25.5%.

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PUM

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Abstract

The invention discloses a novel tet34 resistance gene of tetracycline and application of the novel tet34 resistance gene. The nucleotide sequence of the tetracycline novel tet34 resistance gene is as shown in SEQ ID NO: 1. According to the potential tetracycline resistance gene tet34 found in the application, a database of the tetracycline resistance gene is supplemented, help is provided for understanding the resistance mechanism of tetracycline, and a theoretical support is also provided for subsequent tetracycline action target research.

Description

technical field [0001] The invention relates to the technical field of environment and biological detection, in particular to a novel tetracycline tet34 resistance gene and its application. Background technique [0002] Tetracycline is a broad-spectrum antibiotic that binds to the 30S ribosome and inhibits the entry of aminoacyl tRNA into the receptor site of the ribosome complex of mRNA, thereby ultimately inhibiting the synthesis of proteins in microorganisms, and is effective for both Gram-positive and negative bacteria It has a good inhibitory effect, so it is widely used in the treatment of bacterial infections and in animal husbandry as an additive to promote animal growth. [0003] In recent years, with the extensive use of antibiotics, a large number of antibiotic resistance genes (Antibiotic resistance genes, ARGs) and resistant strains have inevitably been formed. The emergence of tetracycline-resistant strains and resistance genes has led to a significant decline...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869G16B30/00C12N15/11
CPCC12Q1/689C12Q1/6869G16B30/00
Inventor 李炳梁贺彬黄锦
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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