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Rapid quantitative detection method of novel chloramphenicol resistance gene and application thereof

A quantitative detection method and technology of resistance genes, which are applied in the field of rapid quantitative detection of new chloramphenicol resistance genes in environmental samples, can solve the problems of long verification period, inability to detect high-throughput environmental samples, complicated operation, etc., and achieve High accuracy, improved timeliness, high timeliness effect

Pending Publication Date: 2021-08-24
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing new ARGs detection technology mainly relies on the combination of pure culture technology and molecular biology technology. Although this method can achieve accurate qualitative analysis of new ARGs, the verification cycle of this method is long, the operation is relatively complicated, and it is impossible. To achieve high-throughput detection of environmental samples, it is necessary to develop a simple and rapid method for rapid and accurate quantitative analysis of novel chloramphenicol resistance genes in different environmental samples

Method used

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  • Rapid quantitative detection method of novel chloramphenicol resistance gene and application thereof
  • Rapid quantitative detection method of novel chloramphenicol resistance gene and application thereof
  • Rapid quantitative detection method of novel chloramphenicol resistance gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Extraction and sequencing of genomic DNA from different environmental samples

[0030] Surface water and tap water samples were collected in reservoirs and waterworks, respectively, filled into 1.5L sterile glass bottles, and then sent back to the laboratory for subsequent processing. Surface water and tap water samples were filtered with a microporous filter membrane with a pore size of 0.22 μm, and the filter membrane was stored at -80°C until DNA extraction. At the same time, samples of the activated sludge in the reactor were collected and centrifuged at 8000 rpm at 4°C for 10 minutes to collect the precipitate and place it at -80°C for subsequent DNA extraction.

[0031] DNA extraction from different samples was performed using FastDNA TM Spin Kit (MP Biomedicals, USA) kit, specific steps refer to kit instructions. After the genomic DNA was extracted, the integrity and concentration of the genomic DNA were checked by 1% agarose gel electrophoresis and ...

Embodiment 2

[0032] Embodiment 2: Alignment of novel chloramphenicol resistance genes

[0033] 利用实施例1中得到的测序数据进行质控获得cleandata,随后采用DIAMOND软件将双端数据与新型氯霉素抗性基因进行比对,这里以氨基酸序列为SEQNo.1(MWPAGKVLGGGSSINGMMYVRGNRGDYDQWAQLGCKGWSYDDVLPFFNKAETNENGGSRFRGDKGPLRVSNARLSTTLADAFIASGVRAGIPHNPDTNGAEQEGIGPCQATQNKGWRHSTARAYLAKAKRRSNLKVETHFMVSRVLIEKGRAIGVEGVQNGRTVRYLANKEVILCGGALSSPKILMLSGIGPAKHLGEHGIPVVVDSPGVGQNLQEHPGVLMSTHVGIDSLNVEVQSVARIVKHGLNFALFGRGPATACVASALAFIRTRDHLEWPNIQLSFSPIAYDFTPDGVHLYKRAAIGVAINICRPETRGQLLLRSTDPSERPIIQHELLGGDDEIKQLIEGCRIVRKIFRSKPFSEYDKGERLPGKQVETDADWIEYIRQSAFLMYHPTGTCAMGIGPTAVLDPELRVKGVTGLRVADASIMPTLVSANTNAPCIMIGERAADLIRRSH)的新型氯霉 Taking the chloramphenicol resistance gene as an example, obtain a sequence with a similarity higher than 95% with the novel chloramphenicol resistance gene (SEQNo.1) in the comparison result, and the specific parameters are set as follows:

[0034] diamond blastx-d CL5-p 16-q CL0-1_1.fq-f 6-o CL0-1-e 1e-10-k 20--id95.

[0035] The present invention uses 16 threads ...

Embodiment 3

[0036] Embodiment 3: the statistics of bacterial 16S rRNA gene number in different environmental samples

[0037] In order to facilitate the quantitative detection of the new chloramphenicol resistance gene, the 16S rRNA gene in the sample is selected as the internal reference here, and the number of 16S rRNA genes in the paired-end data needs to be counted. The present invention selects and adopts Metaxa2 software to count the number of 16S rRNA genes in each sample, and the specific comparison parameters are as follows:

[0038]metaxa2-1CL0-1_1.fq-2CL0-1_2.fq-o CL0-1

[0039] Such as figure 2 As shown, the result of comparison between the reactor sample in Example 1 and the database shows that a total of 36340 bacterial 16S rRNA genes were detected in the paired-end data of the sample.

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Abstract

The invention relates to the technical field of environmental and biological detection, in particular to a novel rapid quantitative detection method of a chloramphenicol resistance gene and application thereof. The quantitative detection method comprises the following steps: (1) acquiring and pretreating an environmental sample; (2) extracting genome DNA (deoxyribonucleic acid); (3) performing metagenome sequencing; (4) comparing novel chloramphenicol resistance genes and counting the number of bacteria 16S rRNA genes; and (5) making quantitative analysis of the novel chloramphenicol resistance gene. Compared with the prior art, the invention disclosed by the invention is simple and easy to implement, does not need to construct standard plasmids, is not limited by sample types, is relatively high in detection timeliness, is relatively high in comparison result accuracy, can accurately realize quantitative analysis of the gene in different environmental samples, greatly improves the timeliness of accurate detection. And popularization in actual environmental sample monitoring is facilitated.

Description

technical field [0001] The invention relates to the technical field of environmental and biological detection, in particular to a rapid quantitative detection method for a novel chloramphenicol resistance gene in an environmental sample and an application thereof. Background technique [0002] In recent years, the widespread use and even massive abuse of antibiotics has led to an increase in the level of antibiotics in the environment, which has become an important environmental problem. Residues of antibiotics in the environment promote the proliferation of antibiotic-resistant bacteria and the spread of antibiotic resistance genes, seriously threatening human health. [0003] As a class of broad-spectrum antibiotics, chloramphenicol has a good antibacterial effect on bacteria such as Salmonella typhimurium, Escherichia coli and pneumococcus, so it has been widely used in clinic. With the deepening of research and its widespread use, researchers have found that the resista...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2535/122C12Q2537/165
Inventor 李炳梁贺彬张家禹黄锦
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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