Detection method of bacterial drug resistance gen and special kit thereof

A drug-resistant gene and kit technology, applied in the biological field, can solve problems such as difficult and accurate detection

Active Publication Date: 2019-05-17
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Flight mass spectrometry and biochemical detection can only identify pathogens producing carbapenemase and beta lactamase, and it is difficult to accurately detect other drug-resistant bacteria
In addition, bacterial resistance detec

Method used

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  • Detection method of bacterial drug resistance gen and special kit thereof
  • Detection method of bacterial drug resistance gen and special kit thereof
  • Detection method of bacterial drug resistance gen and special kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the acquisition of bacterial resistance gene and the design of detection primers

[0036] First, the acquisition of drug resistance genes

[0037] According to the types of commonly used clinical antibiotics and the types of common clinical pathogens, download from The Comprehensive Antibiotic Resistance Database (CARD; http: / / apcard.mcmaster.ca) and ARDB database (http: / / ardb.cbcb.umd.edu) The relevant fragments of drug resistance genes are sorted according to the types of pathogens, antibiotics, and drug resistance genes. The sorting method is as follows: The related fragments of drug resistance genes downloaded from ARDB database and CARD database are clustered by cd-hit, and 95% As the threshold, redundant sequences were removed; then BLAST was used to align the obtained fragments between species, and with 97% as the threshold, the specific fragments of drug resistance sites were screened to remove redundant sequences. Finally, a total of 68 drug resi...

Embodiment 2

[0054] Example 2. Application of primers for detecting bacterial resistance genes

[0055] (1) Detection of drug resistance genes of pathogens to be tested

[0056] 1. Extraction of pathogen nucleic acid

[0057] The nucleic acids of the pathogens to be detected in Table 4 were extracted by the magnetic bead method using Qiagen's MagAttract HMW DNA Kit. The specific extraction steps were carried out according to the instructions of the kit.

[0058] Table 4. Pathogens to be detected

[0059]

[0060]

[0061] 2. Construction of gene library

[0062] Library construction was performed using the Ion Ampliseq Library Kit 2.0 (Thermo Fisher Scientific, Cat. no. 4480441) based on the Ampliseq technology. Specific steps are as follows:

[0063] 1. Establish a DNA target amplification reaction with 2 primer pools

[0064] Ampliseq primers are divided into primer pool 1 (primer pool 1) and primer pool 2 (primer pool 2). The primers in primer pool 1 consist of the primers m...

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Abstract

The invention discloses a detection method of a bacterial drug resistance gene and a special kit thereof. The kit comprises 296 single-stranded DNAs shown by sequences 1-296. The detection method of the bacterial drug resistance gene comprises the following steps: (1) extracting a genome DNA of a bacterium to be detected, and amplifying the genome DNA by using a complete set of single-stranded DNAs to obtain an amplification product; (2) constructing a sequencing library by using the amplification products based on the Ampliseq technology; (3) sequencing the sequencing library to obtain a sequencing result, and comparing the sequencing result with the drug resistance gene sequence to determine whether the bacterium to be detected carry the drug resistance gene or not. The detection methodand the kit can simultaneously detect multiple drug resistance genes of a sample to be detected with high throughput, rapidness and accuracy, and have a good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting bacterial drug resistance genes and a special kit thereof. Background technique [0002] The detection of bacterial resistance genes is of great significance in clinical diagnosis. At present, the widely used commercial bacterial drug resistance gene detection methods mainly include the following four: Vitek2 of automatic detection system, flight mass spectrometry MALDI-TOF, rapid biochemical detection and real-time image monitoring. Vitek2 showed good consistency in the detection of clinical samples, but the test samples need to be positive cultures, while the general blood samples report positive time for at least 24 hours, which greatly delays the timing of treatment. The same problem exists in real-time image monitoring. Flight mass spectrometry and biochemical detection can only identify pathogens that produce carbapenemase and beta-lactamase, and it is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/14C12Q1/10C12Q1/04C12N15/11
Inventor 刘鹏李倩姜永强律清宇江华郑玉玲
Owner ACADEMY OF MILITARY MEDICAL SCI
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