RPA (recombinase polymerase amplification) reagent and device for detecting antibiotic resistance genes and detection method of RPA reagent and device

A technology of drug-resistant genes and antibiotics, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as dependence, very high requirements for amplification reactions, and inability to adapt to different detection samples

Active Publication Date: 2021-06-01
常州市疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LFD-RPA and EXO-RPA are the main detection modes in the product, but RPA products still rely on laboratory operations, have very high requirements for amplification reactions, and rely on essential instruments such as constant temperature amplification instruments
The requirement of on-site detection on the amplification

Method used

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  • RPA (recombinase polymerase amplification) reagent and device for detecting antibiotic resistance genes and detection method of RPA reagent and device
  • RPA (recombinase polymerase amplification) reagent and device for detecting antibiotic resistance genes and detection method of RPA reagent and device
  • RPA (recombinase polymerase amplification) reagent and device for detecting antibiotic resistance genes and detection method of RPA reagent and device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1: Design of upstream and downstream primers and exo and nfo probes for detecting antibiotic resistance genes

[0104] A kind of RPA reagent for detecting antibiotic resistance gene, described antibiotic resistance gene is bla NDM 、bla KPC , mcr, tetLR, the reagents include: for detecting bla NDM Reagent A for drug resistance gene, for detecting bla KPC The reagent B for the drug resistance gene, the reagent C for detecting the mcr drug resistance gene; also includes: any one of the reagent D for detecting the tetL drug resistance gene and the reagent E for detecting the tetR drug resistance gene.

[0105] bla NDM The gene is an antibiotic resistance gene that produces metallo-beta-lactamase NDM, and the relevant primers and probes follow the sequence bla NDM-1 (KX999121.1), bla NDM-2 (KU510393.1), bla NDM-3 (JQ734687.1), bla NDM-4 (KP772213.1), bla NDM-5 (KP772211.1), bla NDM-6 (NG_049338.1), bla NDM-7 (JX262694.1) and other 22 conserved sequences of ...

Embodiment 2

[0185] Example 2: Verification of the Exo amplification system

[0186] Amplification reaction system and amplification conditions: the experimental groups RPND1 and RPND2 use the Exo-1 probe, and the experimental groups RPND3 and RPND4 use the Exo-2 probe. See Table 1 for details. The exo amplification reaction was monitored by a PCR instrument. The amplification program was as follows: set the isothermal mode, pre-amplify at 40°C for 1.0min, and amplify at 40°C for 31sec for a total of 40 cycles.

[0187] Amplification system sensitivity test: mark the plasmid DNA stock solution carrying the antibiotic resistance gene as P0, dilute it by 10 (P1), 100 (P2), 1000 (P3), 10000 (P4) and 100000 (P5) times respectively, and finally Prepare a serial dilution template 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2 and 20 copies / mL, the RPA amplification experiment was carried out respectively, and this method analyzed the detection limit of the RPA amplification system to the templa...

Embodiment 3

[0196]Embodiment 3: Nfo amplification system verification

[0197] Amplification reaction system and amplification conditions: respectively adopt the primer pair and probe pair described in Example 1 containing bla NDM 、bla KPC , mcr, tetLR gene plasmid sequences for simulated amplification experiments. The amplification reaction was carried out with a PCR instrument, and the amplification program was set to an isothermal mode, with 40°C pre-amplification for 1.0 min, and 40°C amplification for 31 sec for a total of 40 cycles. Then pipette 5 μl of the amplification product and add it to 95 μl of PBST to prepare a reaction diluent, insert it into the lateral flow test strip containing the anti-FAM antibody of the commercial kit, and perform interpretation within 5 minutes.

[0198] Amplification system sensitivity test: mark the plasmid DNA stock solution carrying the antibiotic resistance gene as 1, and dilute 10, 100, 1000, 10 4 、10 5 、10 6 、10 7 、10 8 and 10 9 Times,...

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Abstract

The invention provides an RPA (recombinase polymerase amplification) reagent and device for detecting antibiotic resistance genes and a detection method of the RPA reagent and device. The invention discloses the RPA reagent for detecting antibiotic drug resistance genes. The antibiotic drug resistance genes are blaNDM, blaKPC, mcr and tetLR, and the reagent comprises a reagent A for detecting the blaNDM drug resistance gene, a reagent B for detecting the blaKPC drug resistance gene and a reagent C for detecting the mcr drug resistance gene; the kit further comprises any one of a reagent D for detecting a tetL drug-resistant gene and a reagent E for detecting a tetR drug-resistant gene. According to the reagent, the device and the detection method provided by the invention, a normal-temperature amplification mode is adopted to amplify and detect the antibiotic drug resistance gene, the dependence of large-scale instruments and equipment can be completely eliminated, portable miniaturization and mobility are realized, and the requirement of quick detection in environmental sample detection is met.

Description

technical field [0001] The invention relates to the field of antibiotic resistance gene detection, in particular to an RPA reagent, a device and a detection method for detecting antibiotic resistance genes. Background technique [0002] With the widespread use of antibiotics, clinical microorganisms carrying multiple antibiotic resistance genes (ARGs) have become common. The widespread spread of antibiotic resistance genes (ARGs) will inevitably lead to the proliferation of pan-drug-resistant bacteria in water, soil, air, animals and plants, and the control of antibiotic resistance requires the joint attention and actions of all parties. Compared with the clinical situation, the research on drug-resistant microorganisms in the environment started late, and the scale and depth of research are insufficient. The increase and spread of drug-resistant microorganisms or antibiotic resistance genes is very rapid. It is reported that the biomass of drug-resistant microorganisms can...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2563/107C12Q2565/629Y02A50/30
Inventor 屠博文薛银刚毛旭建杜强唐宏兵
Owner 常州市疾病预防控制中心
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