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CN-122012765-A - Primer pair for detecting enterococcus drug resistance gene poxtA based on RAA-agarose gel electrophoresis and application thereof

CN122012765ACN 122012765 ACN122012765 ACN 122012765ACN-122012765-A

Abstract

The invention belongs to the technical field of microorganism detection, and particularly relates to a primer pair for detecting enterococcus drug-resistant genes poxtA based on RAA-agarose gel electrophoresis and application thereof, wherein the primer pair consists of an upstream primer with a sequence shown in SEQ ID NO.1 and a downstream primer with a sequence shown in SEQ ID NO.2, and can be used for detecting enterococcus drug-resistant or preparing a kit for detecting enterococcus drug-resistant. Based on the primer pair, the invention also provides a method for detecting drug-resistant enterococcus based on RAA-agarose gel electrophoresis development, the method can finish amplification within 15min under the isothermal condition of 30 ℃, and the result can be judged by gel electrophoresis, so that the detection result is accurate and reliable, and the method has the advantages of strong specificity, high sensitivity, rapidness and visualization.

Inventors

  • Xuan Huiyong
  • WANG RUINAN
  • Ji Yuhao
  • ZHANG YUTING
  • HUANG TINGHUANG
  • WANG YINA
  • Cheng Dexiu
  • ZHANG YING
  • ZHANG MINGRUI
  • WANG FANG
  • CUI YAOCHENG
  • LIAN KEXUN
  • QI YAYIN
  • WANG SHU
  • Cao Shuzhu
  • WANG ZEXUAN
  • HAN YALI

Assignees

  • 石河子大学

Dates

Publication Date
20260512
Application Date
20260330

Claims (9)

  1. 1. A primer pair for detecting enterococcus drug resistance gene poxtA based on RAA-agarose gel electrophoresis is characterized in that the primer pair consists of an upstream primer of a sequence shown as SEQ ID NO.1 and a downstream primer of a sequence shown as SEQ ID NO. 2.
  2. 2. Use of a primer pair according to claim 1 in the preparation of a kit for detecting enterococcus resistant bacteria.
  3. 3. A kit for detecting a drug-resistant enterococcus, comprising the primer pair of claim 1.
  4. 4. The kit of claim 3, further comprising buffers, enzyme preparations and deoxyribonucleoside triphosphates required for a recombinase-mediated isothermal amplification reaction.
  5. 5. Use of a primer pair according to claim 1 for detecting and/or identifying a drug-resistant enterococcus.
  6. 6. The method for detecting the drug-resistant enterococcus is characterized by comprising the following steps of: extracting DNA of a sample to be detected; carrying out a recombinase-mediated isothermal amplification reaction by using the primer pair as claimed in claim 1 with the DNA as a template to obtain an amplification product; And (3) carrying out agarose gel electrophoresis on the amplified product, and judging the result according to the electrophoresis result, wherein if a specific band appears at a position of 225bp, the sample to be detected is judged to be the drug-resistant enterococcus or the sample to be detected contains the drug-resistant enterococcus.
  7. 7. The detection method according to claim 6, wherein the temperature of the recombinase-mediated isothermal amplification reaction is 25-41 ℃ and the time is 10-30 min.
  8. 8. The method according to claim 6, wherein the system for the 50. Mu.L recombinase mediated isothermal amplification reaction comprises 25. Mu.L A Buffer, 13.5. Mu.L ddH 2 O, 2. Mu.L 5.0. Mu.mol/L upstream primer, 2. Mu.L 5.0. Mu.mol/L downstream primer, 5. Mu.L DNA, 2.5. Mu.L B Buffer, and RAA reaction universal dry powder tube.
  9. 9. The method according to claim 8, wherein the A Buffer contains 10w/v% polyethylene glycol per 25. Mu.L, the B Buffer contains 280mM magnesium acetate per 2.5. Mu.L, and the RAA reaction universal dry powder comprises dNTPs, recombinase, single-stranded DNA binding protein and DNA polymerase.

Description

Primer pair for detecting enterococcus drug resistance gene poxtA based on RAA-agarose gel electrophoresis and application thereof Technical Field The invention belongs to the technical field of microorganism detection, and particularly relates to a primer pair for detecting enterococcus drug resistance genes poxtA based on RAA-agarose gel electrophoresis and application thereof. Background The prevalence of multi-drug resistance (Multiple drug resistance, MDR) is increasing a threat to human health. Antimicrobial drug resistance (AMR-Microbial Resistance) has become an urgent problem in the fields of human medicine and veterinary medicine. Food-producing animals are generally considered as drug-resistant bacteria and as reservoirs of transferable drug-resistant genes that can be transmitted in different hosts and environmental media. Enterococcus is regarded as a gram-positive intestinal symbiotic bacterium and conditional pathogenic bacterium, and is attracting attention because of its ability to persist in various environments such as water, soil and vegetation, and its high inherent drug resistance. Of particular importance, enterococci exhibit significant genomic plasticity, characterized by the frequent acquisition, maintenance and exchange of antibacterial drug resistance genes by Moving Genetic Elements (MGEs). This genomic flexibility allows the bacterium to quickly adapt to selective stress and promotes the spread of drug-resistant genes such as optrA, poxtA, etc. between humans, animals and environmental reservoirs, thus enhancing its role in the "integrated health" framework. In an intensive culture system, bacteria of the genus enterococcus with multiple drug resistance can be bidirectionally transmitted between people and animal groups through the ways of food chains, occupational exposure, environmental pollution and the like. Transferable drug resistance gene poxtA mediates cross drug resistance of florfenicol and linezolid, and presents serious challenges for veterinary and human medicine. PoxtA gene codes an ABC-F family protein, and can mediate the drug resistance of bacteria to oxazolidinone and amidol drugs through a ribosome protection mechanism. The presence of poxtA reduces not only the sensitivity of the bacteria to oxazolidone and florfenicol, but also to tetracycline. The poxtA gene was found in a clinically isolated strain of methicillin-resistant staphylococcus aureus in 2018 at the earliest, and the amino acid sequence homology with optrA is about 32%. Notably, poxtA is often co-present with other drug-resistant genes such as optrA in the same strain, and can be transferred horizontally between different enterococci by mobile genetic elements such as plasmids, and can stably exist in a non-selective pressure environment even under the condition of no detectable conjugation activity, thereby exacerbating the spread and duration of multiple drug resistance. In view of the increasing frequency of poxtA genes in animal sources, environments and clinical strains, and the serious threat of cross-resistance mediated by poxtA genes to the efficacy of final defensive antibiotics such as linezolid, a high-efficiency and accurate detection means are needed to be established to realize the monitoring of the genes. However, the existing reagent for detecting poxtA genes based on common PCR and fluorescent quantitative PCR has the problems of difficulty in meeting the requirements of on-site rapid detection, long detection period, high technical requirements on operators and dependence on expensive instruments during use. Disclosure of Invention In order to solve the problems, the invention provides a primer pair for detecting enterococcus drug resistance genes poxtA based on RAA-agarose gel electrophoresis and application thereof. The primer pair consists of an upstream primer of a sequence shown as SEQ ID NO.1 and a downstream primer of a sequence shown as SEQ ID NO.2, and can be used for detecting drug-resistant enterococci or preparing a kit for detecting the drug-resistant enterococci, and the detection method has the advantages of rapidness, high efficiency, accurate result, strong specificity, high sensitivity and visualized result. In order to achieve the above purpose, the specific technical scheme of the invention is as follows: The first aspect of the invention provides a primer pair for detecting enterococcus drug resistance gene poxtA based on RAA-agarose gel electrophoresis, which consists of an upstream primer of a sequence shown as SEQ ID NO.1 and a downstream primer of a sequence shown as SEQ ID NO. 2. Further, the detection target of the primer pair is a conserved sequence fragment in the CDS region of the drug resistance gene poxtA, and the accession number of the complete sequence of the CDS region of the drug resistance gene poxtA in GenBank is NG_063824.1. Further, the enterococcus includes enterococcus faecium and enterococcus faecalis. The invention also provides an a