A kit for detecting and / or identifying drug-resistant enterococci and use thereof

Abstract

This invention belongs to the field of microbial detection technology, specifically relating to a kit for detecting and / or identifying drug-resistant enterococci and its application. The kit includes primer pairs as shown in SEQ ID NO.3 and SEQ ID NO.4. Based on the kit, this invention establishes an oxazolidinone resistance gene. oprA The RAA-agarose gel electrophoresis assay can complete amplification within 15 minutes under isothermal conditions at 35°C and exhibits high specificity. The detection limit of this method is 10. 1 With a detection limit of copies / μL, two orders of magnitude lower than that of PCR, and 100-fold higher sensitivity, this invention provides a new option for rapid and visualized detection of drug-resistant enterococci, suitable for clinical practice; it also provides a new method for detecting oxazolidinone resistance genes. oprA It provides a convenient tool for rapid screening, diagnosis and monitoring, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a kit for detecting and / or identifying drug-resistant enterococci and its application. Background Technology

[0002] In recent years, with the increasing demand for meat, antibiotics have been widely used in animal husbandry, especially in poultry. Antibiotic resistance has become a major concern in the livestock sector, and antibiotic resistance genes in bacteria are highly prevalent in poultry. Resistant bacteria can be transmitted to humans via genetic pathways, and these genes can transfer not only within the same species but also between different species, posing a significant threat to poultry and human health. Oxazolidinone antibiotics are a new class of fully synthetic antibiotics that inhibit the growth of various Gram-positive bacteria by interfering with bacterial ribosome function and preventing the initiation of protein synthesis. Linezolid, a representative drug, was the first oxazolidinone antibiotic approved for clinical use and was considered a "last resort" for treating enterococcal infections. However, with the increased use of this class of drugs in veterinary and clinical settings, drug-resistant enterococci are constantly emerging, severely weakening the therapeutic effect of linezolid.

[0003] Currently, detection methods for drug-resistant enterococci mainly include traditional microbial culture and drug susceptibility testing, conventional polymerase chain reaction (PCR), and sequencing technologies. However, these methods have significant drawbacks: they are time-consuming, cumbersome to operate, lack sufficient sensitivity and specificity, and rely on expensive equipment and specialized personnel. Therefore, there is an urgent need to develop a new strategy for the rapid, accurate, and convenient detection and / or identification of drug-resistant enterococci, especially those carrying oxazolidinone resistance genes. Summary of the Invention

[0004] The purpose of this invention is to provide a kit for detecting and / or identifying drug-resistant enterococci, which solves the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: The present invention provides a kit for detecting and / or identifying drug-resistant enterococci, the kit comprising primer pairs as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0006] Preferably, the kit further includes at least one of recombinase, DNA polymerase, dNTPs, and buffer.

[0007] A second aspect of the present invention provides an application of the reagent kit, wherein the application refers to: The kit is used to detect and / or identify the sample by recombinase-mediated isothermal amplification. If a specific band appears after electrophoresis, the sample is a drug-resistant enterococcus.

[0008] Preferably, the method for detecting drug-resistant enterococci is as follows: Extract DNA from the sample to be tested; Using DNA as a template, the primer pair was used to perform a recombinase-mediated isothermal amplification reaction to obtain the amplification product. The amplification products were subjected to electrophoresis. The electrophoresis results were observed. If a specific band appeared at the 157bp position, the sample to be tested was determined to be drug-resistant enterococci.

[0009] Preferably, the conditions for the recombinase-mediated isothermal amplification reaction are: 25℃~41℃, 10min~30min.

[0010] Preferably, the conditions for the recombinase-mediated isothermal amplification reaction are: 35°C for 15 min.

[0011] Preferably, the drug-resistant enterococci refer to enterococci that are resistant to oxazolidinone drugs.

[0012] Preferably, the oxazolidinone drug includes at least one of linezolid, terdizolid, and retazolid.

[0013] Preferably, the enterococcus is Enterococcus faecalis and / or Enterococcus faecium.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a kit for detecting and / or identifying drug-resistant bacteria, the kit comprising the primer pairs shown in SEQ ID NO. 3 and SEQ ID NO. 4. Based on the kit, this invention establishes an oxazolidinone resistance gene. optrA The RAA-agarose gel electrophoresis detection method can complete amplification within 15 minutes under isothermal conditions at 35℃ and has high specificity. After amplification products are subjected to agarose gel electrophoresis for 30 minutes, the detection results can be visually observed using a UV analyzer, providing a possibility for rapid detection in grassroots areas. The detection limit of this method is 10. 1 copies / μL, compared to the detection limit of PCR (10 3 The sensitivity is 100 times higher than that of oxazolidinone resistance genes (copies / μL) with a difference of two orders of magnitude lower. In summary, the kit described in this invention addresses oxazolidinone resistance genes. optrA This provides a new option for rapid, visualized detection, suitable for clinical practice. It is useful for identifying oxazolidinone resistance genes. optrA It provides a convenient tool for rapid screening, diagnosis and monitoring, and has good application prospects. Attached Figure Description

[0015] Figure 1 for optrA Primer screening; A: The gel imaging system presents a grayscale image; B: The image appears as an ultraviolet electrophoresis image under ultraviolet light; Lanes 1 through 4 in A and B: These represent the detection results of primer pairs 1 through 4, respectively; M: DNA relative molecular mass standard; N: Blank control.

[0016] Figure 2 for optrA Specificity test results of the RAA detection method; A: The gel imaging system presents a grayscale image; B: The image appears as an ultraviolet electrophoresis image under ultraviolet light; In A and B, M: relative molecular mass standard of DNA; N: blank control; Lane 1 and Lane 2: Carry optrA Two repeats of Enterococcus faecalis DNA in the gene; Lanes 3 and 4: Carry optrA Two repeats of Enterococcus faecalis DNA in the gene; Lanes 5 through 12 are for swimmers carrying oxazolidinone resistance genes. poxtA Enterococcus faecalis carrying oxazolidinone resistance genes poxtA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying tetracycline resistance genes tetM Enterococcus faecalis carrying amyl alcohol resistance genes fexA Enterococcus faecalis carrying amyl alcohol resistance genes floR Enterococcus faecalis carrying carbapenem resistance genes blaNDM DNA and ddH2O from Escherichia coli.

[0017] Figure 3 The results show the optimized reaction conditions for RAA detection; A: Primer concentration optimization grayscale image, lanes 1 to 5 are respectively: final primer concentrations of 1 μmol / L, 2.5 μmol / L, 5 μmol / L, 7.5 μmol / L and 10 μmol / L; B: Primer concentration optimization UV image, lanes 1 to 5 are respectively: final primer concentrations of 1 μmol / L, 2.5 μmol / L, 5 μmol / L, 7.5 μmol / L and 10 μmol / L; C: Temperature-optimized grayscale image, lanes 1 to 6 are 25℃, 30℃, 35℃, 37℃, 39℃, and 41℃ respectively; D: Temperature-optimized UV images, lanes 1 to 6 are 25℃, 30℃, 35℃, 37℃, 39℃, and 41℃ respectively; E: Time-optimized grayscale images, lanes 1 to 5 are 10 min, 15 min, 20 min, 25 min and 30 min respectively; F: Time-optimized UV images, lanes 1 to 5 are 10 min, 15 min, 20 min, 25 min and 30 min respectively; In A~F, M: relative molecular mass standard of DNA; N: blank control.

[0018] Figure 4 for optrA Sensitivity results of the RAA detection method; A: The RAA test results are presented as grayscale images by the gel imaging system; B: The RAA detection results are presented as ultraviolet electrophoresis images under ultraviolet light; C: Results of sensitivity testing using the PCR method; D: Results of sensitivity testing using the qPCR method; In A through D, 1 through 8 are respectively: optrA Template concentration 10 7 copies / μL~10 0 copies / μL; Lane 9: negative control; M: relative molecular mass standard of DNA; N: blank control.

[0019] Figure 5 for optrA Results of repeatability tests within the RAA group; A: Template concentration is 10 5 At copies / μL, the grayscale image presented by the gel imaging system, lanes 1 to 3 represent 3 biological replicates; N: blank control; B: Template concentration is 10 4 At copies / μL, the grayscale image presented by the gel imaging system, lanes 4 to 6 represent 3 biological replicates; N: blank control; C: Template concentration is 10 3 At copies / μL, the grayscale image presented by the gel imaging system, lanes 7-9 represent 3 biological replicates; N: blank control; D: Template concentration is 10 5 At copies / μL, the UV electrophoresis image presented by the gel imaging system, lanes 1 to 3 represent 3 biological replicates; N: blank control; E: Template concentration is 10 4At copies / μL, the UV electrophoresis image presented by the gel imaging system, lanes 4-6 represent 3 biological replicates; N: blank control; F: Template concentration is 10 3 When copies / μL, the UV electrophoresis image presented by the gel imaging system, lanes 7 to 9 represent 3 biological replicates; N: blank control.

[0020] Figure 6 for optrA Results of the RAA intergroup repeatability trial; A~C: Grayscale images of three biological replicates at different template concentrations; in A~C, lane 1: template concentration is 10. 5 copies / μL; Lane 2: template concentration of 10 copies / μL; 4 copies / μL; Lane 3: template concentration is 10 copies / μL; 3 copies / μL; N: blank control; D~F: UV electrophoresis images of three biological replicates at different template concentrations; in D~F, lane 1: template concentration of 10 5 copies / μL; Lane 2: template concentration of 10 copies / μL; 4 copies / μL; Lane 3: template concentration is 10 copies / μL; 3 copies / μL; N: blank control. Detailed Implementation

[0021] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0022] The inventive concept of this invention is as follows: In recent years, transferable oxazolidinone resistance genes, such as optrA The emergence and rapid spread of the disease have reduced its clinical efficacy. optrA The gene-encoded ATP-binding cassette (ABC)-F protein protects bacterial ribosomes from antibiotic inhibition, thus preventing cross-resistance to linezolid and florfenicol. optrA As a novel transferable resistance gene within the oxazolidinone class, it confers resistance to so-called "last resort" antibiotics through horizontal transmission of mobile genetic elements. Therefore, in order to better detect... optrA For genes, a variety of detection methods have been established, including conventional PCR and quantitative real-time PCR.

[0023] Recombinase-mediated isothermal amplification (RAA) is a novel isothermal nucleic acid amplification technique that enables gene amplification under isothermal conditions in a short time, characterized by its simplicity, speed, and high precision. In recent years, the RAA method has been successfully applied to the detection of various human and animal drug resistance genes. The RAA-agarose gel electrophoresis method allows for interpretation of results via gel electrophoresis and has previously been used to detect other drug resistance genes. Compared to fluorescent RAA, RAA-assisted lateral flow test strips, and RAA-assisted CRISPR-Cas13a / Cas12a methods, the RAA-agarose gel electrophoresis method only requires the design of RAA-specific primers, eliminating the need for additional probes and consumables such as test strips, thus resulting in lower detection costs. This method requires only simple equipment (UV analyzer) for visual observation, making it more suitable for use by grassroots veterinarians. This invention establishes a highly specific, sensitive, rapid, and visually accurate RAA-agarose gel electrophoresis method for the detection of optrA, providing a new tool for the clinical diagnostic assessment of optrA.

[0024] This invention targets chicken-derived Enterococcus. optrA Primers were designed from the conserved regions of the gene. After primer screening, the primer concentration, reaction temperature, and reaction time of the RAA-agarose gel electrophoresis method were further optimized, and the specificity and sensitivity of the method were analyzed. The results showed that under isothermal conditions of 35°C, the RAA amplification reaction targeting the gene could be completed in 15 min; after 30 min of agarose gel electrophoresis, clear specific bands could be directly observed using a UV analyzer. This invention demonstrates that the RAA detection system has high specificity, no cross-reactivity with other common drug-resistant gene strains, good reproducibility, high sensitivity, and a limit of detection of 2.22 × 10⁻⁶. 1 copies / μL.

[0025] To further verify its clinical applicability, parallel tests were performed on 100 chicken anal swab samples using three methods: RAA, polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR). The results showed that the detection concordance rate between RAA and PCR was 99%, and the concordance rate with qPCR was also 99%. In summary, the RAA-agarose gel electrophoresis method established in this invention is simple to operate, highly specific, and highly sensitive. This method is suitable for primary care institutions equipped with basic laboratories to conduct studies on oxazolidinone resistance genes. optrA Diagnostic work has promising applications.

[0026] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0027] The list of abbreviations for this invention is shown in Table 1.

[0028] Table 1 List of Abbreviations The materials and methods used in this invention are as follows: 1. Main reagents.

[0029] RAA nucleic acid amplification reagent (basic type) was purchased from Hangzhou Zhongce Biotechnology Co., Ltd. Gel extraction kit, DNA purification kit, plasmid miniprep kit, and pMD19-T vector were all purchased from Novizan Biotechnology Co., Ltd. A 25:24:1 mixture of phenol, chloroform, and isoamyl alcohol, and Biowest agarose were purchased from Xinjiang Hengchao Biotechnology Co., Ltd. *E. coli* DH5α competent cells were purchased from Weidi Biotechnology Co., Ltd. Ampicillin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. 2× Universal Blue SYBR Green qPCR Master Mix and DNA molecular weight standard markers were purchased from Wuhan Sewell Biotechnology Co., Ltd. Gold View nucleic acid dye was purchased from Beijing Bio-Tech Technology Co., Ltd. 2× Es Taq PCR Master Mix (Dye) and ddH2O were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.

[0030] 2. Main equipment.

[0031] The water bath was purchased from Zhejiang Qun'an Scientific Instruments Co., Ltd. The high-speed benchtop centrifuge was purchased from Nanjing Haidixi Equipment Co., Ltd. The PCR instrument was purchased from Thermo Fisher Scientific. The real-time PCR instrument was purchased from Tianlong Technology Co., Ltd. The electrophoresis gel imaging system was purchased from Bio-Rad (USA).

[0032] 3. Strains and clinical samples.

[0033] Carrying oxazolidinone resistance genes optrA Enterococcus faecalis ( Enterococcus faecium ) and Enterococcus faecalis ( Enterococcus faecalis The DNA of the virus carries an oxazolidinone resistance gene. poxtA Enterococcus faecalis carrying oxazolidinone resistance genes poxtA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying tetracycline resistance genes tetM Enterococcus faecalis carrying amyl alcohol resistance genes fexA Enterococcus faecalis carrying amyl alcohol resistance genes floR Enterococcus faecalis carrying carbapenem resistance genes blaNDMAll E. coli samples were clinical isolates preserved in the laboratory. A total of 100 anal swab samples were collected from chickens in Shihezi City, Xinjiang, and stored at -80°C.

[0034] 4. Nucleic acid extraction.

[0035] DNA templates for Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Klebsiella pneumoniae were extracted according to the instructions of the DNA extraction kit. All DNA samples were stored at -20°C.

[0036] 5. Preparation of standard plasmids.

[0037] Enterococcus amplified by PCR optrA Genes. Enterococci. optrA The primers for full-length gene PCR amplification are shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0038] Forward primer, SEQ ID NO.9: TACTAACGCAAAGGAGGATAT.

[0039] Reverse primer, SEQ ID NO.10: ATTCTCTCATCAACTGTTCCC.

[0040] The PCR amplification reaction system is as follows: 10 μL PCR Mix, 6 μL ddH2O, 1 μL each of forward and reverse primers (10 μmol / L), and 2 μL template DNA.

[0041] The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 32 cycles; final extension at 72℃ for 7 min. After the reaction, the amplified fragment was ligated into the pMD19-T vector and transformed into DH5α competent cells. Positive strains were screened by bacterial culture PCR and sent to Youkang Biotechnology Co., Ltd. for sequencing. Sequencing results were compared with NCBI. Plasmid DNA was extracted using the plasmid extraction kit instructions and stored at -20℃. After PCR and sequencing verification of the correct recombinant plasmid, its concentration was determined using an ultra-micro spectrophotometer, and the copy number was calculated to obtain pMD19-T. optrA Plasmid.

[0042] Example 1 A kit for detecting and / or identifying drug-resistant enterococci, as detailed below: 0. Primer design and establishment of the RAA reaction system.

[0043] (1) Primer design.

[0044] According to information published in GenBank optrAFour pairs of specific primers were designed using Oligo 7.0 software based on the conserved region of the gene (accession number: KP399637.1) and the RAA reaction principle. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 2.

[0045] Table 2 Primer sequences (2) Establishment of the RAA reaction system.

[0046] According to the RAA basic nucleic acid amplification kit instructions, a 50 μL RAA reaction system was prepared, including: 0) Prepare the premixed solution.

[0047] The premixed solution consists of: 25 μL A Buffer, 13.5 μL ddH2O, 2 μL of 10 μmol / L upstream primer, and 2 μL of 10 μmol / L downstream primer.

[0048] 2) RAA reaction.

[0049] After thoroughly mixing the premixed solution in a clean PCR reaction tube, add it to the detection unit tube containing the reaction powder. Then, add 5 μL of the DNA sample to be tested to the detection unit tube and drop 2.5 μL of B Buffer inside the cap. Tightly cap the tube, gently invert it, and tap the tube wall 6 times to mix thoroughly. Centrifuge rapidly for 10 seconds and incubate at 39°C for 30 minutes. After the reaction, add 50 μL of a mixture of phenol, chloroform, and isoamyl alcohol (V:25:24:1) to the detection unit tube, mix thoroughly, and centrifuge at 12000 rpm for 5 minutes. Perform agarose gel electrophoresis on the supernatant. The electrophoresis results are presented as grayscale images using a gel imaging system and as UV electrophoresis images under UV light.

[0050] Using the four primer pairs shown in Table 2 optrA The gene was amplified using the RAA method, and detected by 20 g / L agarose gel electrophoresis. Primer pairs were then selected based on the detection results. Results are as follows: Figure 1 As shown, of the four primer pairs designed, primer pair 2, i.e. optrA -F2 / optrA -R2 primers produce amplified bands with high brightness, good clarity, and minimal non-specific bands. Therefore, they are the preferred choice. optrA -F2 / optrA -R2 was selected as the optimal primer pair for subsequent experiments.

[0051] 2. Specific test results.

[0052] Will carry oxazolidinone resistance genes optrAThe DNA of *Enterococcus faecalis* carries oxazolidinone resistance genes. poxtA Enterococcus faecalis carrying oxazolidinone resistance genes poxtA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying tetracycline resistance genes tetM Enterococcus faecalis carrying amyl alcohol resistance genes fexA Enterococcus faecalis carrying amyl alcohol resistance genes floR Enterococcus faecalis carrying carbapenem resistance genes blaNDM Using E. coli DNA as a template and ddH2O as a negative control, the RAA amplification method was the same as described above. Detection... optrA -F2 / optrA -R2 specificity. Results showed that all carriers optrA Both Enterococcus faecalis and Enterococcus faecium DNA can amplify the target band, while other strains do not carry the gene. optrA No bands were amplified in the DNA of the genes, which is unprecedented. Figure 2 .

[0053] Meanwhile, specificity tests were conducted using primer pairs 1, 3, and 4. Under the same detection conditions and with the same samples, none of these primer pairs amplified any bands. These results indicate that the present invention is based on... optrA -F2 / optrA -R2 established optrA The RAA detection method has good specificity and no cross-reactivity with other common drug resistance genes.

[0054] Example 2 An application of a kit for detecting and / or identifying drug-resistant enterococci is as follows: 1. Optimization of RAA reaction primer concentration, temperature and time.

[0055] Experiment 1: [The text appears to be incomplete and contains several grammatical errors. A more accurate translation would require the full context.] optrA -F2 / optrA -R2 was used for dilution. With other components in the reaction system described in Example 1 unchanged, upstream and downstream primers were added to the reaction system at final concentrations of 1 μmol / L, 2.5 μmol / L, 5 μmol / L, 7.5 μmol / L and 10 μmol / L, respectively, to screen for the optimal primer concentration.

[0056] Experiment 2: Based on the optimal primer concentration, the reaction was carried out at 25℃, 30℃, 35℃, 37℃, 39℃ and 41℃ for 30 min respectively to screen the optimal reaction temperature.

[0057] Experiment 3: The reaction time was set to 10 min, 15 min, 20 min, 25 min and 30 min to optimize the optimal reaction time.

[0058] All RAA amplification products were detected by agarose gel electrophoresis using the method described above. The results are as follows: The results of Experiment 1 are as follows Figure 3 As shown in Figures A and B, the target band brightness increases with increasing primer concentration, indicating enhanced RAA amplification ability. Considering all factors, 7.5 μmol / L was selected as the optimal primer concentration.

[0059] Based on the above optimal primer concentrations, RAA detection was performed at different temperatures to test the optimal reaction temperature. The results are as follows: Figure 3 As shown in C and D, bands can be amplified in the temperature range of 25℃ to 41℃, and the band brightness is high in the range of 35℃ to 41℃; therefore, the lower temperature of 35℃ in this range was selected for subsequent RAA detection.

[0060] Based on the optimal primer concentration and reaction temperature, RAA assays were performed at different time intervals to determine the optimal reaction time. The results are as follows: Figure 3 As shown in E and F, clear target bands can be observed in the reaction time range of 10 min to 30 min, with the band brightness being better in the range of 15 min to 30 min; therefore, the shorter time of 15 min in this range was selected for subsequent experiments.

[0061] 2. Comparison of the sensitivity of RAA, PCR and qPCR.

[0062] pMD19- optrA The plasmid was serially diluted 10-fold to achieve a concentration range of 2.22 × 10⁻⁶. 7 copies / μL up to 2.22×10 0 The samples were taken at a density of 10 copies / μL and used as a template to determine the sensitivity of three methods: RAA, PCR, and qPCR.

[0063] RAA testing was performed under the optimized conditions described above; the primers used for PCR testing were the same as those used for RAA.

[0064] The PCR amplification reaction system is as follows: 10 μL PCR Mix, 6 μL ddH2O, 1 μL each of forward and reverse primers (10 μmol / L), and 2 μL template DNA.

[0065] The PCR reaction procedure is as follows: Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 56.5℃ for 30 s, extension at 72℃ for 30 s, for a total of 32 cycles; final extension at 72℃ for 7 min.

[0066] qPCR detection was performed using SYBR Green qPCR Mix, with the same primers used in PCR. The composition of the 10 μL qPCR reaction system is as follows:

[0067] 5 μL qPCR Mix, 0.2 μL each of forward and reverse primers (10 μM), 3.6 μL sterile water, and 1 μL template DNA.

[0068] The qPCR reaction program is as follows: the reaction solution is first pre-denatured at 95℃ for 30s; then 40 cycles are performed, each cycle including denaturation at 95℃ for 15s, annealing at 60℃ for 10s, and extension at 72℃ for 30s.

[0069] The results are as follows Figure 4 As shown, the limits of detection for RAA, PCR, and qPCR are 2.22 × 10⁻⁶, respectively. 1 copies / μL, 2.22×10 3 copies / μL and 2.22copies / μL.

[0070] 3. Stability test.

[0071] The invention established optrA Nucleic acid detection methods for 2.22 × 10 5 copies / μL, 2.22×10 4 copies / μL, 2.22×10 3 Using recombinant plasmids at three concentrations of copies / μL as templates, three intra- and inter-group repeatability tests were performed to analyze the amplification results.

[0072] Results of intragroup repeatability tests are as follows Figure 5 As shown, the target band was successfully amplified at all concentrations, and the brightness of the amplified bands in each repeated detection at the same concentration was basically the same. The results of the inter-group repeatability test are as follows: Figure 6 As shown, all concentrations successfully amplified the target band in three replicate experiments, and the band brightness was essentially consistent across different replicate groups. This demonstrates the effectiveness of the method established in this invention. optrA The RAA-agarose gel electrophoresis detection method has good stability.

[0073] 4. Clinical sample testing.

[0074] This invention collected 100 anal swab samples from chickens in three broiler farms. The established RAA detection method, PCR method, and qPCR method were used to test all samples, and the concordance rates among the three methods were compared.

[0075] The results showed that the positive rate of RAA detection was 48% (48 / 100); the positive rate of PCR detection was 47% (47 / 100); and the positive rate of qPCR detection was 49% (49 / 100). The concordance rate between RAA and PCR detection was 99%, and the concordance rate between RAA and qPCR detection was 99%. The results are shown in Table 3.

[0076] Furthermore, all 47 samples that tested positive by PCR also tested positive by RAA, meaning that the sensitivity of RAA relative to PCR was 100%; of the 53 samples that tested negative by PCR, 52 also tested negative by RAA, meaning that the specificity of RAA relative to PCR was 97.7%.

[0077] Compared with the PCR method, the kappa value of the RAA detection method established in this invention is 0.98 (K>0.75), as shown in Table 4. These results indicate that the RAA detection method established in this invention can be used in clinical samples. optrA The detection.

[0078] Table 3. Detection results of clinical samples using different methods (n=100) Table 4. RAA detection methods and PCR methods for detecting RAA in clinical samples. optrA Performance comparison (n=100) Note: In Table 4, PPV: Positive predictive value; NPV: Negative predictive value.

[0079] optrA Genes have been confirmed as a key factor in linezolid resistance in Enterococci, and their detection rate in Enterococci is gradually increasing. Although the vast majority of Gram-positive bacteria are sensitive to linezolid and resistance is still uncommon, various antimicrobial surveillance studies or projects show that the number of linezolid-resistant Enterococcal strains is on the rise. Therefore, a simple, rapid, and accurate detection method is crucial. optrA Early clinical diagnosis and prevention of infection are crucial.

[0080] Currently, in most laboratories, PCR and qPCR are still the most common methods. optrAThe main techniques for molecular detection. However, the requirements for specific instruments and the technical skills required of operators limit the application of these methods in the field. RAA detection methods have low requirements for the detection environment and equipment, are easy to operate, can report results in a short time, and make the results visible, thus having significant potential for widespread application. This invention establishes a novel and simple method... optrA The RAA-agarose gel electrophoresis detection method can be completed within 15 minutes at 35℃. The amplified products can be detected after 30 minutes of agarose gel electrophoresis, with a detection limit of 2.22 × 10⁻⁶. 1 More importantly, compared to the fluorescent RPA method, the method established in this invention requires no complex consumables, has lower detection costs, and has greater potential for practical application. Furthermore, this invention found that the sensitivity of the RAA method is 100 times higher than that of the PCR method. In addition, this method does not cross-react with other common drug resistance genes, and repeatability tests all showed good amplification, indicating good specificity and repeatability. Using this method to test 100 clinical samples, the positive rate for RAA detection was 48% (48 / 100); the positive rate for PCR detection was 47% (47 / 100); and the positive rate for qPCR detection was 49% (49 / 100). The concordance rate between RAA and PCR detection was 99%, and the concordance rate between RAA and qPCR detection was 99%, with a kappa value of 0.98 (K>0.75), indicating that the RAA method has high detection accuracy.

[0081] Primers are a key factor in RAA detection. To maximize amplification specificity and efficiency, the primer length used for RAA detection should be controlled between 30bp and 35bp. Therefore, primer screening is necessary for RAA detection. This invention is based on... optrA Four pairs of primers were designed for the conserved regions of the gene, and the results were determined through screening. optrA -F2 / optrA -R2 is optrA The optimal primer pair detected a target band size of 157 bp, and no false positive signals were detected, indicating that the amplified fragment should be controlled within 200 bp when designing RAA primers. Furthermore, the repeatability test of the RAA method is one of the core aspects of its validation. The core objective of the RAA repeatability test is to evaluate the stability and consistency of RAA detection results under the same experimental conditions, and to provide crucial evidence for the reliability, applicability, and subsequent promotion of the method. The intra- and inter-group repeatability tests in this invention successfully amplified the target band, and the band size was consistent with the experimental expectations, with no significant difference, indicating that the method has good reliability and applicability.

[0082] RAA-agarose gel electrophoresis (RAA-ALD) costs only half that of real-time fluorescent RAA and one-third that of RAA-LFD because it does not require additional probe design. Fluorescent RAA and RAA-LFD are more suitable for rapid on-site detection at grassroots levels. It is worth noting that because RAA-ALD requires additional gel electrophoresis equipment compared to fluorescent RAA and RAA-LFD, it is more suitable for grassroots institutions equipped with basic laboratories. optrA Drug resistance gene detection work.

[0083] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A kit for detecting and / or identifying drug-resistant enterococci, characterized in that, The kit includes primer pairs as shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. The kit according to claim 1, characterized in that, The kit also includes at least one of recombinase, DNA polymerase, dNTPs, and buffer.

3. The application of the reagent kit as described in claim 1, characterized in that, The application refers to: The kit is used to detect and / or identify the sample by recombinase-mediated isothermal amplification. If a specific band appears after electrophoresis, the sample is a drug-resistant enterococcus.

4. The application as described in claim 3, characterized in that, The methods for detecting drug-resistant enterococci are as follows: Extract DNA from the sample to be tested; Using DNA as a template, the primer pair was used to perform a recombinase-mediated isothermal amplification reaction to obtain the amplification product. The amplification products were subjected to electrophoresis. The electrophoresis results were observed. If a specific band appeared at the 157bp position, the sample to be tested was determined to be drug-resistant enterococci.

5. The application as described in claim 4, characterized in that, The conditions for recombinase-mediated isothermal amplification reaction are: 25℃~41℃, 10min~30min.

6. The application as described in claim 5, characterized in that, The conditions for recombinase-mediated isothermal amplification reaction were: 35℃ for 15 min.

7. The application as described in claim 3, characterized in that, The drug-resistant enterococci refer to enterococci that are resistant to oxazolidinone drugs.

8. The application as described in claim 7, characterized in that, The oxazolidinone class of drugs includes at least one of linezolid, terdizol, and retazolid.

9. The application as described in claim 7, characterized in that, The enterococci are Enterococcus faecalis and / or Enterococcus faecium.

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