A primer set for detecting drug-resistant bacteria and application thereof

Abstract

The application belongs to the technical field of microorganism detection, and particularly relates to a primer set for detecting drug-resistant bacteria and application thereof, wherein the primer set comprises one upstream primer, one downstream primer and one probe; the sequence of the upstream primer is shown as SEQ ID NO. 1; the sequence of the downstream primer is shown as SEQ ID NO. 2; and the sequence of the probe is shown as SEQ ID NO. 6. Based on the primer set, the application establishes a method for detecting azolane ketone drug-resistant gene optrA The fluorescent RAA detection method can complete amplification within 21 min under isothermal conditions at 37 DEG C, the primer set has high specificity, and the RAA amplification result can be observed by ultraviolet analysis instrument with naked eyes, so that the possibility of rapid detection in basic areas is provided.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a primer set for detecting drug-resistant bacteria and its application. Background Technology

[0002] In recent years, with the increasing demand for meat, antibiotics have been widely used in animal husbandry, and their overuse poses a substantial challenge to human and animal health. Antibiotic resistance genes in bacteria are highly prevalent in poultry, and these genes can be transmitted to humans along with the resistant bacteria. These genes can transfer not only within the same species but also between different species, posing a significant threat to the poultry industry and human health.

[0003] 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. Typical examples include linezolid, terdizolid, and retazolid. However, with the increasing use of these drugs in veterinary and clinical settings, drug-resistant bacteria are emerging, severely diminishing the therapeutic efficacy of linezolid.

[0004] Currently, detection methods for drug-resistant bacteria mainly include traditional microbial culture and antimicrobial susceptibility testing, conventional polymerase chain reaction (PCR), and sequencing technologies. However, these methods have significant drawbacks: they are time-consuming, cumbersome, 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 bacteria, especially those carrying oxazolidinone resistance genes. Summary of the Invention

[0005] The purpose of this invention is to provide a primer set for detecting drug-resistant bacteria, which solves the problems existing in the prior art.

[0006] The technical solution adopted in this invention is: This invention provides a primer set for detecting drug-resistant bacteria, the primer set comprising an upstream primer, a downstream primer, and a probe; The sequence of the upstream primer is shown in SEQ ID NO.1; the sequence of the downstream primer is shown in SEQ ID NO.2; and the sequence of the probe is shown in SEQ ID NO.6.

[0007] Preferably, the probe further includes a fluorescent group and a quenching group.

[0008] Preferably, the fluorescent group is FAM; the quenching group is BHQ1.

[0009] The present invention also provides an application of the primer set, wherein the application refers to: The primer set is used to detect and / or identify the sample to be tested using fluorescent recombinase-mediated isothermal amplification technology. If a fluorescent signal appears after amplification, the sample to be tested is a drug-resistant bacterium.

[0010] Preferably, the method for detecting drug-resistant bacteria is as follows: Extract DNA from the sample to be tested; Using DNA as a template, a fluorescent recombinase-mediated isothermal amplification reaction was performed using the primer set described above; If a fluorescent signal appears after the fluorescent recombinase-mediated isothermal amplification reaction, the sample to be tested is a drug-resistant bacterium.

[0011] Preferably, the conditions for the fluorescent recombinase-mediated isothermal amplification reaction are: 25℃~41℃, 20min~35min.

[0012] Preferably, the conditions for the fluorescent recombinase-mediated isothermal amplification reaction are: 37°C for 21 min.

[0013] Preferably, the system for fluorescent recombinase-mediated isothermal amplification reaction is as follows: 25 μL Buffer A, 12.1 μL ddH2O, 2.2 μL 10 μmol / L upstream primer, 2.2 μL 10 μmol / L downstream primer, 1 μL 10 μmol / L probe, 2.5 μL Buffer B, and 5 μL DNA.

[0014] Preferably, the drug-resistant bacteria are bacteria resistant to oxazolidinone drugs.

[0015] Preferably, the oxazolidinone drug includes at least one of linezolid, terdizolid, and retazolid.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a primer set for detecting drug-resistant bacteria, comprising an upstream primer, a downstream primer, and a probe; the sequence of the upstream primer is shown in SEQ ID NO.1; the sequence of the downstream primer is shown in SEQ ID NO.2; and the sequence of the probe is shown in SEQ ID NO.6. Based on the primer set, this invention establishes an oxadiazonium resistance gene. optrA The fluorescent RAA detection method described in this invention can complete amplification within 21 minutes under isothermal conditions at 37℃ and exhibits high specificity. Furthermore, the RAA amplification 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 10⁻⁶. 1 copies / μL, compared to the detection limit of PCR (10 3The number of copies / μL is two orders of magnitude lower, while the sensitivity is 100 times higher. In summary, azole resistance genes... optrA Rapid and visual detection of fluorescent RAAs is suitable for field testing by grassroots personnel. It is a gene for resistance to azole ketones. optrA It provides a convenient tool for rapid screening, diagnosis and monitoring, and has good application prospects. Attached Figure Description

[0017] Figure 1 for optrA Identification results of gene-positive plasmids. M: DNA standard DL.3000; lanes 1 and 2 are two different positive clones.

[0018] Figure 2 for optrA Results of primer screening. In the figure, NC: negative control.

[0019] Figure 3 The results show the probe dosage screening. A: Detection results through fluorescence signal monitoring; B: Fluorescence RAA grayscale image, where detection unit tubes 1-4 represent the probe addition amounts as follows: 0.4μL, 0.6μL, 0.8μL, and 1.0μL.

[0020] Figure 4 The results show the primer quantity screening. A: Detection results by fluorescence signal monitoring; B: Fluorescent RAA grayscale image, where detection unit tubes 1-4 represent primer addition amounts of 1.8 μL, 2.0 μL, 2.2 μL, and 2.4 μL, respectively.

[0021] Figure 5 The results show the temperature screening results. A: 25℃; B: 35℃; C: 37℃; D: 39℃; E: 41℃; F: Fluorescent RAA grayscale image, with detection units 1-5 representing reaction temperatures of 25℃, 35℃, 37℃, 39℃, and 41℃ respectively.

[0022] Figure 6 Results of fluorescence RAA initiated at body temperature. A: Grayscale image of fluorescence RAA; B: Ultraviolet image of fluorescence RAA; 1-3 in A and B represent 3 biological replicates.

[0023] Figure 7 for optrA Specificity test results of the RAA detection method. A: Detection results monitored by fluorescence signal. Since 3-9 and NC are all negative, no fluorescence curve can be amplified, so a straight line is shown; B: Grayscale image of fluorescent RAA; In A and B, 1: carries optrA 1. Enterococcus faecalis DNA; 2. Carrying optrA Enterococcus faecalis DNA containing genes; 3-9 are, in order: those not carrying oxazolidinone resistance genes. optrAEnterococcus faecalis, without carrying oxazolidinone resistance genes optrA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying oxazolidinone resistance genes poxtA Enterococcus faecalis carrying tetracycline resistance genes tetM Enterococcus faecalis carrying β-lactam antibiotic resistance genes mecA Staphylococcus aureus carrying carbapenem resistance genes bla NDM DNA of Escherichia coli; M: relative molecular mass standard of DNA; 10 / NC: negative control.

[0024] Figure 8 for optrA Sensitivity results of RAA detection methods. A: RAA detection results monitored by fluorescence signal; B: Grayscale image of fluorescent RAA; C: Sensitivity test results of qPCR method; D: Sensitivity test results of PCR method; In A and C, 10 6 ~10 0 represent optrA The template concentration was 2.22 × 10⁻⁶. 6 copies / μL ~2.22×10 0 copies / μL; in B and D, 1~7: optrA The template concentration was 2.22 × 10⁻⁶. 6 copies / μL ~2.22×10 0 copies / μL; 8: negative control; M: relative molecular mass standard of DNA.

[0025] Figure 9 Results of intragroup repeatability tests. A: Detection results via fluorescence signal monitoring; B: Grayscale image of fluorescence RAA; 1-3: Template concentration of 2.22 × 10⁻⁶. 4 Three replicates of copies / μL, 4–6: template concentration 2.22 × 10⁻⁶. 2 Three replicates of copies / μL; NC: blank control.

[0026] Figure 10 This is the result of the inter-group repeatability test. A: Detection results by fluorescence signal monitoring; B: Grayscale image of fluorescence RAA; 1: Template concentration is 2.22 × 10⁻⁶. 4 Three replicates of copies / μL; 2: template concentration of 2.22×10 2 Three replicates of copies / μL; NC: blank control. Detailed Implementation

[0027] 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.

[0028] 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 this disease have reduced the clinical efficacy of oxazolidinone antibiotics. optrA The gene-encoded ATP-binding cassette (ABC)-F protein protects bacterial ribosomes, preventing bacteria from developing cross-resistance to linezolid and florfenicol due to antibiotic inhibition. optrA As a novel transferable resistance gene within the oxazolidinone class, it confers resistance to antibiotics through horizontal transmission of mobile genetic elements. Therefore, in order to better detect... optrA For gene detection, existing technologies have established a variety of detection methods, including conventional PCR and quantitative real-time PCR. However, these detection methods all require professional personnel to operate expensive instruments, which are not suitable for grassroots personnel to conduct on-site testing.

[0029] Recombinase-mediated isothermal amplification (RAA) is a novel isothermal nucleic acid amplification technique that can amplify genes in a short time (20-30 min) under isothermal conditions (37-42°C), 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-derived drug resistance genes. The fluorescent RAA detection method used in this invention allows for visual interpretation of the results using a UV analyzer. This method requires only simple equipment (UV analyzer) for visual observation, making it more suitable for use by grassroots veterinarians. Therefore, this invention establishes a highly specific, sensitive, rapid, and visually perceptible method for detecting multidrug resistance genes in chicken-derived Enterococcus. optrA The fluorescent RAA is optrA This provides new methods for on-site testing at the grassroots level.

[0030] This invention establishes a simple, rapid, and visualized method for identifying oxazolidinone resistance genes based on the aforementioned primer set. optrA This invention relates to a fluorescent recombinase-mediated isothermal amplification detection method. Following the RAA primer design principle, this invention targets oxazolidinone resistance genes. optrAPrimers were designed from the conserved regions of the gene, and standard recombinant plasmids were prepared. After primer screening, the primer dosage, probe dosage, and reaction temperature of the fluorescent RAA detection method were further optimized, and the specificity and sensitivity of the method were analyzed. Results showed that under isothermal conditions of 37°C, the fluorescent RAA amplification reaction targeting the optrA gene could be completed in 21 min, and clear fluorescence could be directly observed using a UV analyzer. Furthermore, the optimized fluorescent RAA detection method can be started at body temperature without the need for sophisticated instruments. Method validation showed that this fluorescent RAA detection system has high specificity, no cross-reactivity with other common drug-resistant gene strains, good reproducibility, and high sensitivity, with a limit of detection of 2.22 × 10⁻⁶. 1 Copies / μL. 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 98%, and the concordance rate with qPCR was 99%. In summary, the fluorescent RAA detection method established in this invention is simple to operate, highly specific, and highly sensitive. This method is suitable for field investigation of oxazolidinone resistance genes at the grassroots level. optrA Diagnostic work has promising applications.

[0031] 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.

[0032] The list of abbreviations for this invention is shown in Table 1.

[0033] Table 1. List of abbreviations for this invention The materials and methods used in this invention are as follows: 1. Materials.

[0034] 1.1. Strains.

[0035] The strain used in this invention carries an oxazolidinone resistance gene. optrA Enterococcus faecalis carrying oxazolidinone resistance genes optrA Enterococcus faecalis, without carrying oxazolidinone resistance genes optrA Enterococcus faecalis, without carrying oxazolidinone resistance genes optrA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying oxazolidinone resistance genes poxtA Enterococcus faecalis carrying tetracycline resistance genes tetM Enterococcus faecalis carrying β-lactam antibiotic resistance genesmecA Staphylococcus aureus carrying carbapenem resistance genes bla NDM Escherichia coli.

[0036] 1.2 Clinical samples.

[0037] A total of 100 anal swab samples were collected from the chicken farm and stored at -80°C for future use.

[0038] 1.3. Main reagents and instruments.

[0039] RAA nucleic acid amplification reagent (fluorescent 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.; Biowestagarose agarose was purchased from Xinjiang Hengchao Biotechnology Co., Ltd.; Escherichia 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 Saiwei Biotechnology Co., Ltd.; Gold View nucleic acid dye was purchased from Beijing Bio-Top Technology Co., Ltd.; 2× Es Taq PCR Master Mix (Dye) and ddH2O were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; water bath was purchased from Zhejiang Qun'an Scientific Instruments Co., Ltd.; high-speed benchtop centrifuge was purchased from Nanjing Haidixi Equipment Co., Ltd.; PCR instrument was purchased from Thermo Fisher Scientific Co., Ltd.; real-time PCR instrument was purchased from Tianlong Technology Co., Ltd.; electrophoresis gel imaging system was purchased from Bio-Rad, USA.

[0040] 2. Methods.

[0041] 2.1 Nucleic acid extraction.

[0042] 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.

[0043] 2.2 Preparation of standard plasmids.

[0044] The Enterococcus optrA gene was amplified by PCR. The primers for the full-length PCR amplification of the Enterococcus optrA gene are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0045] Forward primer, SEQ ID NO.7: TACTAACGCAAAGGAGGATAT.

[0046] Reverse primer, SEQ ID NO.8: ATTCTCTCATCAACTGTTCCC.

[0047] The PCR amplification reaction system is as follows: 10 μL PCR Mix, 6 μL ddH2O, 1 μL of 10 μmol / L forward primer, 1 μL of 10 μmol / L reverse primer, and 2 μL template DNA.

[0048] 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; 72℃ final extension for 7 min.

[0049] After the reaction, the amplified fragment was ligated into the pMD19-T vector. The 10 μL ligation system consisted of: 4 μL of the gel-recovered optrA gene, 1 μL of the pMD19-T vector, and 5 μL of Solution I ligase.

[0050] The ligation system was incubated at 4°C for 12 h. E. coli DH5α competent cells stored at -80°C were thawed on ice. In a clean bench, 10 μL of the ligation product was transferred into the competent cells, gently shaken, and incubated on ice for 30 min. A heat shock at 42°C for 90 s was then performed, followed by an immediate ice incubation for 3 min. 1 mL of Amp-free LB liquid medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. The culture was centrifuged at low speed for 1 min, the supernatant was removed, and 200 μL of the bacterial culture was evenly spread on Amp-resistant LB solid medium. After complete absorption, the culture was inverted and incubated for 16 h. Single colonies were picked from the medium and transferred to 5 mL of Amp-resistant LB liquid medium, and incubated at 37°C and 220 rpm for 12 h. Positive strains were screened by colony PCR using universal primers M13F and M13R for pMD19-T. The reaction conditions were: 95℃ for 5 min; 30 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min); extension at 72℃ for 7 min. The samples were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were compared with NCBI. Plasmid DNA was extracted using a plasmid miniprep kit and stored at -20℃. The recombinant plasmid pMD19-T was verified by PCR and sequencing. optrA Once the result is correct, the concentration is determined using an ultra-micro spectrophotometer, and the copy number is calculated.

[0051] M13F, SEQ ID NO.9: CGCCAGGGTTTTCCCAGTCACGAC.

[0052] M13R, SEQ ID NO. 10: AGCGGATAACAATTTCACACAGGA.

[0053] like Figure 1 As shown, the PCR identification results of the positive bacterial culture revealed a target band of 1925 bp. Plasmids were extracted from the positive bacterial culture and sequenced; the sequence obtained matched that in GenBank. optrA The sequence homology reached 99.9%.

[0054] Example 1 A primer set for detecting drug-resistant bacteria is as follows: 1. Primer design and screening.

[0055] According to information published in GenBank optrA Based on the conserved regions of the gene and the RAA reaction principle, one upstream primer, four downstream primers, and one probe were designed using Oligo 7.0 software. The primers and probe were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 2.

[0056] optrA The gene's accession number is KP399637.1.

[0057] Table 2 Primer sequences Note: In Table 2, FAMdT: deoxythymidine linked to the FAM fluorescent group; THF: Tetrahydrofuran; BHQ1-dT: Deoxythymidine linked to a BHQ1 quencher group; C3spacer: a spacer arm consisting of three carbon atoms.

[0058] 2. Establishment of the RAA reaction system.

[0059] According to the RAA fluorescent nucleic acid amplification kit instructions, a 50 μL RAA reaction system was prepared, including: 1) Prepare the premixed solution.

[0060] 25 μL A Buffer, 12.9 μL ddH2O, 2 μL upstream primer (10 μmol / L) and 2 μL downstream primer (10 μmol / L), 0.6 μL probe (10 μmol / L).

[0061] 2) RAA reaction.

[0062] 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 pMD19- to the detection unit tube. optrAAdd 2.5 μL of B Buffer to the inside of the tube cap. Tighten the cap, gently invert the tube and tap the tube wall 6 times to mix thoroughly, centrifuge quickly for 10 seconds, and place it in a real-time PCR instrument. The reaction procedure is shown in Table 3. After the reaction, observe the amplification results visually at 470 nm using an electrophoresis gel imaging system.

[0063] Table 3 Fluorescent RAA Reaction Procedure Four sets of primers were used for amplification using fluorescent RAA, and primer pairs were selected based on the detection results. The four sets of primers are as follows:

[0064] Primer set 1: optrA -F1+ optrA -R1+ optrA -5 probes; Primer set 2: optrA -F1+ optrA -R2+ optrA -5 probes; Primer set 3: optrA -F1+ optrA -R3+ optrA -5 probes; Primer set 4: optrA -F1+ optrA -R4+ optrA -5 probe.

[0065] The results showed that among the four primer sets, the cycle numbers of primer sets 1 through 4 were 7, 8, 8, and 10, respectively. This indicates that primer set 1, when paired, exhibited better RAA detection performance and higher amplification efficiency. (See above results). Figure 2 Therefore, primer set 1 was chosen as the optimal primer pair for subsequent experiments.

[0066] Example 2 An application of a primer set for detecting drug-resistant bacteria is as follows: 1. Optimization of reaction conditions.

[0067] In order to optimize the primer concentration, probe concentration and reaction temperature in the reaction system, the following experiments were first conducted in this invention.

[0068] Experiment 1: Using pMD19- in a 50 μL RAA reaction system optrA The plasmid was used as a template (concentration 2.22 × 10⁻⁶). 4 (Copies / μL) Using the primer and probe concentration recommended by the kit at 10 μmol / L, the probe addition amounts were set to 0.4 μL, 0.6 μL, 0.8 μL, and 1.0 μL, respectively, to determine the optimal probe amount.

[0069] Experiment 2: After determining the optimal probe dosage, the amounts of upstream and downstream primers added were set to 1.8 μL, 2.0 μL, 2.2 μL and 2.4 μL, respectively, to determine the optimal amounts of upstream and downstream primers.

[0070] Experiment 3: After determining the optimal amount of upstream and downstream primers, the reaction temperature was set to 25℃, 35℃, 37℃, 39℃ and 41℃ respectively to determine the optimal reaction temperature, with ddH2O water as a negative control.

[0071] The results are as follows: Depend on Figure 3 As shown, the amplification efficiency of fluorescent RAA increases with increasing probe dosage, reaching its highest level when the probe dosage is increased to 1 μL. Therefore, 1 μL is selected as the optimal probe dosage.

[0072] Depend on Figure 4 As shown, the amplification efficiency of fluorescent RAA increases with increasing primer dosage. The amplification efficiency is highest when the primer dosage reaches 2.2 μL. Further increasing the primer dosage does not improve amplification efficiency but instead increases the risk of non-specific amplification. Therefore, the optimal primer dosage is 2.2 μL.

[0073] like Figure 5 As shown, within the range of 25℃ to 37℃, the enzymatic amplification efficiency increased with increasing temperature. However, within the range of 37℃ to 41℃, the amplification efficiency began to decrease with further increases in temperature. These experimental results indicate that the peak time is earliest and the amplification efficiency is best at 37℃. Therefore, 37℃ was selected as the optimal reaction temperature for subsequent fluorescent RAA.

[0074] Additionally, in the absence of heating equipment, simply hold the centrifuged test unit tube in your palm for 21 minutes; the results are shown below. Figure 6 This allows for efficient amplification and accurate detection.

[0075] 2. Specificity test.

[0076] Carrying oxazolidinone resistance genes optrA Enterococcus faecalis ( Enterococcus faecium ), carrying oxazolidinone resistance genes optrA Enterococcus faecalis ( Enterococcus faecalis ), does not carry oxazolidinone resistance genes optrA Enterococcus faecalis, without carrying oxazolidinone resistance genes optrA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying oxazolidinone resistance genes poxtA Enterococcus faecalis carrying tetracycline resistance genes tetMEnterococcus faecalis carrying β-lactam antibiotic resistance genes mecA Staphylococcus aureus carrying carbapenem resistance genes bla NDM Using DNA from strains such as Escherichia coli as templates and ddH2O as a negative control, the RAA fluorescence method established in this invention was used for detection, with ddH2O water as a negative control, to evaluate the specificity of the method.

[0077] The results show that... Figure 7 All carrying optrA The DNA of all strains carrying the gene showed obvious amplification curves and fluorescence, while those not carrying the gene showed similar results. optrA No obvious amplification curves or fluorescence were detected in any of the Enterococcus strains carrying the target drug resistance gene. The experimental results indicate that the fluorescent RAA detection method established in this invention can only detect strains carrying the target drug resistance gene. optrA strains, while those not carrying the target drug resistance gene optrA None of the strains were detected, indicating that this detection method has good specificity.

[0078] 3. Sensitivity tests for fluorescent RAA, PCR, and qPCR.

[0079] pMD19- optrA The plasmid was serially diluted 10-fold to obtain pMD19 -optrA The plasmid concentration range was adjusted to 2.22 × 10⁻⁶. 6 copies / μL up to 2.22×10 0 Copies / μL were used as a template, with ddH2O water as a negative control. The sensitivity of three methods—RAA fluorescence assay, PCR, and qPCR—was determined.

[0080] Fluorescent RAA detection was performed under the optimized conditions described above; the primers used for PCR detection were the same as those used for RAA. The PCR amplification reaction system is as follows:

[0081] 10 μL PCR Mix, 6 μL ddH2O, 1 μL each of forward and reverse primers (10 μmol / L), and 2 μL template DNA. 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.

[0082] qPCR detection was performed using SYBR Green qPCR Mix, and the primers used were the same as those used in fluorescent RAA. The composition of a 10 μL qPCR reaction system is as follows:

[0083] The reaction mixture consisted of 5 μL qPCR Mix, 0.2 μL each of forward and reverse primers (10 μM), 0.2 μL template, 3.6 μL sterile water, and 1 μL template DNA. The reaction program was as follows: the reaction mixture was pre-denatured at 95 °C for 30 s; then 40 cycles were performed, each cycle consisting of denaturation at 95 °C for 15 s, annealing at 60 °C for 10 s, and extension at 72 °C for 30 s.

[0084] The results show that... Figure 8 As shown, the limits of detection for fluorescent RAA, PCR, and qPCR are 2.22 × 10⁻⁶, respectively. 1 copies / μL, 2.22×10 3 copies / μL and 2.22copies / μL.

[0085] 4. Repeatability test.

[0086] The RAA detection method established in this invention is used for 2.22 × 10⁻⁶ Ω·cm. 4 copies / μL, 2.22×10 2 Using recombinant plasmids at two concentrations of copies / μL as templates, three intra- and inter-group repeatability tests were performed to analyze the amplification results.

[0087] Results of intragroup repeatability tests are as follows Figure 9 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.

[0088] Results of intergroup repeatability tests as follows Figure 10 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 detection method has good stability.

[0089] 5. Clinical sample testing.

[0090] The established fluorescent RAA detection method, PCR method, and qPCR method were used to detect all samples, and the concordance among the three methods was compared. To evaluate the methods established in this invention... optrA The concordance rate between the fluorescent RAA nucleic acid detection method and PCR and qPCR methods was measured when the same batch of samples was tested using all three methods. Results showed:

[0091] The positive rate of fluorescent RAA detection was 52% (52 / 100); the positive rate of PCR detection was 50% (50 / 100); and the positive rate of qPCR detection was 53% (53 / 100). The concordance rate between fluorescent RAA and PCR detection was 98%, and the concordance rate between fluorescent RAA and qPCR detection was 99%. The results are shown in Table 4.

[0092] Furthermore, all 50 samples that tested positive by PCR also tested positive for fluorescent RAA, indicating that the sensitivity of fluorescent RAA compared to PCR was 100%. Of the 50 samples that tested negative by PCR, 48 also tested negative for RAA, indicating that the specificity of fluorescent RAA compared to PCR was 96%. Compared to the PCR method, the kappa value of the fluorescent RAA detection method established in this invention was 0.96 (K>0.75), as shown in Table 5. These results demonstrate that the RAA detection method established in this invention can be used to detect the drug resistance gene optrA in clinical samples.

[0093] Table 4. Detection results of clinical samples using different methods (n=100) Table 5. RAA detection methods and PCR methods for detecting RAA in clinical samples. optrA Performance comparison (n=100) Note: In Table 5, PPV: Positive predictive value; NPV: Negative predictive value.

[0094] 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.

[0095] 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 primer set for detecting drug-resistant bacteria, characterized in that, The primer set includes an upstream primer, a downstream primer, and a probe; The sequence of the upstream primer is shown in SEQ ID NO.1; The sequence of the downstream primer is shown in SEQ ID NO.2; The probe sequence is shown in SEQ ID NO.

6.

2. The primer set as described in claim 1, characterized in that, The probe also includes fluorescent groups and quenching groups.

3. The primer set as described in claim 2, characterized in that, The fluorescent group is FAM; The quenching group is BHQ1.

4. The application of the primer set as described in claim 1, characterized in that, The application refers to: The primer set is used to detect and / or identify the sample to be tested using fluorescent recombinase-mediated isothermal amplification technology. If a fluorescent signal appears after amplification, the sample to be tested is a drug-resistant bacterium.

5. The application as described in claim 4, characterized in that, The methods for detecting drug-resistant bacteria are as follows: Extract DNA from the sample to be tested; Using DNA as a template, a fluorescent recombinase-mediated isothermal amplification reaction was performed using the primer set described above; If a fluorescent signal appears after the fluorescent recombinase-mediated isothermal amplification reaction, the sample to be tested is a drug-resistant bacterium.

6. The application as described in claim 5, characterized in that, The conditions for fluorescent recombinase-mediated isothermal amplification reaction are: 25℃~41℃, 20min~35min.

7. The application as described in claim 6, characterized in that, The conditions for fluorescent recombinase-mediated isothermal amplification reaction were: 37℃, 21 min.

8. The application as described in claim 5, characterized in that, The system for fluorescent recombinase-mediated isothermal amplification reaction is as follows: 25 μL Buffer A, 12.1 μL ddH2O, 2.2 μL 10 μmol / L upstream primer, 2.2 μL 10 μmol / L downstream primer, 1 μL 10 μmol / L probe, 2.5 μL Buffer B, and 5 μL DNA.

9. The application as described in claim 4, characterized in that, The drug-resistant bacteria are those resistant to oxazolidinone drugs.

10. The application as described in claim 9, characterized in that, The oxazolidinone class of drugs includes at least one of linezolid, terdizol, and retazolid.

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