Primer pair for detecting enterococcus drug-resistant gene poxtA based on RAA-agarose gel electrophoresis and application of primer pair
By using RAA-agarose gel electrophoresis combined with specific primer pairs, the problems of speed, accuracy, and cost in the detection of the enterococcal drug resistance gene poxtA in existing technologies have been solved, achieving efficient and low-cost detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHIHEZI UNIVERSITY
- Filing Date
- 2026-03-30
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies are insufficient for the rapid and accurate detection of the enterococcal drug resistance gene poxtA. Furthermore, existing methods are costly, rely on expensive instruments, and are complex to operate, making it difficult to meet the needs for rapid on-site testing.
The detection method based on RAA-agarose gel electrophoresis was adopted. A recombinase-mediated isothermal amplification reaction was performed using specific primer pairs (SEQ ID NO.1 and SEQ ID NO.2). The amplification products were analyzed by agarose gel electrophoresis, and the detection was completed within 15 minutes under isothermal conditions of 30℃.
It achieves rapid, accurate, and low-cost detection of the enterococcal drug resistance gene poxtA, with high sensitivity, strong specificity, and visualized detection results. It also has a high concordance rate with conventional PCR and qPCR methods, making it suitable for rapid on-site detection.
Smart Images
Figure CN122012765A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial detection technology, specifically relating to a method for detecting enterococcal drug resistance genes based on RAA-agarose gel electrophoresis. pixA Primer pairs and their applications. Background Technology
[0002] The prevalence of multiple drug resistance (MDR) poses an increasing threat to human health. Antimicrobial resistance (AMR) has become a pressing issue in human medicine and veterinary medicine. Food production animals are widely considered reservoirs of drug-resistant bacteria and transferable resistance genes that can spread across different hosts and environmental media. Enterococci, as Gram-positive gut commensal bacteria and opportunistic pathogens, are of particular concern due to their ability to persist in various environments, including water, soil, and vegetation, and their high inherent resistance. Crucially, Enterococci exhibit significant genomic plasticity, characterized by the frequent acquisition, maintenance, and exchange of antimicrobial resistance genes through mobile genetic elements (MGEs). This genomic flexibility allows the bacteria to rapidly adapt to selective pressures and promotes… optrA , pixA The spread of drug-resistant genes among humans, animals, and environmental reservoirs reinforces their role in the "integrated health" framework. In intensive farming systems, multidrug-resistant Enterococci can spread bidirectionally between human and animal populations via the food chain, occupational exposure, and environmental contamination. Transferable drug resistance genes... pixA The cross-resistance between florfenicol and linezolid poses a serious challenge to both veterinary and human medicine.
[0003] pixA The gene encodes an ABC-F family protein that mediates bacterial resistance to oxazolidinones and ammonium alcohols through ribosomal protection mechanisms. pixA The presence of it not only reduces the sensitivity of bacteria to oxazolidinone and florfenicol, but also reduces the sensitivity of bacteria to tetracycline. pixA The gene was first discovered in 2018 in a clinically isolated methicillin-resistant Staphylococcus aureus, and is related to... optrA The amino acid sequence homology is approximately 32%. It is worth noting that... pixA Often with optrA Other drug resistance genes coexist in the same strain and can be horizontally transferred between different enterococci via mobile genetic elements such as plasmids. They can even remain stable in non-selective stress environments without detectable conjugation activity, exacerbating the spread and persistence of multidrug resistance.
[0004] Given pixA The frequency of this gene in animal-derived, environmental, and clinical strains is increasing, and the cross-resistance it mediates seriously threatens the efficacy of last-line antibiotics such as linezolid. Therefore, there is an urgent need to establish efficient and accurate detection methods for monitoring this gene. However, existing detection methods based on conventional PCR and quantitative real-time PCR are insufficient. pixA Gene reagents have several drawbacks when used, including difficulty in meeting the needs of rapid on-site testing, long testing cycles, high technical requirements for operators, and reliance on expensive instruments. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a method for detecting enterococcal antibiotic resistance genes based on RAA-agarose gel electrophoresis. pixA Primer pairs and their applications. The primer pairs consist of an upstream primer of the sequence shown in SEQ ID NO.1 and a downstream primer of the sequence shown in SEQ ID NO.2. They can be used to detect drug-resistant enterococci or to prepare kits for detecting drug-resistant enterococci. The detection method has the advantages of being rapid and efficient, accurate, specific, sensitive, and providing visualized results.
[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a method for detecting enterococcal antibiotic resistance genes based on RAA-agarose gel electrophoresis. pixA The primer pair consists of an upstream primer of the sequence shown in SEQ ID NO.1 and a downstream primer of the sequence shown in SEQ ID NO.2.
[0007] Furthermore, the detection target of the primer pair is a drug resistance gene. pixA Conserved sequence fragments in the CDS region, the drug resistance gene pixA The complete sequence of the CDS region has the accession number NG_063824.1 in GenBank.
[0008] Furthermore, the enterococci include Enterococcus faecium and Enterococcus faecalis.
[0009] A second aspect of the present invention provides the application of the primer pair described above in the preparation of a kit for detecting drug-resistant enterococci.
[0010] A third aspect of the present invention provides a kit for detecting drug-resistant enterococci, the kit comprising the primer pairs described above.
[0011] Furthermore, the kit also includes buffer, enzyme preparation and deoxyribonucleoside triphosphates (dNTPs) required for recombinase-mediated isothermal amplification reaction.
[0012] A fourth aspect of the present invention provides the use of the primer pair described above in the detection and / or identification of drug-resistant enterococci.
[0013] The fifth aspect of this invention provides a method for detecting drug-resistant enterococci, characterized by comprising the following steps: Extract DNA from the sample to be tested; Using the DNA as a template, a recombinase-mediated isothermal amplification reaction was performed using the primer pair described above to obtain the amplification product; The amplification products were subjected to 2 w / v% agarose gel electrophoresis. The agarose gel was placed in a gel imaging system for observation and result interpretation. If a specific band appeared at the 225bp position, the sample was determined to be drug-resistant enterococci or contain drug-resistant enterococci.
[0014] Furthermore, the enterococci include Enterococcus faecium and Enterococcus faecalis.
[0015] Furthermore, the recombinase-mediated isothermal amplification reaction is carried out at a temperature of 25℃~41℃ for a time of 10min~30min.
[0016] Furthermore, the recombinase-mediated isothermal amplification reaction is carried out at a temperature of 30℃~41℃ for a time of 15min~30min.
[0017] Furthermore, the recombinase-mediated isothermal amplification reaction is carried out at a temperature of 30°C for 15 minutes.
[0018] Furthermore, each 50 μL recombinase-mediated isothermal amplification reaction system contains: 25 μL A Buffer, 13.5 μL ddH2O, 2 μL 5.0 μmol / L upstream primer, 2 μL 5.0 μmol / L downstream primer, 5 μL DNA, 2.5 μL B Buffer, and one tube of RAA reaction general dry powder.
[0019] Furthermore, each 25 μL of A Buffer contains 10 w / v% polyethylene glycol; each 2.5 μL of B Buffer contains 280 mM magnesium acetate; the RAA reaction universal dry powder includes dNTPs, recombinase, single-stranded DNA binding protein and DNA polymerase.
[0020] Furthermore, the drug-resistant enterococci contain drug resistance genes. pixA The conserved sequence of the CDS region, the drug resistance gene pixA The accession number of the conservative sequence in the CDS region in GenBank is NG_063824.1.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for detecting enterococcal antibiotic resistance genes based on RAA-agarose gel electrophoresis. pixA The invention discloses a primer pair and its application. The primer pair consists of an upstream primer of the sequence shown in SEQ ID NO.1 and a downstream primer of the sequence shown in SEQ ID NO.2. This primer pair can be used to detect drug-resistant enterococci or to prepare kits for detecting drug-resistant enterococci. Based on the primer pair, the invention also provides a method for detecting drug-resistant enterococci based on RAA-agarose gel electrophoresis. This method can complete amplification within 15 minutes under isothermal conditions at 30℃. The amplified products are detected after 30 minutes of agarose gel electrophoresis. The detection results are accurate and reliable, and have the advantages of high specificity, high sensitivity, simplicity, speed, and visualization.
[0022] The detection limit of this method is 2.35 × 10⁻⁶. 0 Copies / μL. More importantly, compared to the fluorescent RPA method, this method requires no complex consumables, has lower detection costs, and has greater potential for practical application. Meanwhile, the detection method provided by this invention has a sensitivity 1000 times higher than the traditional PCR method. Furthermore, this method does not cross-react with other common drug resistance genes, and repeatability tests all show good amplification, indicating good specificity and repeatability. The positive rate when using this method to test 100 clinical samples was 11%, while the positive rate of conventional PCR was 10%, and the positive rate of qPCR was 11%. The detection method provided by this invention has a 99% concordance rate with conventional PCR and a 100% concordance rate with qPCR, with a kappa value of 0.95 (K>0.75), demonstrating high detection accuracy. This method is used to detect oxazolidinone resistance genes. pixA It provides a convenient tool for the rapid detection and monitoring of drug-resistant enterococci and has good application prospects. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] tú1 To amplify drug resistance genes pixA Primer screening. A: Grayscale image presented by gel imaging system; B: Ultraviolet electrophoresis image presented under ultraviolet light. Lanes 1, 2, 3, and 4 in A and B correspond to primer pairs 1 through 4 respectively; M: DNA relative molecular mass standard; N: Blank control.
[0025] tú2The following are the optimization results of the RAA detection reaction conditions: A: Primer concentration optimization grayscale image, lanes 1-5 correspond to final primer concentrations of 1 μmol / L, 2.5 μmol / L, 5 μmol / L, 7.5 μmol / L, and 10 μmol / L, respectively. B: Primer concentration optimization UV image, lanes 1-5 correspond to final primer concentrations of 1 μmol / L, 2.5 μmol / L, 5 μmol / L, 7.5 μmol / L, and 10 μmol / L, respectively. C: Temperature optimization grayscale image, lanes 1-6 correspond to temperatures of 25℃, 30℃, 35℃, 37℃, 39℃, and 41℃, respectively. D: Temperature optimization UV image, lanes 1-6 correspond to temperatures of 25℃, 30℃, 35℃, 37℃, 39℃, and 41℃, respectively. E: Time optimization grayscale image, lanes 1-5 correspond to times of 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. F: Time-optimized UV images, lanes 1-5 correspond to times of 10 min, 15 min, 20 min, 25 min and 30 min respectively; M: DNA relative molecular mass standard; N: Blank control.
[0026] tú3 Results of RAA initiated by body temperature. A: RAA grayscale image; B: RAA ultraviolet image; 1-3: Samples initiated by body temperature, where 1 and 2 are samples initiated by body temperature, 3 is the negative control, N: blank control.
[0027] tú4 The results are for the specificity of the detection method. A: Grayscale image presented by the gel imaging system; B: Ultraviolet electrophoresis image presented under ultraviolet light. Lanes 1-2 in A and B: Carrying pixA Enterococcus faecalis DNA; lanes 3-4: carrying pixA Enterococcus faecalis DNA of the gene; lanes 5-12 are, in order: those not carrying oxazolidinone resistance genes. pixA Enterococcus faecalis, without carrying oxazolidinone resistance genes pixA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying oxazolidinone resistance genes optrA Enterococcus faecalis carrying tetracycline resistance genes tetM Enterococcus faecalis carrying amyl alcohol resistance genes fexA Enterococcus faecalis carrying amyl alcohol resistance genes flora Enterococcus faecalis carrying carbapenem resistance genes blah NDM DNA of Escherichia coli; M: relative molecular mass standard of DNA; N: blank control.
[0028] tú5The results show the sensitivity of the detection methods. A: RAA detection results are presented as grayscale images on a gel imaging system; B: RAA detection results are presented as UV electrophoresis images under UV light; C: Sensitivity test results of the PCR method; D: Sensitivity test results of the qPCR method. The template concentrations corresponding to 1-8 are 2.35 × 10⁻⁸. 7 copies / μL, 2.35×10 6 copies / μL, 2.35×10 5 copies / μL, 2.35×10 4 copies / μL, 2.35×10 3 copies / μL, 2.35×10 2 copies / μL, 2.35×10 1 copies / μL, 2.35×10 0 copies / μL; 9: negative control with ddH2O template; M: relative molecular mass standard of DNA; N: blank control.
[0029] tú6 This is the result of an intra-group repeatability test for the detection method. A: Grayscale image presented by the gel imaging system; B: UV electrophoresis image presented under UV light. The template concentration for lanes 1-3 is 2.35 × 10⁻⁶. 4 copies / μL; the template concentration in lanes 4-6 is 2.35×10⁻⁶. 3 copies / μL; the template concentration in lanes 7-9 is 2.35×10⁻⁶. 2 copies / μL; M: relative molecular mass standard of DNA; N: blank control.
[0030] tú7 This is the result of the inter-group repeatability test of the detection method. A: Grayscale image presented by the gel imaging system; B: Ultraviolet electrophoresis image presented under ultraviolet light. Lane 1: Template concentration is 2.35 × 10⁻⁶. 4 copies / μL; template concentration in lane 2 was 2.35 × 10⁻⁶. 3 copies / μL; template concentration in lane 3 was 2.35 × 10⁻⁶. 2 copies / μL; M: relative molecular mass standard of DNA; N: blank control. Detailed Implementation
[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0032] The list of abbreviations for this invention is shown in Table 1.
[0033] Table 1. List of Abbreviations The materials and methods used in this invention are as follows: 1. Main reagents 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 Vazyme Biotechnology Co., Ltd. V (phenol): V (Chloroform): V Isoamyl alcohol (25:24:1) and Biowest agarose were 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 Sewell 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.
[0034] 2. Main equipment 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).
[0035] 3. Strains and clinical samples Carrying oxazolidinone resistance genes pixA The DNA of *Enterococcus faecalis* does not carry oxazolidinone resistance genes. pixA Enterococcus faecalis, without carrying oxazolidinone resistance genes pixAEnterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying oxazolidinone resistance genes optrA Enterococcus faecalis carrying tetracycline resistance genes tetM Enterococcus faecalis carrying amyl alcohol resistance genes fexA Enterococcus faecalis carrying amyl alcohol resistance genes flora Enterococcus faecalis carrying carbapenem resistance genes blah NDM The E. coli samples were donated by the Key Laboratory of New Drug Research and Development for Herbivorous Animals, Xinjiang Agricultural University. A total of 100 anal swab samples were collected from pigs in Shihezi City, Xinjiang, and stored at -80℃.
[0036] 4. Nucleic acid extraction 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.
[0037] Example 1: Primer Design and Screening According to information published in GenBank pixA The complete CDS region sequence of the gene (accession number: NG_063824.1), combined with the RAA reaction principle, was used to design four pairs of specific amplifications using Oligo 7.0 software. pixA Primers for conserved fragments in the CDS region. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 2.
[0038] Table 2 Primer Sequences Four primer pairs were used to amplify the amplification reaction. pixA Genes were detected by 2w / v% agarose gel electrophoresis, and primer pairs were screened based on the detection results. The results showed that among the four designed primer pairs, primer pair 1 produced bands with high brightness, good clarity, and the fewest non-specific bands. tú1 Therefore, primer pair 1 was selected as the optimal primer pair for subsequent experiments.
[0039] Example 2: Establishment of the RAA reaction system Following the instructions for the RAA basic nucleic acid amplification kit, a 50 μL RAA reaction system was prepared. The premixed solution included 25 μL A Buffer, 13.5 μL ddH2O, and 2 μL... pixA -F1 and 2 μL pixA-R1. Thoroughly mix the premixed solution in a clean PCR reaction tube, then add it to a detection unit tube pre-filled with RAA reaction universal dry powder (including dNTPs, recombinase, single-stranded DNA binding protein, and DNA polymerase). Next, 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 tube cap. Each 25 μL of A Buffer contains 10 w / v% polyethylene glycol; each 2.5 μL of B Buffer contains 280 mM magnesium acetate. Tightly cap the tube, gently invert and tap the tube wall 5 times to thoroughly mix, centrifuge rapidly for 10 s, and incubate at an incubator. After the reaction is complete, add 50 μL of [unspecified ingredient] to the detection unit tube. V (phenol): V (Chloroform): V A mixture of isoamyl alcohol (25:24:1) was thoroughly mixed and centrifuged at 12,000 rpm for 5 min. The supernatant was then subjected to 2 w / v agarose gel electrophoresis. The electrophoresis results were presented as grayscale images using a gel imaging system and as UV electrophoresis images under UV light.
[0040] Optimization of RAA reaction primer concentration, temperature, and time: (1) pixA -F1 and pixA -R1 was diluted, and while keeping other components of the reaction system unchanged, primers were added to the above 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. pixA -F1 and pixA -R1, incubate in a 39℃ constant temperature water bath for 30 min, and screen for the optimal primer concentration.
[0041] The results are as follows tú2 As shown, the target band brightness increases with increasing primer concentration, indicating enhanced RAA amplification ability. Considering all factors, 5.0 μmol / L was selected as the optimal primer concentration.
[0042] (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.
[0043] As shown in Figure 2, bands can be amplified within the temperature range of 25℃ to 41℃, and the band brightness is high in the range of 30℃ to 41℃. Therefore, the lower temperature of 30℃ in this range was selected for subsequent RAA detection.
[0044] (3) Based on the above optimal primer concentration and optimal reaction temperature, the reaction time was set to 10 min, 15 min, 20 min, 25 min and 30 min to screen the optimal reaction time.
[0045] The results are as follows tú2 As shown, clear target bands could be observed within a reaction time of 10 min to 30 min, with the bands exhibiting better brightness in the 15 min to 30 min range. Therefore, the shorter time of 15 min within this range was selected for subsequent experiments.
[0046] Additionally, in the absence of heating equipment, simply hold the centrifuged test unit tube in your palm for 15 minutes. tú3 This allows for efficient amplification and accurate detection.
[0047] Example 3: A method for detecting drug-resistant enterococci based on RAA-agarose gel electrophoresis, comprising the following steps: Extract DNA from the sample to be tested; Using DNA as a template, a recombinase-mediated isothermal amplification reaction was performed using primers with the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 to obtain the amplification product; Each 50 μL recombinase-mediated isothermal amplification reaction system contains: 25 μL A Buffer, 13.5 μL ddH2O, and 2 μL 5.0 μmol / L... pixA -F1, 2μL 5.0 μmol / L pixA -R1, 5μL DNA, 2.5μL B Buffer, and one tube of RAA reaction general dry powder.
[0048] The conditions for recombinase-mediated isothermal amplification reaction are: 25℃~41℃, 15min~30min.
[0049] The preferred conditions for recombinase-mediated isothermal amplification reaction are: 30℃, 15 min.
[0050] The amplification products were subjected to 2 w / v agarose gel electrophoresis, and the electrophoresis results were observed using a UV analyzer. If a specific band appeared at the position corresponding to 225 bp, the sample was determined to contain an oxazolidinone resistance gene. poxtA, The sample to be tested contains or is drug-resistant enterococci.
[0051] Example 4: Specificity Experiment To evaluate the specificity of the detection method established in Example 3 of this invention, samples carrying oxazolidinone resistance genes were tested. pixA The DNA of *Enterococcus faecalis* does not carry oxazolidinone resistance genes. pixA Enterococcus faecalis, without carrying oxazolidinone resistance genes pixA Enterococcus faecalis carrying tetracycline resistance genes tetA Klebsiella pneumoniae carrying oxazolidinone resistance genes optrA Enterococcus faecalis carrying tetracycline resistance genes tetM Enterococcus faecalis carrying amyl alcohol resistance genes fexA Enterococcus faecalis carrying amyl alcohol resistance genes flora Enterococcus faecalis carrying carbapenem resistance genes blah NDM Using E. coli DNA as a template and ddH2O as a negative control, amplification was performed according to the method established in Example 3 (the amplification reaction conditions were 30℃ for 15 min), and the specificity of the method was tested.
[0052] The results are as follows tú4 As shown, all carrying pixA Both Enterococcus faecalis and Enterococcus faecium DNA can amplify the target band, while other strains that do not carry the gene can. pixA No DNA bands were amplified in any of the genes. This indicates that the method established in this invention has good specificity and no cross-reaction with other common pathogens.
[0053] Example 5: Sensitivity tests of RAA, PCR and qPCR 1. Recombinant plasmid pMD19- pixA Preparation Enterococcus amplified by PCR pixA Genes. Enterococci. pixA The sequences of the primers for full-length gene PCR amplification are shown in SEQ ID NO.9 and SEQ ID NO.10.
[0054] Upstream primer: 5'-TGCTGAGTTTCAAATCGGAGA-3', SEQ ID NO.9; Downstream primer: 5'-TACTCAAGCAGCTCACGGGAA-3', SEQ ID NO.10.
[0055] Each 20 μL PCR amplification reaction system contains: 10 μL PCR Mix, 6 μL ddH2O, 1 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, and 2 μL template DNA.
[0056] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60.0℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ final extension for 7 min.
[0057] After the reaction was completed, the amplification product was subjected to agarose gel electrophoresis, the target band was cut off and purified.
[0058] The amplified product fragment was ligated into the pMD19-T vector. Each 10 μL ligation system contained: gel-recovered fragments... pixA 4 μL of gene, 1 μL of pMD19-T vector, and 5 μL of Solution I ligase were added. The ligation system was incubated at 4°C for 12 h to obtain the ligation product.
[0059] Thaw E. coli DH5α competent cells stored at -80℃ on ice. In a clean bench, transfer 10 μL of ligation product into the competent cells, gently shake, incubate on ice for 30 min, perform heat shock at 42℃ for 90 s, and then quickly incubate on ice for 3 min. Add 1 mL of LB liquid medium without Amp resistance and incubate at 37℃ and 200 rpm for 1 h. Take the culture, centrifuge at low speed for 1 min, remove the supernatant, and take 200 μL of the bacterial culture to spread evenly on LB solid medium (Amp resistant). After the bacterial culture is completely absorbed, incubate upside down for 12 h. Pick a single colony from the medium and transfer it to 5 mL of LB liquid medium containing Amp resistance. Incubate at 37℃ and 220 rpm for 12 h.
[0060] Positive strains were screened by bacterial culture PCR verification. The sequences of the universal primers M13F and M13R for pMD19-T are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. M13F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3', SEQ ID NO.11; M13R: 5'-AGCGGATAACAATTTCACACAGGA-3', SEQ ID NO. 12.
[0061] Each 20 μL PCR amplification reaction system contains: 10 μL PCR Mix, 6 μL ddH2O, 1 μL 10 μmol / L M13F, 1 μL 10 μmol / L M13R, and 2 μL bacterial culture.
[0062] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; extension at 72℃ for 7 min. The reaction products 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 (to obtain pMD19-). pixAThe plasmid was stored at -20°C. The correct recombinant plasmid pMD19- was obtained after PCR and sequencing verification. pixA The concentration was determined using an ultra-micro spectrophotometer, and the copy number was calculated.
[0063] 2. Sensitivity determination of the detection method Recombinant plasmid pMD19- pixA Perform a 10-fold serial dilution to a concentration of 2.35 × 10⁻⁶. 7 copies / μL, 2.35×10 6 copies / μL, 2.35×10 5 copies / μL, 2.35×10 4 copies / μL, 2.35×10 3 copies / μL, 2.35×10 2 copies / μL, 2.35×10 1 copies / μL, 2.35×10 0 copies / μL, with different concentrations of recombinant plasmid pMD19- pixA Using a template, the sensitivity of three methods—RAA, PCR, and qPCR—was determined.
[0064] The RAA detection method is the same as in Example 3 (the amplification reaction conditions are 30℃, 15min).
[0065] The primers used for PCR detection were the same as those used in RAA. The PCR amplification reaction system was as follows: 10 μL PCR mixture, 6 μL ddH2O, 1 μL each of forward and reverse primers (10 μmol / L), and 2 μL template DNA. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62.5℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; and a 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 qPCR reaction system (10 μL) consisted of: 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. The reaction program was as follows: the reaction solution was pre-denatured at 95℃ for 30 s; then 40 cycles were performed, each cycle consisting of denaturation at 95℃ for 15 s, annealing at 60℃ for 10 s, and extension at 72℃ for 30 s.
[0067] The results are as follows tú5As shown, the limits of detection for RAA, PCR, and qPCR are 2.35 × 10⁻⁶. 0 copies / μL, 2.35×10 3 copies / μL and 2.35 copies / μL.
[0068] Example 6: Repeatability test of the detection method The detection method established in Example 3 (amplification reaction conditions: 30°C, 15 min) was used with a concentration of 2.35 × 10⁻⁶. 4 copies / μL, 2.35×10 3 copies / μL, 2.35×10 2 The three concentrations of recombinant plasmid pMD19- were [copies / μL]. pixA Using the template, three intra-group and inter-group repeatability tests were performed to analyze the amplification results.
[0069] Results of intragroup repeatability tests are as follows tú6 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.
[0070] Results of intergroup repeatability tests as follows tú7 As shown, the target band was successfully amplified in all three replicate experiments at all concentrations, and the band brightness was basically consistent across different replicate groups. This indicates that the detection method established in this study has good stability.
[0071] Example 7: Performance Measurement in Practical Applications The test samples in the method for detecting drug-resistant enterococci can be samples collected from any environment. In this embodiment, pig anal swab samples were used as the test samples: 100 anal swab samples were collected from pigs in two pig farms in Shihezi City, Xinjiang Uygur Autonomous Region. The detection method established in Example 3 above (amplification reaction conditions: 30℃, 15 min), PCR method, and qPCR method were used to test all samples, and the concordance rates among the three methods were compared.
[0072] Table 3. Detection results of clinical samples using different methods (n=100) Note: In Table 3, "-" indicates that the corresponding indicator is not included in the statistics.
[0073] The results showed that the positive rate of the detection method provided by this invention was 11% (11 / 100); the positive rate of PCR detection was 10% (10 / 100); and the positive rate of qPCR detection was 11% (11 / 100). The detection method provided by this invention had a 99% concordance rate with PCR detection and a 100% concordance rate with qPCR detection (Table 3).
[0074] Table 4. Detection of samples by the detection method provided by this invention and the PCR method. pixA The result (n=100) Table 5. Detection of drug resistance genes in samples using the detection method provided by this invention and the PCR method. pixA Performance evaluation (n=100) Furthermore, as shown in Tables 4 and 5, all 10 samples that were positive by PCR were also detected as positive by the detection method established in Example 3, indicating that the sensitivity of the detection method provided by this invention relative to PCR is 100%. Of the 90 samples that were negative by PCR, 89 were also negative by RAA-agarose gel electrophoresis, indicating that the specificity of the detection method provided by this invention relative to PCR is 98.9%. Compared with the PCR method, the kappa value of the detection method established by this invention is 0.95 (K>0.75).
[0075] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0076] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A method for detecting enterococcal drug resistance genes based on RAA-agarose gel electrophoresis poxtA The primer pair is characterized in that, The primer pair consists of an upstream primer of the sequence shown in SEQ ID NO.1 and a downstream primer of the sequence shown in SEQ ID NO.
2.
2. The use of the primer pair of claim 1 in the preparation of a kit for detecting drug-resistant enterococci.
3. A kit for detecting drug-resistant enterococci, characterized in that, The kit contains the primer pair as described in claim 1.
4. The reagent kit according to claim 3, characterized in that, The kit also includes buffer, enzyme preparation, and deoxyribonucleoside triphosphate required for recombinase-mediated isothermal amplification reaction.
5. The use of the primer pair of claim 1 in the detection and / or identification of drug-resistant enterococci.
6. A method for detecting drug-resistant enterococci, characterized in that, Includes the following steps: Extract DNA from the sample to be tested; Using the DNA as a template, a recombinase-mediated isothermal amplification reaction was performed using the primer pair described in claim 1 to obtain the amplification product; The amplification products were subjected to agarose gel electrophoresis, and the results were interpreted based on the electrophoresis results: if a specific band appeared at the 225bp position, the sample was determined to be drug-resistant enterococci or contain drug-resistant enterococci.
7. The detection method according to claim 6, characterized in that, The recombinase-mediated isothermal amplification reaction is carried out at a temperature of 25℃ to 41℃ for a time of 10 min to 30 min.
8. The detection method according to claim 6, characterized in that, Each 50 μL recombinase-mediated isothermal amplification reaction system contains: 25 μL A Buffer, 13.5 μL ddH2O, 2 μL 5.0 μmol / L upstream primer, 2 μL 5.0 μmol / L downstream primer, 5 μL DNA, 2.5 μL B Buffer, and one tube of RAA reaction general dry powder.
9. The detection method according to claim 8, characterized in that, Each 25 μL A Buffer contains 10 w / v% polyethylene glycol; each 2.5 μL B Buffer contains 280 mM magnesium acetate; the RAA reaction universal dry powder contains dNTPs, recombinase, single-stranded DNA binding protein and DNA polymerase.