Method for rapidly screening methicillin-resistant staphylococcus aureus in oral cavity of wild animal
By selective culture using a culture medium composition of a specific composition and combined with molecular biological detection, the problem of time-consuming and costly screening of MRSA in the prior art is solved, and a fast, accurate and efficient MRSA screening effect is achieved.
Patent Information
- Application Number
- CN202411716240.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art faces problems of long time, high cost, low detection throughput and low detection efficiency when screening MRSA in oral samples of wild animals.
Selective culture of medium compositions including sodium chloride, polymyxin b, nystatin and oxacillin were used to perform selective culture and combined with molecular biological detection, identified by PCR amplification and agarose gel electrophoresis. , filter out MRSA.
It realizes rapid, accurate and efficient screening of enriched MRSA from a large number of samples, significantly improving detection efficiency and being able to detect MRSA within as fast as three days.
Smart Images
Figure CN119432669A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for screening wild animal oral samples for Methicillin-Resistant Staphylococcus aureus (MRSA). Background Art
[0002] MRSA is a subspecies of Staphylococcus aureus, which has broad-spectrum resistance to β-lactam antibiotics. It has similar biological characteristics to Staphylococcus aureus: typical Staphylococcus aureus is spherical, with a diameter of about 0.8μm, arranged in grape-like clusters under a microscope, without spores and flagella, most of which have no capsules, and are Gram-positive. MRSA growth does not require high nutrition, grows well on ordinary culture media, aerobic or facultative anaerobic, with an optimal growth temperature of 37°C and an optimal growth pH of 7.4. The colonies on the plate are thick, shiny, round and raised, with a diameter of 1 to 2mm. It has a high degree of salt tolerance and can grow in 10 to 15% NaCl broth. It can decompose glucose, maltose, lactose, and sucrose, and produce acid but not gas. Methyl red reaction is positive, and VP (acetylmethylcarbinol test) reaction is weakly positive. It can decompose arginine, hydrolyze urea, reduce nitrates, and liquefy gelatin.
[0003] At present, the detection methods of MRSA include paper diffusion method, agar screening method, etc. Due to the heterogeneous drug resistance of MRSA, its detection brings certain difficulties. The traditional screening and culture method is to culture the colonies and then conduct morphological observations. This cannot clearly determine whether it is MRSA, and it must be confirmed by antigen-antibody reaction. This method faces problems such as long time consumption, high cost, and low detection throughput. Moreover, the detection rate of MRSA is affected by many factors such as incubation temperature, time, pH and NaCl concentration of the culture medium, and the amount of bacterial solution, and the detection efficiency needs to be improved.
[0004] MRSA is a zoonotic disease that can be transmitted through biting or scratching, or even direct or indirect contact between the two parties. If you want to quickly understand the situation of wildlife populations being infected or carrying MRSA, you need to quickly screen for MRSA. When collecting samples from wild animals, researchers are easily attacked by wild animals, and cannot easily collect blood, hair, feces and other samples. There are many uncertainties. Collecting samples from the mouths of wild animals can quickly, simply and effectively obtain high-quality samples. Summary of the invention
[0005] In view of the shortcomings of the prior art, the present invention provides a method for screening MRSA in oral samples of wild animals. By using a culture medium with a specific composition for selective culture and combining it with molecular biological detection, MRSA can be screened and enriched from a large number of samples quickly, accurately and efficiently.
[0006] In a first aspect, the present invention provides a selective culture medium composition for MRSA, comprising a basal culture medium and an additive component, wherein the additive component comprises sodium chloride, polymyxin B, nystatin and oxacillin.
[0007] In some embodiments, each 1 L of the culture medium composition comprises the following additive components: 50-200 g of sodium chloride, 50-100 g of polymyxin b, 5-20 g of nystatin, and 2-6.4 g of oxacillin.
[0008] In some embodiments, each 1 L of the culture medium composition comprises the following additive components: 70 g sodium chloride, 75 g polymyxin b, 12.5 g nystatin, and 6 g oxacillin.
[0009] The culture medium composition of the present invention contains high concentration of salt. In a high concentration of salt environment, some bacteria lose a large amount of water due to cell osmosis and die. However, some other bacteria, such as Staphylococcus aureus, have a high degree of salt tolerance and can grow in 10-15% NaCl broth, have the ability to adapt to a high salt environment, and can survive and reproduce in a high salt environment, thereby screening out bacteria that can survive in a high salt environment.
[0010] Nystatin is a polyene antibiotic that exerts its antifungal effect mainly by inhibiting the synthesis of fungal cell membranes. It can bind to ergosterol on the fungal cell membrane, increase the permeability of the cell membrane, and cause the leakage of cell contents, thereby inhibiting the growth and reproduction of fungi.
[0011] Polymyxin B contains free amino groups with positive charges, which can combine with negatively charged phosphate groups on the cell membrane of Gram-negative bacteria. The formed lipophilic chains are inserted between the fatty chains in the bacterial cell membrane, causing the structure of the bacterial cell membrane to be depolymerized, resulting in increased permeability of the bacterial cell membrane, leakage of substances in the bacterial cells, and ultimately leading to the death of the bacteria.
[0012] Oxacillin is a penicillin antibiotic that exerts its antibacterial effect mainly by interfering with the synthesis of bacterial cell walls, resulting in defects in bacterial cell walls and ultimately causing bacterial lysis and death. Oxacillin has strong antibacterial activity against a variety of Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae. It has a better effect on penicillinase-producing bacteria because these bacteria can produce penicillinase, which hydrolyzes and destroys penicillin drugs, making them lose their ability to kill bacteria. Oxacillin is suitable for the treatment of various infections caused by penicillin-resistant Staphylococci, such as pneumonia, sepsis, endocarditis, skin and soft tissue infections, etc.
[0013] The culture medium composition of the present invention can effectively inhibit the growth of non-halophilic bacteria, Gram-negative bacteria, fungi and penicillinase-producing Staphylococcus by combining the above additive components, and is conducive to the growth of methicillin-resistant Staphylococcus aureus, making it a dominant strain, thereby achieving the purpose of selectively culturing methicillin-resistant Staphylococcus aureus.
[0014] In some embodiments, the basal culture medium is Tryptone Soy Broth medium.
[0015] In a second aspect, the present invention provides a use of the aforementioned culture medium composition in the cultivation or screening of MRSA.
[0016] In a third aspect, the present invention provides a method for screening MRSA from a wild animal oral sample set, comprising: using the aforementioned culture medium composition to culture each sample in the sample set to obtain a preliminary screened selective culture product; performing PCR amplification of MRSA-specific genes on the preliminary screened selective culture product and further screening positive samples therefrom.
[0017] In some embodiments, the following method is used to screen for MRSA in a collection of oral specimens from wild animals:
[0018] Each sample of the wild animal oral sample set was inoculated into a basic culture medium containing sodium chloride, polymyxin B, nystatin and oxacillin, and cultured at 36-38°C and 200-250 rpm for 18-24 hours. The culture products that were turbid after culture were selected and recorded as the culture product set;
[0019] The nuc gene and the mecA gene were PCR amplified for each culture product in the culture product set, and the amplified products were identified by gel electrophoresis. The culture products with both positive nuc gene and mecA gene were selected and purified to obtain MRSA.
[0020] In some embodiments, the following method is used to screen for MRSA in a collection of oral specimens from wild animals:
[0021] Each sample of the wild animal oral sample set was inoculated into a basic culture medium containing sodium chloride, polymyxin B and nystatin, and cultured at 36-38°C and 200-250 rpm for 22-26 hours, and the culture products that were turbid after culture were selected and recorded as the first culture product set;
[0022] PCR amplification of the nuc gene is performed on each culture product of the first culture product set, and the amplified products are identified by gel electrophoresis, and culture products with positive nuc gene are selected and recorded as the second culture product set;
[0023] The culture products of the second culture product set were transferred to a new basal medium containing oxacillin for culture at 36-38° C. and 200-250 rpm for 24-48 hours, and the culture products that were turbid after culture were selected and recorded as the third culture product set;
[0024] The nuc gene and mecA gene were PCR amplified for each culture product of the third culture product set, and the amplified products were identified by gel electrophoresis. The culture products with positive results for both the nuc gene and the mecA gene were selected and purified to obtain MRSA.
[0025] The screening method provided by the present invention, on the one hand, uses the above-mentioned culture medium composition of specific composition for selective culture of MRSA, and on the other hand, uses a molecular biological detection method to perform PCR amplification and agarose gel electrophoresis identification of nuc gene and mecA gene in MRSA, nuc gene and mecA gene are respectively Staphylococcus aureus heat-resistant nuclease and penicillin binding protein 2a encoding genes, and screen positive samples. The above two operations can significantly improve the screening efficiency of MRSA.
[0026] In some embodiments, the PCR amplification reaction system is 30 μl and comprises the following components: 10XDMSO: 3 μl, 2XPCRmix: 15 μl, primers: 3 μl, 5X Buffer: 6 μl, turbid culture product: 1 μl, and sterile water 2 μl; the PCR reaction program is: pre-denaturation at 98°C for 10 min, denaturation at 98°C for 1 min, annealing temperature of nuc gene at 57.5°C, annealing temperature of mecA gene at 55°C, annealing for 30 s, extension at 72°C for 1 min, 35 cycles in total, 72°C for 10 min, and insulation at 16°C.
[0027] In some embodiments, purification is performed by single colony screening using Baird-Parker agar plates.
[0028] In some embodiments, the method further comprises subjecting the screened single colonies to PCR amplification of the nuc gene and the mecA gene again, and subjecting the amplified products to electrophoresis identification to verify the screening results.
[0029] The beneficial effects of the present invention are:
[0030] The culture medium composition provided by the present invention contains additive components including sodium chloride, polymyxin B, nystatin and oxacillin. By using these components in combination, the growth of non-halophilic bacteria, Gram-negative bacteria, fungi and penicillinase-producing staphylococci can be effectively inhibited, while the growth of MRSA is favorable. The culture medium composition has high selectivity for MRSA and can realize rapid screening of MRSA.
[0031] The screening method provided by the present invention uses the above-mentioned culture medium composition of specific composition to selectively culture MRSA, and combines it with the molecular biological detection method to screen MRSA from the oral samples of wild Tibetan macaques for the first time. The screening method of the present invention greatly improves the screening efficiency of MRSA, and can quickly, accurately and efficiently screen and enrich MRSA from a large number of samples (in actual work, it can be detected within three days from the inoculation of the sample at the fastest, the normal sample can become turbid within 12-20 hours of inoculation, and the electrophoresis time of the PCR product is within three hours), providing technical support for the experimental research of MRSA. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 : Flow chart of the screening method of the present invention;
[0033] Figure 2 : Electrophoresis of nuc gene after adding polymyxin B and nystatin to TSB medium, nuc gene 269bp; M: 5000bp Maker; 0: sterile water; 1: MRSA quality inspection strain; 2: NM1; 3: BM1; 4: BM2; 5: TXH1; 6: TXH2; 7: TXH3; 8: TXH4; 9: TQL1; 10: YXM1; 11: TQS1; 12: TQS2; 13: YXK1; 14: YQT1; 15: CX1; 16: TQY1; 17: TQY2; 18: TQY3; 19: GX1; 20: GX2; 21: TQR1; 22: TXX1; 23: TXX2; 24: TFJ1; 25: TFJ2; 26: TFJ3; 27: YL1; 28: YL2; 29: 11; 30: 12; 31: 13;
[0034] Figure 3: Electrophoresis of nuc gene after TSB medium was added with polymyxin B and nystatin, nuc gene 269bp; M: 5000bp Maker; 0: sterile water; 1: MRSA quality inspection strain; 32:14; 33:21; 34:22; 35:23; 36:24; 37:25; 38:31; 39:32; 40:33; 41:34; 42:35; 43:41; 44:42; 45:43; 46:44; 47:51; 48:52; 49:53; 50:54; 51:55; 52:YXM; 53:CX; 54:YQT; 55:4;
[0035] Figure 4 : Electrophoresis of nuc gene after adding oxacillin to TSB medium, nuc gene 269bp; M: 5000bp Maker; 0: sterile water; 1: MRSA quality inspection strain; 2: BM1; 3: BM2; 4: TXH2; 5: YXM1; 6: TQS2; 7: YXK1; 8: YQT1; 9: CX1; 10: TQY1; 11: TQY3; 12: GX2; 13: TXX1; 14: TXX2; 15: TFJ1; 16:11; 17:12; 18:13; 19:14; 20:21; 21:22; 22:23; 23:31; 24:32; 25:33; 26:34; 27:35; 28:41; 29:43; 30:51; 31:53;
[0036] Figure 5 : Electrophoresis of nuc gene after adding oxacillin to TSB medium, nuc gene 269bp; M: 5000bp Maker; 0: sterile water; 1: MRSA quality inspection strain; 32: 54; 33: 55; 34: YQT; 35: 4;
[0037] Figure 6 :The electrophoresis of mecA gene after adding oxacillin to TSB medium, mecA gene 310bp; M: 5000bpMaker; 0: sterile water; 1: MRSA quality inspection strain; 2: BM1; 3: BM2; 4: TXH2; 5: YXM1; 6: TQS2; 7: YXK1; 8: YQT1; 9: CX1; 10: TQY1; 11: TQY3; 12: GX2; 13: TXX1; 14: TXX2; 15: TFJ1; 16:11; 17:12; 18:13; 19:14; 20:21; 21:22; 22:23; 23:31; 24:32; 25:33; 26:34; 27:35; 28:41; 29:43; 30:51; 31:53;
[0038] Figure 7: Electrophoresis of mecA gene after adding oxacillin to TSB medium, mecA gene 310bp; M: 5000bpMaker; 0: sterile water; 1: MRSA quality inspection strain; 32: 54; 33: 55; 34: YQT; 35: 4. DETAILED DESCRIPTION
[0039] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0040] 1. Materials and Methods
[0041] 1.1 Sample situation
[0042] Table 1 Sample collection information in this study
[0043]
[0044]
[0045] 1.2 Materials and culture procedures
[0046] TSB was purchased from Oxoid. Sodium chloride and other biological reagents such as nystatin, polymyxin B, and oxacillin were purchased from a reagent company. General MRSA operation techniques were performed according to standard procedures. Primer synthesis was completed by Sangon Biotech (Shanghai) Co., Ltd.
[0047] Table 2 Primers used in this study
[0048]
[0049] 1.3 Bacterial liquid PCR amplification system and reaction procedure
[0050] Bacterial liquid PCR amplification system: a total of 30μl system, including: 10X DMSO: 3μl; 2X PCR mix: 15μl; forward primer: 1.5μl; reverse primer 1.5μl; 5X Buffer: 6μl; bacterial liquid: 1μl; sterile water 2μl.
[0051] The PCR reaction procedure of the bacterial solution was as follows: pre-denaturation at 98°C for 10 min, then denaturation at 98°C for 1 min, annealing temperature of nuc gene at 57.5°C, annealing temperature of mecA gene at 55°C, annealing for 30 seconds, extension at 72°C for 1 min, a total of 35 cycles, 72°C for 10 min, and insulation at 16°C to complete the PCR reaction.
[0052] Example 1 Screening MRSA from oral samples of wild animals
[0053] Screening flow chart Figure 1 shown.
[0054] 1. One-time screening
[0055] A total of 81 samples were collected, including 52 oral samples, 5 human oral samples, and 24 environmental samples. The TSB culture medium was sterilized and poured into the centrifuge tubes containing the sample swabs, and cultured overnight at 37°C and 220 rpm. After culture, 20 μl of culture solution was pipetted with a pipette and added to the newly prepared sterile 7.5% NaCl TSB culture medium, and 75 μg / l polymyxin B and 12.5 μg / l nystatin solution were added. Cultured overnight at 37°C and 220 rpm, 54 turbid bacterial solutions were obtained, indicating that bacteria grew in the samples, and the target strains we need to identify may exist.
[0056] 2. Secondary screening
[0057] 54 turbid bacterial solutions were subjected to bacterial solution PCR, and the nuc gene, a specific gene fragment of Staphylococcus aureus, was PCR-treated. The amplified product was subjected to 1% agarose gel electrophoresis and photographed with a gel imaging system, and 34 turbid bacterial solutions with positive nuc gene were obtained. Figure 2 , Figure 3 ), BM1;BM2;TXH2;YX M1;TQS2;YXK1;YQT1;CX1;TQY1;TQY3;GX2;TXX1;TXX2;TFJ1;11;12;13;14;21;22;23;31;32;33;34;35;41;43;51;53;54;55;YQT;4; all were nuc gene positive.
[0058] 3. Three-time screening
[0059] The 34 turbid bacterial liquids that were positive for the nuc gene were re-inoculated into TSB medium, and 6 μg / l of oxacillin was added. The culture medium was cultured at 37°C and 220 rpm for 24-48 hours. The turbidity of the culture medium was observed every 12 hours to obtain 34 turbid bacterial liquids for use.
[0060] 4. Four-time screening
[0061] PCR was performed on 34 turbid bacterial solutions, and PCR and electrophoresis were performed to identify the nuc gene and mecA gene of Staphylococcus aureus. Eight bacterial solutions with both nuc gene and mecA gene positive were obtained ( Figure 4-Figure 7), BM1; TXH2; YXK1; TQY3; GX2; TFJ1; 11; 21; nuc gene and mecA gene were both positive.
[0062] 5. Verification of screening results
[0063] The double-positive bacterial solution was diluted in a gradient manner, and each sample was diluted 10 -1 ~10 -6 Dilution 6, 10 -1 ~10 -6 Five plates were coated for each dilution, and Baird-Parker medium containing potassium tellurite was added. MRSA strain 25138 was used as a positive control. Single colonies containing transparent circles and precipitation rings were picked and cultured at 37°C for 45-48 hours. Every 12 hours, the culture medium was observed to see if black colonies with transparent circles and precipitation rings grew. Three single colonies of Staphylococcus aureus were obtained, and MRSA strain 25138 was used as a positive control. PCR amplification and electrophoresis identification of its products were performed using primers for nuc gene and mecA gene. The results showed that a single colony TQY1 from monkey sample TQY was a positive band, which was MRSA.
[0064] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.
Claims
1. A selective culture medium composition for methicillin-resistant Staphylococcus aureus, characterized in that: The medium composition comprises a basal medium and an additive component, wherein the additive component comprises sodium chloride, polymyxin B, nystatin and oxacillin.
2. The culture medium composition according to claim 1, characterized in that Each 1L of the culture medium composition contains the following additive components: 50-200g of sodium chloride, 50-100g of polymyxin b, 5-20g of nystatin and 2-6.4g of oxacillin.
3. The culture medium composition according to claim 2, characterized in that Each 1 L of the culture medium composition contains the following additive components: 70 g of sodium chloride, 75 g of polymyxin b, 12.5 g of nystatin and 6 g of oxacillin.
4. The culture medium composition according to claim 3, characterized in that The basic culture medium is tryptone soy broth medium.
5. Use of the culture medium composition according to any one of claims 1 to 4 in the cultivation or screening of methicillin-resistant Staphylococcus aureus.
6. A method for screening methicillin-resistant Staphylococcus aureus from oral samples of wild animals, characterized in that: The method comprises: Using the culture medium composition according to any one of claims 1 to 4 to culture each sample in the sample set to obtain a preliminary screened selective culture product; The selective culture products of the preliminary screening were subjected to PCR amplification of the methicillin-resistant Staphylococcus aureus-specific genes and positive samples were further screened from them.
7. The method according to claim 6, characterized in that The method comprises: Each sample of the wild animal oral sample set was inoculated into a basic culture medium containing sodium chloride, polymyxin B, nystatin and oxacillin, and cultured at 36-38°C and 200-250 rpm for 18-24 hours. The culture products that were turbid after culture were selected and recorded as the culture product set; The nuc gene and the mecA gene were amplified by PCR in each culture product of the culture product set, and the amplified products were identified by gel electrophoresis. The culture products with both positive nuc gene and mecA gene were selected and purified to obtain methicillin-resistant Staphylococcus aureus.
8. The method according to claim 6, characterized in that The method comprises: Each sample of the wild animal oral sample set was inoculated into a basic culture medium containing sodium chloride, polymyxin B and nystatin, and cultured at 36-38°C and 200-250 rpm for 22-26 hours, and the culture products that were turbid after culture were selected and recorded as the first culture product set; PCR amplification of the nuc gene is performed on each culture product of the first culture product set, and the amplified products are identified by gel electrophoresis, and culture products with positive nuc gene are selected and recorded as the second culture product set; The culture products of the second culture product set were transferred to a new basal medium containing oxacillin for culture at 36-38° C. and 200-250 rpm for 24-48 hours, and the culture products that were turbid after culture were selected and recorded as the third culture product set; The nuc gene and mecA gene were amplified by PCR in each culture product of the third culture product set, and the amplified products were identified by gel electrophoresis. The culture products with positive results for both the nuc gene and the mecA gene were selected and purified to obtain methicillin-resistant Staphylococcus aureus.
9. The method according to claim 7 or 8, characterized in that: The PCR amplification reaction system, calculated as 30 μl, comprises the following components: 10X DMSO: 3 μl, 2X PCRmix: 15 μl, primer: 3 μl, 5X Buffer: 6 μl, turbid culture product: 1 μl, and sterile water 2 μl; The PCR reaction program is: 98°C pre-denaturation for 10 min, 98°C denaturation for 1 min, nuc gene annealing temperature of 57.5°C, mecA gene annealing temperature of 55°C, annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles, 72°C for 10 min, and 16°C insulation.
10. The method according to claim 7 or 8, characterized in that: The purification was performed by single colony screening on Baird-Parker agar plates.