A method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin

Abstract

The present invention discloses a method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin. The nucleic acid extraction method for methicillin-resistant Staphylococcus aureus (MRSA) disclosed by the present invention firstly proposes to use nisin to lyse MRSA, that is, incubate the cultured methicillin-resistant Staphylococcus aureus with nisin, and then use conventional DNA extraction operations, such as using a ready-made commercial kit for genomic DNA extraction. Experimental data shows that after pretreatment with nisin, the extraction efficiency of genomic DNA of MRSA can be significantly improved. The genomic DNA extracted by the method of the present invention has high purity and large concentration, can meet the requirements of research such as identification and sequencing of specific genes, and is time-saving, efficient, low-cost and simple to operate.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial genome extraction, and particularly relates to a method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin. Background Art

[0002] Aerobic Gram-positive cocci are important pathogenic bacteria for bacterial infections, including bacteria such as Staphylococcus, Streptococcus pneumoniae, hemolytic streptococci, viridans streptococci, Enterococcus, etc. The infection rate is increasing year by year, accounting for about 40% of all bacterial infections. In hospital-acquired bloodstream infections, coagulase-negative Staphylococcus, Staphylococcus aureus, and Enterococcus rank among the top three. In recent years, the drug resistance of Gram-positive cocci has become increasingly serious, especially methicillin-resistant Staphylococcus aureus (MRSA). Whether it is the molecular diagnostic technology for detecting pathogenic microorganisms or the transcriptome research technology for bacterial molecular biology research, the first step is the preparation of template nucleic acids, that is, the extraction of nucleic acids (including RNA and DNA) in the sample, which directly affects the results of these detection technologies. Staphylococcus aureus is a Gram-positive bacterium with a thick cell wall that is difficult to break. More than 90% of Staphylococcus aureus contains protein A. Some Staphylococcus aureus itself also secretes a heat- and nuclease-resistant protein that can degrade nucleic acids. Therefore, the effect of its nucleic acid extraction has not been very satisfactory.

[0003] Currently, the nucleic acid extraction of Gram-positive bacteria mainly uses the method of extracting nucleic acids after lysing cells. Among them, mechanical fragmentation methods and enzymatic lysis methods are commonly used to break the cell wall of bacteria. Mechanical fragmentation methods such as the glass bead method and ultrasonic fragmentation have limitations in that they require additional equipment and the fragmentation process is intense, which may cause physical breakage of genomic DNA. The enzymatic lysis method is relatively mild. Lysozyme is the most commonly used cell wall hydrolase in the laboratory for extracting nucleic acids from Gram-positive bacteria, mainly hydrolyzing the β-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid that connects the cell wall of G+ bacteria. However, due to the presence of protein A in Staphylococcus aureus, which is covalently cross-linked with peptidoglycan in the cell wall and is insensitive to lysozyme treatment, the lysis effect is not obvious.

[0004] Due to the great difficulty in extracting genomic DNA of MRSA, there is currently also a Lysostaphin product on the market. Lysostaphin is an endopeptidase, and its mechanism of action is to cleave the glycine pentapeptide cross-linking bridge in the peptidoglycan of the staphylococcal cell wall, thereby destroying the integrity of the cell wall and lysing the bacteria, and improving the extraction rate of S. aureus genomic DNA. However, Lysostaphin has two limitations. On the one hand, previous studies have reported that due to the structural changes in the peptidoglycan cross-linking bridge of the bacterial cell wall, Staphylococcus aureus strains resistant to this enzyme have emerged. On the other hand, Lysostaphin is relatively expensive (600 - 800 yuan / mg, and the single extraction costs about 3 - 4 yuan). The prices of these kits are expensive, the operation steps are cumbersome, and the extraction effect is not ideal. Therefore, how to efficiently and quickly extract nucleic acids from MRSA has become a bottleneck in the popularization and application of current molecular diagnostic technologies and transcriptome research and other technologies.

[0005] Nisin is a bacteriocin produced by a group of Gram-positive bacteria of the genera Lactococcus and Streptococcus. It can act on the cell wall and cell membrane of bacteria, showing a destructive effect on the cell wall and cell membrane, and inhibiting the vast majority of Gram-positive bacteria that cause food spoilage and human infections. Currently, nisin is mainly applied to food preservation and antibacterial research, and no researchers have reported on nisin as a nucleic acid extraction reagent for Gram-positive pathogenic bacteria. Summary of the Invention

[0006] In order to overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin, which can solve the technical problems that the enzymes used in the existing extraction methods are prone to drug resistance, high in price, and the extraction operation is complex, resulting in low extraction efficiency.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin disclosed by the present invention includes the following steps:

[0009] 1) Add nisin to the liquid medium for culturing methicillin-resistant Staphylococcus aureus, incubate, centrifuge to remove the supernatant, and collect the cell precipitate;

[0010] 2) Use a DNA extraction kit to lyse the cell precipitate, remove RNA and proteins, and obtain methicillin-resistant Staphylococcus aureus DNA.

[0011] Preferably, in step 1), MRSA is inoculated into tryptone soy broth medium (TSB liquid medium) and cultured overnight at 37°C with shaking at 200 rpm on a shaker.

[0012] Preferably, the nisin is a commercially available biological grade reagent with an activity > 1000000 IU / g.

[0013] More preferably, the dosage of nisin is as follows: Add a sterile nisin solution to every 2 - 3 mL of liquid medium until the final concentration of nisin is 1 mg / mL.

[0014] Even more preferably, the method for preparing the sterile nisin solution is as follows: Take 50 mg of nisin, add 1 mL of hydrochloric acid solution with a pH of 2, vortex to dissolve, centrifuge at 2500 rpm for 1 min, and filter the supernatant through a 0.22 μm filter to remove bacteria, obtaining a 50 mg / mL sterile nisin solution.

[0015] Preferably, in step 1), add nisin to the overnight - cultured MRSA liquid culture, mix well, incubate at 37°C in an incubator for 1 - 2 h, and after incubation, centrifuge to remove the supernatant.

[0016] Preferably, in step 2), use the lysis buffer and proteinase K in the kit to lyse the cell pellet and remove proteins; continue to treat with RNase A in the kit to remove RNA.

[0017] More preferably, the genomic DNA can be extracted from the cell pellet according to the conventional bacterial DNA extraction steps or using a commercially available conventional bacterial DNA extraction kit, including resuspending the cell pellet, lysing, removing RNA and proteins, and precipitating to obtain genomic DNA.

[0018] More preferably, the specific operation is as follows: Add a sterile nisin solution to 2-3 ml of the culture medium to a final concentration of 1 mg / ml, incubate at 37 °C for 1 h. After incubation, centrifuge to remove the supernatant, add 100 μL of TE buffer, and mix well by shaking; add 100 μL of the lysis solution in the genomic DNA extraction kit and 20 μL of proteinase K, mix well by shaking, and incubate at 55 °C for 0.5 h. This step is to completely lyse the cells and remove proteins; add 5 μL of RNase A, gently invert and mix well, and let stand at room temperature for 5 min; centrifuge at 10000 g for 2 min, transfer the supernatant to a new EP tube, add 2 volumes of cold absolute ethanol to the supernatant from which RNA and proteins have been removed, gently mix the EP tube, and the visible filamentous suspended precipitate is genomic DNA; centrifuge at 10000 g for 2 min to collect the precipitate, and wash the precipitate with 70% pre-cooled ethanol. The precipitate is dried in a laminar flow hood and dissolved in 50 μL of sterile water or TE buffer.

[0019] Furthermore, the proteinase K is provided in the kit and is a commercially available product. Removing RNA and proteins from the lysed system can improve the purity of the genome.

[0020] The present invention also discloses the application of nisin as an enhancer for extracting genomic DNA of methicillin-resistant Staphylococcus aureus.

[0021] The present invention also discloses the application of nisin in the preparation of a kit for extracting genomic DNA of methicillin-resistant Staphylococcus aureus.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The nucleic acid extraction method for methicillin-resistant Staphylococcus aureus (MRSA) disclosed by the present invention first proposes to use nisin to lyse MRSA, that is, incubate the cultured methicillin-resistant Staphylococcus aureus with nisin, and then use conventional DNA extraction operations, such as using a ready-made commercially available kit for genomic DNA extraction. Experimental data show that after pretreatment with Nisin, the extraction efficiency of genomic DNA of MRSA can be significantly improved. The genomic DNA extracted by the method of the present invention has high purity and high concentration (up to 60 times the DNA concentration extracted by the ordinary kit method), can meet the requirements of research such as identification and sequencing of specific genes, and is time-saving, efficient, low-cost, and simple to operate. Therefore, the method of the present invention effectively solves the technical problems that the enzymes used in the existing extraction methods are prone to drug resistance and high in price, and the extraction operation is complex resulting in low extraction efficiency.

[0024] The present invention opens up a new use for nisin, which can be used as a synergist in the MRSA nucleic acid extraction kit. Without using lysostaphin or lysozyme to treat cells, it can significantly improve the efficiency of MRSA nucleic acid extraction, and greatly improve the working efficiency of clinical detection, molecular diagnosis, transcriptome analysis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shows the change in the concentration of MRSA DNA extracted under different nisin concentrations and treatment times;

[0026] Figure 2 Is the gel electrophoresis diagram of MRSA DNA extracted under different nisin concentrations and treatment times; among them, A is nisin incubated for 0.5 h; B is nisin incubated for 1 h; C is nisin incubated for 2 h;

[0027] Figure 3 Is the sensitivity result diagram of qPCR detection of MRSA pure culture; among them, A is the kit method; B is the nisin method;

[0028] Figure 4 Is the standard curve of the qPCR detection system for MRSA;

[0029] Figure 5 Is the amplification efficiency result of the qPCR system using the DNA of MRSA extracted by the nisin method as a template. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0031] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above drawings are used to distinguish similar objects, and do not have to be used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units does not have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0032] The present invention will be further described in detail below with reference to the accompanying drawings:

[0033] I. Experimental materials

[0034] 1. Strains

[0035] The tested MRSA strains used were clinical isolates from a certain disease control and prevention center; Staphylococcus aureus ATCC 25923 was the standard strain and was stored in this laboratory. All strains were stored in a -80°C refrigerator in the form of glycerol cryopreservation. Staphylococcus aureus was cultured in TSB liquid medium (overnight culture in a 37°C constant temperature shaking incubator) or on solid plates (cultured in a 37°C constant temperature incubator for 24 h).

[0036] 2. Reagents and reagent kits

[0037] Nisin was a commercially available biological-grade reagent (activity > 1000000 IU / g, Nanjing Dulai Biotechnology Co., Ltd., production batch number N2202062153), lysozyme was a commercially available molecular biology purity-grade reagent (activity ≥ 20000 U / mg), and lysostaphin was a commercially available biological-grade reagent (activity ≥ 1200 U / mg).

[0038] Randomly select the OMEGA bacterial DNA extraction kit as the extraction control.

[0039] Prepare the Nisin solution: Weigh 50 mg of nisin, add 1 mL of hydrochloric acid solution with pH = 2, vortex to dissolve, centrifuge at 2500 rpm for 1 min, and filter the supernatant through a 0.22 μm filter to sterilize, obtaining a 50 mg / mL sterile Nisin solution.

[0040] II. Experimental methods

[0041] 1. Conditions for extracting genomic DNA after treating MRSA with Nisin

[0042] Take 3 mL of the overnight-cultured MRSA Yn2020070 bacterial solution, and add 0, 30, 60, 90, 150, 300, 450 μL of the above-prepared Nisin solution with different volumes to the 3 mL bacterial solution respectively. Then the working concentrations of Nisin after adding the samples are: 0, 0.5, 1, 1.5, 2.5, 5, 7.5 mg / mL. Incubate statically at 37°C for 0.5, 1, 2 h respectively. Among them, the bacterial solution not treated with Nisin was used as the control group. After the incubation ended, the treatment group (i.e., the Nisin group) was not treated with lysozyme and lysostaphin, while the control group was treated with lysozyme and lysostaphin respectively according to the kit instructions. Except for this, the remaining extraction steps were carried out according to the instructions of the OMEGA bacterial DNA extraction kit.

[0043] The concentration and purity of the extracted genomic DNA of MRSA Yn2020070 were determined using Nanodrop, and the results are shown in Table 1.

[0044] Table 1 Concentrations and purities of the extracted MRSA DNA under different Nisin concentrations and treatment times

[0045]

[0046]

[0047] As can be seen from Table 1, in the negative control group, where MRSA DNA was extracted directly using the OMEGA bacterial DNA extraction kit without Nisin incubation, the DNA concentration was between 7.9 - 9.1 ng / μL, and the A 260 / A 280 、A 260 / A 230 values were both low. For a pure DNA sample, A 260 / A 280 is greater than 1.8. A ratio lower than 1.8 indicates the influence of proteins or phenolic substances. A 230 indicates the presence of some contaminants such as carbohydrates and salts (guanidine salts) in the sample. For relatively pure nucleic acids, the A 260 / A 230 ratio is greater than 2.0. Meanwhile, the test results show that MRSA is resistant to lysozyme, and the lysis effect of lysozyme on MRSA is not obvious.

[0048] Compared with the negative control group, the extraction effect of the treatment group showed an increase in the concentration and purity of DNA. As Figure 1 can be seen, within a certain range, as the Nisin concentration increases, the concentration of the extracted MRSA genomic DNA increases. However, when the Nisin concentration is too high, the DNA concentration decreases as the Nisin concentration increases. When the Nisin concentration is greater than 1.5 mg / mL, as the Nisin concentration increases, the extracted DNA concentration decreases, which may be related to the electrostatic repulsion between Nisin molecules.

[0049] 2. Agarose gel electrophoresis of genomic DNA extracted from MRSA after Nisin treatment

[0050] The quality of the extracted DNA was detected by 1% agarose gel electrophoresis. The electrophoresis parameters were set as follows: 85 V, 350 mA, 1 h 10 min, sample loading volume 1 μL, and DNA ladder loading volume 3 μL. The Nisin concentrations for lanes 1 - 7 were: 0, 0.5, 1, 1.5, 2.5, 5, 7.5 mg / mL.

[0051] It can be seen from Figure 2 that the concentration of MRSA DNA directly extracted by the kit method is too low, and when electrophoresis is carried out, its bands are almost invisible ( Figure 2 the three lanes 1 of A, B, and C in Figure 2 ). After incubation with Nisin and then using the kit (without lysozyme and lysostaphin) to extract the DNA of MRSA, the electrophoretic bands of the sample DNA are bright, clear, and there is almost no serious damage such as DNA breakage and degradation. In addition, the electrophoresis pattern also shows that when the concentration of Nisin is too high, the DNA bands are almost invisible or become darker ( Figure 2 lanes 6-7 in Figure A of Figure 2 lanes 6-7 in Figure B of

[0052] and lane 7 in Figure C of

[0053] ). In short, within a certain concentration range of action, Nisin can be used to improve the yield of the extracted MRSA DNA.

[0054] Adjust the initial bacterial solution to OD 600 =1. After diluting the bacterial solution by 10-fold gradient, the genomic DNA of the MRSA clinical isolate Yn2020070 was extracted according to the above method. Three groups of parallel qPCR reactions were carried out to quantitatively detect the specific gene mecA of the MRSA strain (upstream primer sequence F: 5’GTA GAA ATG ACT GAA CGT AAG ATA A 3’; downstream primer sequence R: 5’CCA ATTCCA CAT TGT TTC GGT CTA A 3’, amplification fragment length 310bp); the PCR reaction system was: 10μL SYBR High-Sensitivy qPCR SuperMix plus, the final concentrations of the upstream and downstream primers were both 1.0μΜ, 1μl of template DNA, and RNase Free Water was added to make up the total volume to 20μL; the reaction conditions were: pre-denaturation at 95℃ for 1min, denaturation at 95℃ for 20sec - annealing at 55℃ for 20sec - extension at 72℃ for 30sec for a total of 40 cycles), and the qPCR reaction was completed on a Roche fluorescence quantitative PCR instrument (Light cycler 96).

[0055] The concentration range from 2.15CFU / mL to 2.15×10 9A pure culture of MRSA at CFU / mL was used to extract DNA by the kit method and the Nisin method respectively. Using this as a template, qPCR was performed on mecA. Taking the logarithm of the cell concentration (CFU / mL) as the abscissa and the corresponding average cycle threshold (Ct) as the ordinate, the standard curves of each system were constructed. Calculate the R 2 value, and obtain the reaction efficiency according to the curve slope (Slope): E=(10 (-1 / Slope) -1)×100%. Judge the reaction efficiency of each system and the detection range of cell concentration ( Figure 3 , Figure 3 The MRSA concentrations in were 2×10 9 CFU / mL, curve a; 2×10 8 CFU / mL, curve b; 2×10 7 CFU / mL, curve c; 2×10 6 CFU / mL, curve d; 2×10 5 CFU / mL, curve e; 2×10 4 CFU / mL, curve f; 2×10 3 CFU / mL, curve g; 2×10 2 CFU / mL, curve h; 2×10 CFU / mL, curve i).

[0056] According to the results, a graph was made. As shown in Figure 4 , the standard curve of the kit method was: y=-2.377x + 34.277, R 2 =0.9506, Efficiency = 167.07%; the standard curve of the Nisin method was: y=-2.6586x + 32.6, R 2 =0.9165, Efficiency = 137.76%. Both of the two standard curves showed a good linear relationship between the logarithm of the cell concentration and the Ct value.

[0057] However, the qPCR standard curve requires the slope to be within the range of -2.6 to -3.6. The Nisin method meets the slope requirement (-2.6586), while the kit method does not meet the slope requirement (-2.377). Therefore, the genomic DNA extracted by the Nisin treatment method can meet the requirements of downstream qPCR experiments and is superior to the ordinary kit method.

[0058] 4. Detection of the amplification efficiency of qPCR by extracting genomic DNA after treating MRSA with Nisin

[0059] The E value, the amplification efficiency value of qPCR, is mainly judged by the linear relationship equation of the standard curve Cq=-k lgX0 + b. When the amplification efficiency E is between 90% and 110%, it is considered that the amplification is close to the ideal situation; the parameter R2 The requirement is ≥ 0.98, R 2 The closer it is to 1, the higher the correlation between the Log values of Cq and X0. A conventional PCR reaction system includes dNTP, template, primers, Mg+, amplification buffer, polymerase, etc. Changing any minor parameter in the amplification reaction may cause an increase or decrease in the PCR amplification product. The primers for PCR reactions do not need to be highly purified, but contamination by DNA polymerase inhibitors, proteases, nucleases, polysaccharide substances that can bind to DNA, and proteins during the nucleic acid extraction process should be avoided. Some impurities such as SDS may inhibit the activity of DNA polymerase, thereby inhibiting the PCR reaction and causing a decrease in amplification efficiency.

[0060] The genomic DNA of MRSA strain Yn2020070 was extracted using the Nisin method, and its concentration (ng / μL) and purity (A 260 / A 280 、A 260 / A 230 ) were determined by a NanoDrop micro-spectrophotometer. The genomic DNA was serially diluted 5-fold and used as a template for qPCR. Three sets of parallel qPCR reactions were performed to quantitatively detect the 16S rDNA V3-V4 region fragment of the MRSA strain (using universal primers, upstream primer sequence F: 5’CCTACGGNGGCWGCAG 3’; downstream primer sequence R: 5’GACTACHVGGGTATCTAATCC 3’, amplification fragment length 464bp); the PCR reaction system was: 10μl SYBR High-Sensitivy qPCRSuperMix plus, the final concentrations of the upstream and downstream primers were both 1.0 μΜ, 1 μL of template DNA, and RNase Free Water was added to make up the total volume to 20 μL; the reaction conditions were: pre-denaturation at 95°C for 1 min, denaturation at 95°C for 20 sec - annealing at 55°C for 20 sec - extension at 72°C for 30 sec for a total of 40 cycles), and the qPCR reaction was completed on a Roche LightCycler 96 fluorescence quantitative PCR instrument.

[0061] A standard curve was plotted based on the genomic DNA concentration and the Ct values obtained at different concentrations. The results are as Figure 5 shown. It can be seen that when the DNA extracted by the Nisin method was used as a template for qPCR, the amplification efficiency E value was 91.38%, and the effect was ideal.

[0062] 5. Extraction of genomic DNA of other MRSA isolates by the Nisin treatment method

[0063] The genomic DNA of the MRSA clinical isolates extracted by the above method was used with the OMEGA bacterial DNA extraction kit as a control, and the concentration and purity of the genomic DNA were measured by Nanodrop. The results are shown in Table 2.

[0064] The genomic DNA of 7 MRSA isolates was extracted by comparing the kit method and the Nisin method. The concentration and purity of the DNA extracted by the kit method were lower than those of the Nisin treatment group.

[0065] Table 2 Concentration and purity of DNA of MRSA isolates extracted by two methods

[0066]

[0067] From the above comparison, it can be seen that using nisin to replace lysozyme and lysostaphin to treat MRSA cells can fully rupture the cell wall, obtain genomic DNA with high concentration and high purity, with low cost and good effect. The extracted DNA is suitable for downstream detection experiments such as PCR amplification.

[0068] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin, characterized in that, Comprising the following steps: 1) Add nisin to the liquid medium for culturing methicillin-resistant Staphylococcus aureus, incubate, centrifuge to remove the supernatant, and collect the cell precipitate; the dosage of nisin is: add a sterile nisin solution to every 2 - 3 mL of the liquid medium until the final concentration of nisin is 1 mg / mL; the activity of nisin > 1000000 IU / g; 2) Use a DNA extraction kit to lyse the cell precipitate, remove RNA and proteins, and obtain methicillin-resistant Staphylococcus aureus DNA.

2. The method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin according to claim 1, characterized in that, The nisin is a commercially available biological grade reagent.

3. The method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin according to claim 1, wherein The preparation method of the sterile nisin solution is as follows: Take 50 mg of nisin, add 1 mL of hydrochloric acid solution with a pH of 2, vortex to dissolve, then centrifuge at 2500 rpm for 1 min, and filter and sterilize the supernatant through a 0.22 μm filter to obtain a 50 mg / mL sterile nisin solution.

4. The method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin according to claim 1, wherein In step 1), the operation of culturing methicillin-resistant Staphylococcus aureus is as follows: Inoculate methicillin-resistant Staphylococcus aureus into tryptic soy broth medium (TSB liquid medium), and culture overnight at 37°C with a shaking speed of 200 rpm on a shaker.

5. The method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin according to claim 1, characterized in that, In step 1), incubate statically at 37°C for 1 - 2 h.

6. The method for extracting genomic DNA of methicillin-resistant Staphylococcus aureus using nisin according to claim 1, wherein In step 2), use the lysis solution and proteinase K in the kit to lyse the cell precipitate and remove proteins; continue to treat with RNase A in the kit to remove RNA.

7. Application of nisin as a synergist for extracting genomic DNA of methicillin-resistant Staphylococcus aureus.

8. Application of nisin in the preparation of a kit for extracting genomic DNA of methicillin-resistant Staphylococcus aureus.

Images