CN115851699A - Method for extracting methicillin-resistant staphylococcus aureus genome DNA (deoxyribonucleic acid) by adopting nisin - Google Patents
Method for extracting methicillin-resistant staphylococcus aureus genome DNA (deoxyribonucleic acid) by adopting nisin Download PDFInfo
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Abstract
The invention discloses a method for extracting methicillin-resistant staphylococcus aureus genome DNA (deoxyribonucleic acid) by adopting Nisin, and the method is used for extracting nucleic acid of methicillin-resistant staphylococcus aureus (MRSA), firstly proposes that Nisin is used for cracking MRSA, namely, the cultured methicillin-resistant staphylococcus aureus is incubated by adopting Nisin, and then conventional DNA extraction operation is used, for example, a ready-made commercial kit is used for extracting genome DNA, and experimental data shows that the extraction efficiency of the genome DNA of MRSA can be obviously improved after the Nisin is pretreated in advance. The genomic DNA extracted by the method has high purity and concentration, can meet the requirements of researches such as identification, sequencing and the like of specific genes, and has the advantages of time saving, high efficiency, low cost and simple operation.
Description
Technical Field
The invention belongs to the technical field of microbial genome extraction, and particularly relates to a method for extracting methicillin-resistant staphylococcus aureus genome DNA by using nisin.
Background
Gram-positive aerobic cocci are important pathogenic bacteria of bacterial infections, including bacteria of the genera staphylococcus, streptococcus pneumoniae, hemolytic streptococcus, streptococcus viridans, streptococcus, enterococcus, etc., and the infection rate is increasing year by year, accounting for about 40% of all bacterial infections. In hospital-acquired bloodstream infections, coagulase-negative staphylococci, staphylococcus aureus and enterococci were three at the top, respectively. In recent years, the resistance of gram-positive cocci, in particular methicillin-resistant staphylococcus aureus MRSA, has become more severe. Whether it is a molecular diagnostic technique for the detection of pathogenic microorganisms or a transcriptome study technique for the molecular biology study of bacteria, the first step is the preparation of template nucleic acids, i.e., the extraction of nucleic acids (including RNA and DNA) from a sample, which directly affects the results of these detection techniques. The staphylococcus aureus is gram-positive bacteria, the cell wall is thick and difficult to break, and more than 90 percent of the staphylococcus aureus contains staphylococcus protein A; some staphylococcus aureus also secretes a protein which is heat-resistant and nuclease-resistant and can degrade nucleic acid, so the effect is not ideal in the aspect of nucleic acid extraction.
At present, the extraction of nucleic acid from gram-positive bacteria mainly adopts a method of extracting nucleic acid after cell lysis, wherein the cell wall of the bacteria is usually destroyed by a mechanical disruption method and an enzymatic lysis method. Mechanical disruption methods such as glass bead method, ultrasonication, etc. have limitations in that additional equipment is required and the disruption process is severe, which may cause physical disruption of genomic DNA. The enzyme cleavage principle is mild, and Lysozyme (Lysozyme) is the most commonly used cell wall hydrolase in laboratories for the extraction of gram-positive bacterial nucleic acids, which primarily hydrolyzes the β -1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid that are attached to the G + bacterial cell wall. However, since S.aureus contains staphylococcal protein A, covalently cross-linked with peptidoglycan in the cell wall, it is not sensitive to lysozyme treatment and therefore the lytic effect is not significant.
Because of the difficulty in extracting MRSA genomic DNA, a Lysostaphin (Lysostaphin) product is also available on the market. Lysostaphin is an endopeptidase, and its action mechanism is to crack the cross-linked glycine pentapeptide bridge in the peptidoglycan of staphylococcus cell wall, so as to destroy the integrity of cell wall and dissolve thallus, and raise the extraction rate of staphylococcus aureus genome DNA. Lysostaphin, however, has two limitations, and on the one hand, studies have reported that a staphylococcus aureus strain resistant to this enzyme appears due to structural changes in the peptidoglycan cross-linking bridges of the bacterial cell wall. On the other hand, lysostaphin has a high selling price (600-800 yuan/mg, which costs about 3-4 yuan per extraction). These kits are expensive, complicated in operation steps, and unsatisfactory in extraction effect. Therefore, how to efficiently and quickly extract nucleic acid from MRSA has become a bottleneck for the popularization and application of the current molecular diagnosis technology, transcriptome research and other technologies.
Nisin (nisin) is a bacterial peptide produced by a group of gram-positive bacteria of the genus lactococcus and streptococcus, and acts on the cell walls and cell membranes of the bacteria to exert a destructive effect on the cell walls and cell membranes and inhibit most of the gram-positive bacteria causing food spoilage and human infection. At present, nisin is mainly applied to food preservation and bacteriostatic research, and no research report on Nisin as a nucleic acid extraction reagent of gram-positive pathogenic bacteria exists by researchers.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for extracting methicillin-resistant staphylococcus aureus genome DNA by adopting nisin, which can solve the technical problems that the enzyme used in the existing extraction method is easy to generate drug resistance, the price is high, the extraction operation is complex and the extraction efficiency is low.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a method for extracting methicillin-resistant staphylococcus aureus genome DNA by adopting nisin, which comprises the following steps:
1) Adding nisin into a liquid culture medium for culturing methicillin-resistant staphylococcus aureus, incubating, centrifuging to remove supernatant, and collecting thallus precipitate;
2) And (3) cracking the thallus precipitate by adopting a DNA extraction kit, and removing RNA and protein to obtain methicillin-resistant staphylococcus aureus DNA.
Preferably, in step 1), MRSA is inoculated into tryptone soy broth TSB liquid medium, cultured at 37 ℃ and 200rpm with shaking on a shaker overnight.
Preferably, the nisin is a commercially available biological grade agent with an activity of >1000000IU/g.
Further preferably, nisin is used in an amount of: sterile nisin solution was added to 2-3mL of liquid medium until the final concentration of nisin in the milk was 1mg/mL.
Still more preferably, the sterile nisin solution is formulated as follows: taking 50mg of nisin, adding 1mL of hydrochloric acid solution with the pH value of 2, dissolving by vortex, centrifuging at 2500rpm for 1min, and filtering and sterilizing the supernatant by a 0.22 mu m filter to obtain a sterile nisin solution of 50 mg/mL.
Preferably, in the step 1), nisin is added into the overnight-cultured MRSA liquid culture, the mixture is mixed evenly, the culture box is kept still and incubated for 1 to 2 hours at 37 ℃, and after the incubation is finished, the supernatant is centrifuged.
Preferably, in the step 2), the lysate and the proteinase K in the kit are adopted to perform cracking treatment on the thallus precipitate to remove protein; and (4) adopting RNase A in the kit to continue processing to remove RNA.
Further preferably, the bacterial pellet is extracted according to conventional bacterial DNA extraction procedures or by using a conventional commercially available bacterial DNA extraction kit, and the steps include cell pellet resuspension, lysis, RNA and protein removal, and pellet to obtain genomic DNA.
Still more preferably, the specific operations are: adding sterile Nisin (Nisin) solution into 2-3ml of culture solution until the final concentration is 1mg/ml, incubating for 1h at 37 ℃, centrifuging to remove supernatant after incubation is finished, adding 100 mu L of TE buffer solution, and shaking and mixing uniformly; adding 100 mu L of lysate and 20 mu L of proteinase K in the genome extraction kit, oscillating, uniformly mixing, and incubating at 55 ℃ for 0.5h, wherein the step is to completely lyse cells and remove proteins; adding 5 μ L RNase A, gently reversing, mixing, and standing at room temperature for 5min; centrifuging at 10000g for 2min, transferring the supernatant to a new EP tube, adding 2 times of cold absolute ethanol into the supernatant without RNA and protein, and gently mixing the EP tube to obtain filamentous suspension precipitate as genome DNA; the precipitate was collected by centrifugation at 10000g for 2min and washed with 70% precooled ethanol. The pellet was air-dried in a clean bench and dissolved in 50. Mu.L of sterile water or TE buffer.
Further, the proteinase K is provided in a kit, and belongs to a commercially available product. The purity of the genome can be improved by removing RNA and protein in the system after cracking.
The invention also discloses application of nisin as a synergist for extracting methicillin-resistant staphylococcus aureus genome DNA.
The invention also discloses application of nisin in preparing a kit for extracting methicillin-resistant staphylococcus aureus genome DNA.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a nucleic acid extraction method for methicillin-resistant staphylococcus aureus (MRSA), which is characterized in that the MRSA is cracked by Nisin for the first time, namely, the cultured methicillin-resistant staphylococcus aureus is incubated by Nisin, and then conventional DNA extraction operation is used, for example, the existing commercial kit is used for extracting genome DNA. The genomic DNA extracted by the method has high purity and high concentration (the highest concentration can reach 60 times of the concentration of the DNA extracted by a common kit method), can meet the requirements of researches such as identification, sequencing and the like of specific genes, and has the advantages of time saving, high efficiency, low cost and simple operation. Therefore, the method effectively solves the technical problems that the enzyme used in the existing extraction method is easy to generate drug resistance and has high price, and the extraction efficiency is low due to complex extraction operation.
The invention opens up a new application for nisin, can be used as a synergist in an MRSA nucleic acid extraction kit, does not need lysostaphin and lysozyme to treat cells, obviously improves the nucleic acid extraction efficiency of MRSA, and can greatly improve the working efficiency of clinical detection, molecular diagnosis, transcriptome analysis and the like.
Drawings
FIG. 1 shows the variation of the concentration of MRSA DNA under different nisin action concentrations and treatment times;
FIG. 2 is a diagram of the electrophoresis of MRSA DNA gel at different nisin action concentrations and treatment times; wherein A is nisin for incubation for 0.5h; b is nisin incubation for 1h; c is nisin for incubation for 2h;
FIG. 3 is a graph showing the sensitivity results of qPCR detection of pure MRSA cultures; wherein, A is a kit method; b is nisin method;
FIG. 4 is a standard curve for qPCR detection of MRSA system;
FIG. 5 shows the result of the amplification efficiency of qPCR system using MRSA DNA obtained by nisin method as a template.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
1. experimental Material
1. Bacterial strains
The MRSA strains to be tested are clinical isolates of a disease control center; staphylococcus aureus ATCC 25923 was a standard strain and was stored in this laboratory. All strains were stored in a-80 ℃ freezer by means of glycerol cryopreservation. The staphylococcus aureus is cultured in TSB liquid culture medium (37 ℃ constant temperature shaking incubator overnight culture) or solid plate (37 ℃ constant temperature incubator for 24 h).
2. Reagent and kit
Nisin Nisin is a commercial biological-grade reagent (the activity is more than 1000000IU/g, nanjing Doulele biotechnology limited, production batch number is N2202062153), lysozyme is a commercial molecular biological purity-grade reagent (the activity is more than or equal to 20000U/mg), and lysostaphin is a commercial biological-grade reagent (the activity is more than or equal to 1200U/mg).
An OMEGA bacterial DNA extraction kit was randomly selected as an extraction control.
Preparing a Nisin solution: weighing Nisin 50mg, adding hydrochloric acid solution with pH = 21 mL, dissolving by vortex, centrifuging at 2500rpm for 1min, and filtering and sterilizing the supernatant through a 0.22 μm filter to obtain sterile Nisin solution with 50 mg/mL.
2. Experimental method
1. Conditions for extracting genomic DNA after Nisin treatment of MRSA
Taking 3mL of overnight-cultured MRSA Yn2020070 bacterial liquid, adding 0, 30, 60, 90, 150, 300 and 450 muL of the prepared Nisin solution with different volumes into 3mL of bacterial liquid respectively, wherein the action concentration of Nisin after sample addition is as follows: 0. 0.5, 1, 1.5, 2.5, 5, 7.5mg/mL. Standing and incubating at 37 ℃ for 0.5, 1 and 2 hours respectively. The control group was a bacterial solution not treated with Nisin. After the incubation, the treatment group (i.e., nisin group) was not treated with lysozyme and lysostaphin, while the control group was treated with lysozyme and lysostaphin according to the kit instructions, respectively, except that the remaining extraction steps were performed according to the instructions of the OMEGA bacterial DNA extraction kit.
The concentration and purity of the extracted MRSA Yn2020070 genomic DNA were determined by Nanodrop and the results are shown in Table 1.
TABLE 1 concentration and purity of MRSA DNA extracted at different concentrations of Nisin and treatment times
As can be seen from Table 1, the negative control group was extracted MRSA DNA directly using OMEGA bacterial DNA extraction kit without Nisin incubation, and the DNA concentration was 7.9-9.1 ng/. Mu.L, and A 260 /A 280 、A 260 /A 230 The values are all lower. Pure DNA sample A 260 /A 280 Above 1.8, a ratio below 1.8 indicates the effect of the presence of protein or phenolic material. A. The 230 Indicating the presence of some contaminants such as carbohydrates, salts (guanidinium) etc., relatively pure nucleic acids and A 260 /A 230 The ratio is greater than 2.0. Meanwhile, the test result shows that the MRSA has resistance to lysozyme, and the lysozyme isThe cracking effect of MRSA was not significant.
The extraction effect of the treatment group was improved in the concentration and purity of DNA compared to the negative control group. As can be seen from fig. 1, in a certain range, the concentration of the proposed MRSA genomic DNA increases as the Nisin concentration increases, whereas when Nisin concentration is too high, it appears that the DNA concentration decreases as the Nisin concentration increases. When the action concentration of Nisin is more than 1.5mg/mL, the DNA concentration is reduced with the increase of Nisin concentration, and the DNA concentration is possibly related to the electrostatic repulsion between Nisin molecules.
2. Agarose gel electrophoresis of genomic DNA extracted after Nisin treatment of MRSA
The quality of the extracted DNA was checked by electrophoresis on 1% agarose gel. Setting electrophoresis parameters: 85V,350mA,1h, 10min, sample loading amount of 1. Mu.L, and DNA ladder loading amount of 3. Mu.L. Nisin action concentration of Lane 1-7: 0. 0.5, 1, 1.5, 2.5, 5, 7.5mg/mL.
As can be seen from FIG. 2, the concentration of MRSA DNA extracted directly by the kit method was too low, and the bands were hardly visible by electrophoresis (three lanes 1 of A, B, C in FIG. 2). After Nisin incubation is adopted, the kit (without lysozyme and lysostaphin) is used for DNA extraction of MRSA, the electrophoresis band of the sample DNA is bright and clear, and severe damages such as DNA breakage, degradation and the like are hardly caused. In addition, the electrophoretogram showed that the DNA bands were hardly visible or darkened when the concentration of Nisin was too high (lane 6-7 in Panel A of FIG. 2, lane6-7 in Panel B of FIG. 2, and lane7 in Panel C of FIG. 2). In summary, nisin can be used to increase the yield of extracted MRSA DNA within a certain range of action concentration.
Therefore, the optimal conditions for extracting MRSA genomic DNA are selected with the Nisin treatment concentration of 1mg/mL and the treatment time of 1 h.
3. Sensitivity detection of qPCR (quantitative polymerase chain reaction) by extracting genome DNA after MRSA (methicillin-resistant staphylococcus aureus) treatment by Nisin
OD adjustment of initial bacterial liquid 600 =1, 10-fold gradient bacterial suspension, and the genomic DNA of MRSA clinical isolate Yn2020070 extracted according to the above method. 3 sets of parallel qPCR reactions were performed to quantitatively detect the specific gene mecA of the MRSA strain (upstream primer sequence F:5'GTA GAA ATG ACT GAA CGT AAG ATA A3'; the sequence of the downstream primer R:5'CCA ATT CCA CAT TGT TTC GGT CTA A3', the length of the amplified fragment is 310 bp); the PCR reaction system is as follows: 10 μ L
SYBR High-sensitvy qPCR Supermix plus, final concentrations of upstream and downstream primers are both 1.0 μm, template DNA is 1 μ L, RNase Free Water makes up the total volume to 20 μ L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 20 sec-annealing at 55 ℃ for 20 sec-extension at 72 ℃ for 30sec for 40 cycles), qPCR reactions were performed on a roche fluorescence quantitative PCR instrument (Light cycler 96).
The concentration ranges from 2.15CFU/mL to 2.15X 10 9 CFU/mL MRSA pure culture, extracting DNA by kit method and Nisin method respectively, and performing qPCR on mecA as template. Taking the logarithm of the thallus concentration (CFU/mL) as an abscissa, and taking the average cycle threshold (Ct) corresponding to the concentration as an ordinate, constructing a standard curve of each system. Calculation of R 2 And the reaction efficiency is obtained from the Slope of the curve (Slope): e = (10) (-1/Slope) -1) × 100%. The reaction efficiency and the detection interval of the cell concentration were determined for each system (FIG. 3, MRSA concentration in FIG. 3 was 2X 10, respectively) 9 CFU/mL, curve a; 2X 10 8 CFU/mL, curve b; 2X 10 7 CFU/mL, curve c; 2X 10 6 CFU/mL, curve d; 2X 10 5 CFU/mL, curve e; 2X 10 4 CFU/mL, curve f; 2X 10 3 CFU/mL, curve g; 2X 10 2 CFU/mL, curve h; 2X 10CFU/mL, curve i).
Plotted according to the results, as shown in fig. 4, the standard curve for the kit method is: y = -2.377x + 34.277R 2 =0.9506, efficiency =167.07%; the standard curve for the Nisin method is: y = -2.6586x +32.6 2 =0.9165, efficacy =137.76%. All 2 standard curves show good linear relationship between the logarithm value of the thallus concentration and the Ct value.
However, the slope of the qPCR standard curve is required to be within the range of-2.6 to-3.6, the Nisin method meets the slope requirement (-2.6586), and the kit method does not meet the slope requirement (-2.377). Therefore, the genome DNA extracted by the Nisin treatment method can meet the requirements of downstream qPCR experiments and is superior to the common kit method.
4. Amplification efficiency detection of qPCR (quantitative polymerase chain reaction) by extracting genome DNA after Nisin treatment of MRSA (methicillin-resistant staphylococcus aureus)
The E value, the amplification efficiency value of qPCR, is determined mainly by the linear relation equation Cq = -k lgX of the standard curve 0 + b, when the amplification efficiency E is between 90% and 110%, the amplification is considered to be close to the ideal condition; parameter R 2 The requirement is that R is more than or equal to 0.98 2 The closer to 1, cq and X are indicated 0 The higher the correlation between Log values of. Conventional PCR reaction systems include dNTPs, templates, primers, mg +, amplification buffer, polymerase, etc., and changing any one of the minor parameters of the amplification reaction may result in an increase or decrease in the PCR amplification product. Primers for PCR reaction need not be highly purified, but contamination with DNA polymerase inhibitors, proteases, nucleases, polysaccharides capable of binding to DNA, and proteins during extraction of nucleic acids should be avoided, and some impurities such as SDS may inhibit the activity of DNA polymerase to inhibit the reduction of amplification efficiency caused by PCR reaction.
The genomic DNA of MRSA Yn2020070 strain was extracted by Nisin method, and its concentration (ng/. Mu.L) and purity (A) were measured by NanoDrop microspectrophotometer 260 /A 280 、A 260 /A 230 ) Then, genomic DNA was serially diluted 5 times, and qPCR was performed using this as a template. 3 groups of parallel qPCR reactions, quantitatively detecting 1696 rDNA V3-V4 region segment of MRSA strain (using universal primer, upstream primer sequence F:5'CCTACGGNGGCWGCAG 3'; downstream primer sequence R:5'GACTACHVGGGTATCTAATCC 3', amplified segment length 464 bp); the PCR reaction system is as follows: 10 μ l
SYBR High-sensitvy qPCR Supermix plus, final concentrations of upstream and downstream primers are both 1.0 μm, template DNA is 1 μ L, RNase Free Water makes up the total volume to 20 μ L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 20 sec-annealing at 55 ℃ for 20 sec-extension at 72 ℃ for 30sec for 40 cycles), qPCR reaction was performed on a roche fluorescence quantitative PCR instrument (Light cycler 96).
And (5) drawing according to the genomic DNA concentration and the Ct values obtained under different concentrations to obtain a standard curve. As shown in FIG. 5, it was found that the amplification efficiency E of qPCR using the DNA obtained by the Nisin method as a template was 91.38%, and the effect was satisfactory.
5. Nisin treatment method for extracting genome DNA of other MRSA isolates
Genomic DNA of clinical isolates of MRSA extracted according to the above method was determined for concentration and purity using Nanodrop using an OMEGA bacterial DNA extraction kit as a control, and the results are shown in Table 2.
And (3) extracting the genome DNA of the 7 MRSA isolates by comparing a kit method and a Nisin method, wherein the concentration and the purity of the DNA extracted by the kit method are lower than those of the DNA extracted by the Nisin treatment group.
TABLE 2 concentration and purity of DNA extracted from MRSA isolates by two methods
According to the comparison, the nisin is adopted to replace lysozyme and lysostaphin to treat MRSA cells, so that the cell walls can be fully broken, high-concentration and high-purity genome DNA can be obtained, the cost is low, the effect is good, and the DNA is suitable for detection experiments such as downstream PCR amplification.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (9)
1. A method for extracting methicillin-resistant staphylococcus aureus genome DNA by adopting nisin is characterized by comprising the following steps:
1) Adding nisin into a liquid culture medium for culturing methicillin-resistant staphylococcus aureus, incubating, centrifuging to remove supernatant, and collecting thallus precipitate;
2) And (3) cracking the thallus precipitate by adopting a DNA extraction kit, and removing RNA and protein to obtain methicillin-resistant staphylococcus aureus DNA.
2. The method for extracting methicillin-resistant staphylococcus aureus genomic DNA using nisin according to claim 1, wherein nisin is a commercially available biological grade reagent with activity >1000000IU/g.
3. The method for extracting methicillin-resistant staphylococcus aureus genomic DNA using nisin according to claim 1, wherein nisin is used in an amount of: sterile nisin solution was added to each 2-3mL of liquid medium until the final nisin concentration was 1mg/mL.
4. The method for extracting methicillin-resistant staphylococcus aureus genomic DNA using nisin according to claim 3, wherein the sterile nisin solution is formulated as follows: taking 50mg of nisin, adding 1mL of hydrochloric acid solution with the pH value of 2, dissolving by vortex, centrifuging at 2500rpm for 1min, and filtering and sterilizing the supernatant by a 0.22 mu m filter to obtain a sterile nisin solution of 50 mg/mL.
5. The method for extracting methicillin-resistant Staphylococcus aureus genomic DNA using nisin according to claim 1, wherein the step 1) of culturing methicillin-resistant Staphylococcus aureus is performed by: inoculating methicillin-resistant staphylococcus aureus to tryptone soy broth TSB liquid culture medium, culturing at 37 ℃ and 200rpm with shaking table overnight.
6. The method for extracting methicillin-resistant staphylococcus aureus genomic DNA using nisin according to claim 1, wherein in step 1), the genomic DNA is incubated at 37 ℃ for 1-2 h.
7. The method for extracting methicillin-resistant staphylococcus aureus genome DNA by using nisin according to claim 1, wherein in the step 2), the lysate and proteinase K in the kit are used for carrying out lysis treatment on the bacterial precipitation to remove proteins; and (4) adopting RNase A in the kit to continue processing to remove RNA.
8. The application of nisin as a synergist for extracting methicillin-resistant staphylococcus aureus genome DNA.
9. The application of nisin in preparing a kit for extracting methicillin-resistant staphylococcus aureus genome DNA.
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