CN115820627A - Method for extracting bacterial genome DNA and application thereof - Google Patents
Method for extracting bacterial genome DNA and application thereof Download PDFInfo
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Abstract
The invention discloses a method for extracting bacterial genome DNA and application thereof, comprising the steps of sampling a sample solution and mixing the sample solution with a component A, wherein the component A comprises Tris, EDTA and water; adding a component B, wherein the component B is formed by mixing glass beads with different particle sizes; placing in a vortex mixer, shaking, mixing, and denaturing protein at high temperature in a water bath to obtain nucleic acid extractive solution; and cooling the nucleic acid extracting solution to room temperature to obtain the bacterial genome DNA. The method is simple, convenient, rapid and easy to operate, the extraction method has few operation steps and simple operation, and the whole process can be completed in 15 minutes; the extraction flux is high, the sample requirement is low, a plurality of samples can be extracted at one time, and the samples can be single bacterial colony thalli or enriched liquid of a culture dish.
Description
Technical Field
The invention belongs to the field of in-vitro diagnostic reagents, and particularly relates to a method for extracting bacterial genome DNA and application thereof.
Background
At present, the conventional methods for extracting nucleic acid in clinical or scientific fields include physical methods, chemical methods, enzymatic methods, and combinations thereof. Physical methods include freeze-thawing, sonication, boiling, grinding, and the like. The chemical method is to extract after cracking Sodium Dodecyl Sulfate (SDS), cetyl Trimethyl Ammonium Bromide (CTAB), trixon, chelating resin, tween 20 and the like. The enzyme method adopts protease K, plant protease, lysozyme and the like for extraction after cracking.
Documents 1 (CN 103820431A) and 2 (CN 104450684A) disclose a kit and an extraction method for extracting nucleic acid from viruses or bacteria by a magnetic bead method. The method mainly comprises the steps of crushing viruses or bacteria through lysis solution, adsorbing free nucleic acid through magnetic beads to form a magnetic bead-nucleic acid compound after crushing, and completing purification of the nucleic acid under the action of a matching magnetic field. The nucleic acid extracted by the method has high purity and is complete, and can be directly used for subsequent detection, but the method has the problems of long nucleic acid extraction time, complex operation steps, high cost, nucleic acid loss in the process of repeatedly purifying the nucleic acid by using a magnetic field-magnetic beads and the like.
The enzymatic method requires digestion of the cell wall peptidoglycan component with an enzyme followed by extraction with a solvent such as phenol/chloroform. The DNA adsorption material mainly adopts siliceous materials, anion exchange resin, magnetic beads and the like. The method has the advantages of high material consumption and high cost. And the steps are multiple, time is consumed, protease residue is easily generated in the operation process, so that the PCR reaction is inhibited, organic solvents such as phenol, chloroform and the like easily cause environmental pollution, and the health of experiment operators is damaged.
The methods for extracting nucleic acid by using the magnetic bead method and the enzymatic hydrolysis method, as well as other conventional methods for extracting nucleic acid, such as freeze thawing, ultrasound, boiling, grinding and the like, are used for breaking cell walls of bacteria, and have the general limitations of complicated steps, complex operation, long time consumption, severe slowing of detection speed, high price, harsh reagent storage conditions, incapability of directly using the nucleic acid extract for subsequent nucleic acid amplification, need of special instruments and the like.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.
Therefore, the present invention aims to overcome the shortcomings of the prior art and provide a method for extracting bacterial genomic DNA.
In order to solve the technical problems, the invention provides the following technical scheme: a method for extracting bacterial genome DNA, comprising,
taking a sample solution, and mixing the sample solution with a component A, wherein the component A comprises Tris, EDTA and water;
adding a component B, wherein the component B is formed by mixing glass beads with different particle sizes;
placing in a vortex mixer, shaking, mixing, and denaturing protein at high temperature in a water bath to obtain nucleic acid extractive solution;
and cooling the nucleic acid extracting solution to room temperature to obtain the bacterial genome DNA.
As a preferred embodiment of the method of the present invention, wherein: in the component A, the concentration of Tris is 5-20mM, the pH value of Tris is 8.0-9.0, and the concentration of EDTA is 1-5 mM.
As a preferred embodiment of the method of the present invention, wherein: in the component B, the particle size of the glass beads is 300-600 μm.
As a preferable aspect of the method of the present invention, wherein: the shaking and mixing time is 8-15 min.
As a preferable aspect of the method of the present invention, wherein: the protein denaturation temperature is 93-97 ℃.
As a preferable aspect of the method of the present invention, wherein: the component A is 70-90 mu L, and the sample liquid is 10-20 mu L;
0.08-0.12g of component B;
the rotating speed range of the vortex mixer is 2000-3000 rpm.
The water bath time is 5-10 min.
It is still another object of the present invention to overcome the deficiencies of the prior art and to provide a kit for rapid extraction of bacterial genomic DNA.
In order to solve the technical problems, the invention provides the following technical scheme: a kit for rapidly extracting bacterial genome DNA comprises a component A and a component B, wherein,
the component A contains 1-5 mM EDTA and 5-20 mM Tris water solution, and the pH value is 8.0-9.0;
the component B is a glass bead mixture with the particle size of 300-600 mu m;
bacterial genomic DNA is used for LAMP amplification.
As a preferable embodiment of the kit of the present invention, wherein: 0.08-0.12g of the component B of the kit is used; the kit is used by enriching the bacteria to 10-20 μ L.
As a preferable embodiment of the kit of the present invention, wherein: when the kit is used, the mixture is uniformly mixed for 8-15 min by a vortex mixer with the rotation number ranging from 2000 rpm to 3000rpm, and then the mixture is heated to 93-97 ℃ in a water bath or a metal bath for 5-10 min.
The invention has the beneficial effects that:
(1) The extraction method has the advantages of few operation steps and simple operation, and the whole process can be completed in 15 minutes.
(2) The extraction flux is high, the sample requirement is low, a plurality of samples can be extracted at one time, and the samples can be single bacterial colony thalli or enriched liquid of a culture dish.
(3) The method has wide applicable thallus range, adopts glass beads with different grain diameters, can be suitable for bacteria with different diameters, and has reliable and efficient extraction effect; low cost, safety and reliability, no need of special instruments and expensive reagents, no toxic and harmful substances and high safety.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a graph showing the results of the test using the kit in example 1 of the present invention, (a) is the test result obtained by kit I, (b) is the test result obtained by kit II, and (c) is the test result obtained by kit III.
FIG. 2 is a graph showing the test results obtained by the reagent kit IV in example 2 of the present invention.
FIG. 3 is a graph showing the results of the test using the kit in example 4 of the present invention, (d) is the test result obtained by kit VI, and (e) is the test result obtained by kit VII.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, the references herein to "one embodiment" or "an embodiment" refer to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were all reagents purchased from conventional biochemical reagent stores.
Pseudomonas aeruginosa: CMCC strain number (B) 10104, china Medical bacteria Collection (National Center for Medical C. Mu. Lture Collections).
Acinetobacter baumannii: american Type culture Collection (American Type C. Mu. Lture Collection), ATCC strain number 19606.
And (4) staphylococcus aureus: china National Center for Medical C. Mu. Ltube Collections, CMCC strain number (B) 26003.
LAMP: a loop-mediated isothermal amplification technology is a sensitive, specific, convenient and quick nucleic acid amplification technology, and in 2000, japanese researchers Notomi and the like invented a novel in-vitro isothermal nucleic acid amplification technology, namely the loop-mediated isothermal amplification technology, and the technology relies on BstDNA polymerase with a strand displacement function and two pairs of specific primers designed aiming at 6 different regions of a target gene, wherein one pair of inner primers are combined and extended, and the other pair of outer primers are stripped simultaneously to enable two ends of a template to have a dumbbell-shaped single-stranded structure, so that the specific primers can be smoothly combined with the template under the isothermal condition of about 65 ℃ to generate a strand displacement amplification reaction.
General DNA nucleic acid detection kit (freeze-drying/constant temperature fluorescence method): guangzhou Huafeng Biotech, inc., lot number 20040102.
Magnetic bead method nucleic acid (DNA/RNA) extraction kit: wuxi Kouza science and technology Co., ltd (Techstar Kouza), lot No. 20211001.
DNA/RNA rapid extraction kit: changzhou Bai Dynasty Biotechnology Ltd, lot number: 51029.
human upper respiratory tract pharynx swab: a small amount of secretion was dipped from the pharynx of the human body with a medical cotton swab.
Example 1:
extracting pseudomonas aeruginosa genome DNA:
(1) Preparation of the kit
Three kits were prepared separately, each consisting of liquid A and component B packaged separately.
In the kit I, the solution A is an aqueous solution containing 1mM of EDTA and 5mM of Tris; the component B is glass beads with the grain diameter of 300-400 μm.
In the kit II, the solution A is an aqueous solution containing 5mM of EDTA and 20mM of Tris; the component B is glass beads with the particle size of 400-600 mu m.
In the kit III, the solution A is an aqueous solution containing 5mM of EDTA and 20mM of Tris; the component B is glass beads with the particle diameter of 300-400 μm and 400-600 μm, and the ratio of 1:1 and mixing.
(2) Application of kit
Preparing sample liquid to be tested
Respectively taking three branches with the concentration of 1.0 multiplied by 10 6 1000 mu L of CFU/mL pseudomonas aeruginosa bacterial liquid, centrifuging at 1200r, then discarding supernatant, and reserving 10 mu L of pseudomonas aeruginosa bacterial liquid as sample liquid to be detected.
Adding 80 mu L of the solution A in the kit I into three sample solutions to be detected respectively to obtain a reaction system I; adding 80 mu L of solution A in the kit II to obtain a reaction system II; adding 80 mu L of A liquid in the kit III to obtain a reaction system III.
Respectively adding 0.1g of the component B in the kit I into the three reaction systems to obtain reaction liquid I; adding 0.1g of the component B in the kit II to obtain a reaction solution II; and adding 0.1g of the component B in the kit III to obtain a reaction solution III.
Placing the three reaction liquids in a vortex mixer to vibrate and mix uniformly, wherein the revolution range of the vortex mixer is 2500rpm; mixing time is 10min.
And heating the three uniformly mixed reaction liquids in a water bath for 6min, wherein the water bath temperature is 95 ℃. Cooling to obtain nucleic acid extractive solution (nucleic acid extractive solution I, nucleic acid extractive solution II, nucleic acid extractive solution III)
mu.L of the nucleic acid extract (nucleic acid extract I; nucleic acid extract II or nucleic acid extract III) obtained in step 5 was taken and mixed with 22. Mu.L of a constant temperature LAMP amplification reagent, and then LAMP amplification reaction was carried out (reaction conditions: 65 ℃ C.; 1min per cycle, 50 cycles).
The isothermal LAMP amplification reagent is a component in a universal DNA nucleic acid detection kit (freeze-drying type/isothermal fluorescence method) of Guangzhou Huafeng biological technology limited company.
Three replicates were set up simultaneously with each kit.
(3) As shown in FIG. 1, the test results obtained with kit I are shown in FIG. 1 (a), the test results obtained with kit II are shown in FIG. 1 (b), and the test results obtained with kit III are shown in FIG. 1 (c).
In the three groups of test curves, the three repeatedly processed test curves in each group of sample liquid to be tested are basically consistent, which shows that the pseudomonas aeruginosa genome DNA extracted by the reagent component and the extraction method thereof provided by the invention can be directly used for isothermal LAMP amplification reaction.
Example 2:
extracting upper respiratory tract pharynx swab.
(1) Preparation of the kit:
and preparing a kit IV, which consists of the liquid A and the component B which are independently packaged.
In the kit IV, the solution A is an aqueous solution containing 1mM of EDTA and 5mM of Tris; the component B is glass beads with the particle size of 300-400 μm.
(2) Application of kit
Preparing sample liquid to be tested
Human upper respiratory tract pharynx swab: a small amount of secretion is dipped from the pharynx of a human body by a medical cotton swab and soaked in physiological saline. Taking 1000 mu L, centrifuging at 1200r, and then discarding the supernatant, and reserving 10 mu L as the sample liquid to be detected.
80 mu L of the solution A in the kit IV is added into the sample solution to be detected.
Then 0.1g of the component B in the kit IV is added to obtain reaction liquid.
Placing the reaction liquid in a vortex mixer to vibrate and mix uniformly, wherein the revolution range of the vortex mixer is 2500rpm; mixing time is 10min.
And heating the three uniformly mixed reaction liquids in a water bath for 6min, wherein the water bath temperature is 95 ℃. Cooling to obtain nucleic acid extract IV.
And (3) mixing 3 mu L of the nucleic acid extracting solution IV obtained in the step (5) with 22 mu L of constant-temperature LAMP amplification reagent, and then carrying out LAMP amplification reaction (the reaction conditions are 65 ℃, each cycle is 1min, and 50 cycles).
The isothermal LAMP amplification reagent is a component in a universal DNA nucleic acid detection kit (freeze-drying type/isothermal fluorescence method) of Guangzhou Huafeng biological technology limited company.
Three replicates were performed.
The results are shown in fig. 2, and the results show that the reagent component extraction method provided by the invention can be used for extracting the middle-tube family gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in the pharyngeal swab of the upper respiratory tract of the human body, and the extracted product can be directly used for the subsequent LAMP amplification reaction.
Example 3: extraction of pathogenic microorganisms
Preparation of the kit:
and preparing a kit V, which consists of the independently packaged A liquid and the B component.
In the kit V, the solution A is an aqueous solution containing 1mM of EDTA and 5mM of Tris; the component B is glass beads with the particle diameter of 300-400 μm and 400-600 μm, and the ratio of 1:1 and mixing.
The application of the kit comprises the following steps:
1. preparing sample liquid to be tested
Respectively taking the concentration of 1.0 × 10 6 CFU/mL Acinetobacter baumannii bacterial liquid 1000. Mu.L with concentration of 1.0 × 10 6 1000 mu L of CFU/mL staphylococcus aureus liquid, centrifuging 1200r, then discarding supernatant, and reserving 10 mu L of the liquid as sample liquid to be detected.
2. 80 mu L of solution A in the kit V is added into the sample solution to be detected.
3. Then 0.1g of the component B in the kit V is added to obtain a reaction solution.
4. Placing the reaction liquid in a vortex mixer to vibrate and mix uniformly, wherein the revolution range of the vortex mixer is 2500rpm; mixing time is 10min.
5. And heating the three uniformly mixed reaction liquids in a water bath for 6min, wherein the water bath temperature is 95 ℃. And cooling to obtain a nucleic acid extracting solution V and a nucleic acid extracting solution VI.
6. Detection of
Detecting nucleic acid by using an ultraviolet spectrophotometer:
firstly, the ultraviolet spectrophotometer is respectively zeroed by reagents for dissolving nucleic acid, 2 mu L of corresponding nucleic acid extracting solution is added to the ultraviolet spectrophotometer after the zero adjustment, and a measuring button is clicked to measure the concentration of the nucleic acid.
The results are shown in table 1:
table 1 shows the concentration of nucleic acid extracted from pathogenic microorganism and A260/A280 value
According to the detection result of the spectrophotometer, the method provided by the invention has the advantage that the purity and concentration of extracted nucleic acid are superior to those of a magnetic bead method nucleic acid (DNA/RNA) extraction kit of the Techstar family Zhida which is widely used at present and a DNA/RNA rapid extraction kit of hundred generations of organisms in Hezhou in the aspect of extracting pathogenic microorganisms, namely gram-negative bacteria Acinetobacter baumannii and gram-positive bacteria Staphylococcus aureus. Moreover, the method for extracting nucleic acid simultaneously has simple operation and high extraction speed.
Example 4
Effect of liquid A formulation on the overall formulation
Preparation of the kit
Two kits are prepared respectively, and each kit consists of independently packaged A solution and B component.
In the kit VI, the solution A is an aqueous solution containing 5mM of EDTA and 10mM of Tris and has a pH value of 9.0; the component B is glass beads with the particle diameter of 300-400 μm and 400-600 μm, and the ratio of 1:1 and mixing.
In the kit VII, the solution A is a Tris aqueous solution containing 12% Chelex-100 and 10mM, and the pH value is 9.0; the component B is glass beads with the particle diameter of 300-400 μm and 400-600 μm, and the ratio of 1:1 and mixing.
The application of the kit comprises the following steps:
preparing sample liquid to be tested
Taking two branches with concentration of 1.0 × 10 6 1000 mu L of CFU/mL staphylococcus aureus liquid, centrifuging 1200r, then discarding supernatant, and reserving 10 mu L of the liquid as sample liquid to be detected.
Adding 80 mu L of solution A in the kit VI into the two sample solutions to be detected respectively to obtain a reaction system VI; and adding 80 mu L of the solution A in the kit VII to obtain a reaction system VII.
Respectively adding 0.1g of the component B in the kit VI into the two reaction systems to obtain a reaction solution VI; and adding 0.1g of the component B in the kit VII to obtain a reaction liquid VII.
Placing the two reaction liquids in a vortex mixer to vibrate and mix uniformly, wherein the revolution range of the vortex mixer is 2500rpm; mixing time is 10min.
Heating the two uniformly mixed reaction solutions in water bath for 6min, wherein the water bath temperature is 95 ℃. Cooling to obtain nucleic acid extractive solution (nucleic acid extractive solution VI; nucleic acid extractive solution VII)
mu.L of the nucleic acid extract (nucleic acid extract VI; nucleic acid extract VII) obtained in step 5 was taken and mixed with 22. Mu.L of the isothermal LAMP amplification reagent, and then LAMP amplification reaction was carried out (reaction conditions: 65 ℃ C.; 1min per cycle, 50 cycles).
The isothermal LAMP amplification reagent is a component in a universal DNA nucleic acid detection kit (freeze-drying type/isothermal fluorescence method) of Guangzhou Huafeng biological technology limited company.
Three replicates were set up simultaneously with each kit.
(3) As a result, the results are shown in FIG. 3, and the results obtained with kit VI are shown in FIG. (d) and the results obtained with kit VII are shown in FIG. (e).
In the three groups of test curves, three repeatedly processed test curves in each group of sample liquid to be tested are basically consistent, and the Ct value obtained by the kit VII is obviously greater than that obtained by the kit VI, which shows that the influence of EDTA in the component A on the whole formula and the synergistic effect is obviously superior to 12 percent of Chelex-100.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A method for extracting bacterial genomic DNA, comprising: comprises the steps of (a) preparing a substrate,
taking a sample solution, and mixing the sample solution with a component A, wherein the component A comprises Tris, EDTA and water;
adding a component B, wherein the component B is formed by mixing glass beads with different particle sizes;
placing in a vortex mixer, shaking, mixing, and denaturing protein at high temperature in a water bath to obtain nucleic acid extractive solution;
and cooling the nucleic acid extracting solution to room temperature to obtain the bacterial genome DNA.
2. The method for extracting genomic DNA from bacteria according to claim 1, wherein: in the component A, the concentration of Tris is 5-20mM, the pH value of Tris is 8.0-9.0, and the concentration of EDTA is 1-5 mM.
3. The method for extracting bacterial genomic DNA as claimed in claim 1 or 2, wherein: in the component B, the particle size of the glass beads is 300-600 μm.
4. The method for extracting genomic DNA from bacteria according to claim 3, wherein: and the shaking and uniform mixing time is 8-15 min.
5. The method for extracting genomic DNA from bacteria according to claim 1, wherein: the protein denaturation temperature is 93-97 ℃.
6. The method for extracting genomic DNA from bacteria according to claim 1, wherein: the component A is 70-90 mu L, and the sample liquid is 10-20 mu L;
0.08-0.12g of component B;
the rotating speed range of the vortex mixer is 2000-3000 rpm.
The water bath time is 5-10 min.
7. A kit for rapidly extracting bacterial genome DNA is characterized in that: comprises a component A and a component B, wherein,
the component A contains 1-5 mM EDTA and 5-20 mM Tris water solution, and the pH value is 8.0-9.0;
the component B is a glass bead mixture with the particle size of 300-600 mu m;
bacterial genomic DNA is used for LAMP amplification.
8. The kit of claim 7, wherein: 0.08-0.12g of the component B of the kit is used; the kit is used by enriching the bacteria to 10-20 μ L.
9. The kit of claim 7 or 8, wherein: when the kit is used, the mixture is uniformly mixed for 8-15 min by a vortex mixer with the rotation number ranging from 2000 rpm to 3000rpm, and then the mixture is heated to 93-97 ℃ in a water bath or a metal bath for 5-10 min.
10. Use of the extraction method of any one of claims 1 to 6 in the detection of bacterial genomic DNA.
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