CN111808850A - Bacterial nucleic acid extraction lysate, preparation method and application - Google Patents
Bacterial nucleic acid extraction lysate, preparation method and application Download PDFInfo
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- CN111808850A CN111808850A CN202010866693.8A CN202010866693A CN111808850A CN 111808850 A CN111808850 A CN 111808850A CN 202010866693 A CN202010866693 A CN 202010866693A CN 111808850 A CN111808850 A CN 111808850A
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 82
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 79
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 79
- 239000006166 lysate Substances 0.000 title claims abstract description 39
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 29
- 238000000605 extraction Methods 0.000 title claims description 45
- 238000002360 preparation method Methods 0.000 title description 6
- 239000000243 solution Substances 0.000 claims abstract description 22
- 230000009089 cytolysis Effects 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 16
- 102000016943 Muramidase Human genes 0.000 claims abstract description 8
- 108010014251 Muramidase Proteins 0.000 claims abstract description 8
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims abstract description 8
- 229960000274 lysozyme Drugs 0.000 claims abstract description 8
- 235000010335 lysozyme Nutrition 0.000 claims abstract description 8
- 239000004325 lysozyme Substances 0.000 claims abstract description 8
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 7
- 239000003945 anionic surfactant Substances 0.000 claims abstract description 6
- 230000003196 chaotropic effect Effects 0.000 claims abstract description 6
- 239000002738 chelating agent Substances 0.000 claims abstract description 6
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 6
- 150000003839 salts Chemical class 0.000 claims abstract description 6
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims description 7
- 210000003608 fece Anatomy 0.000 claims description 7
- 229920002401 polyacrylamide Polymers 0.000 claims description 7
- 210000003296 saliva Anatomy 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- JLFNLZLINWHATN-UHFFFAOYSA-N pentaethylene glycol Chemical compound OCCOCCOCCOCCOCCO JLFNLZLINWHATN-UHFFFAOYSA-N 0.000 claims description 6
- 239000012472 biological sample Substances 0.000 claims description 5
- 239000013504 Triton X-100 Substances 0.000 claims description 4
- 229920004890 Triton X-100 Polymers 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical group OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 239000003002 pH adjusting agent Substances 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 23
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 230000002496 gastric effect Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 39
- 238000010438 heat treatment Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 10
- 239000002184 metal Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 239000011324 bead Substances 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000002550 fecal effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000003260 vortexing Methods 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- APLNAFMUEHKRLM-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(3,4,6,7-tetrahydroimidazo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)N=CN2 APLNAFMUEHKRLM-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000003172 anti-dna Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a lysis solution for extracting bacterial nucleic acid, which comprises the following components in parts by weight: 1-15 mM of metal ion chelating agent, 0.05-5% of anionic surfactant, 0.5-5% of nonionic surfactant, 5-30 mM of buffer solution, 0.05-0.2 mg/mL of lysozyme, 5-8M of high chaotropic salt, a pH regulator and a solvent, wherein the pH regulator regulates the pH of the lysate to be 7-8. The invention has simple and convenient operation, rapidness and low cost, can provide a basis for nucleic acid amplification detection, provides support for rapidly diagnosing gastrointestinal related diseases, reduces time cost and economic cost in the process of diagnosis, has wide market prospect and larger economic and social benefits, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a bacterial nucleic acid extraction lysate, a preparation method and application.
Background
The nucleic acid extraction method comprises conventional phenol-chloroform method, salting-out method, filter membrane centrifugal column method, etc. The traditional nucleic acid extraction methods have the advantages and disadvantages, but the extraction effect is not good enough. The current mainstream nucleic acid extraction methods include a silica gel membrane adsorption column method, an immune affinity method of an anti-DNA monoclonal antibody, a magnetic bead method widely applied to an automated platform, and the like. Nucleic acids are biomacromolecules synthesized by the polymerization of many nucleotides, and are one of the most basic substances of life. Nucleic acid extraction refers to a process of separating nucleic acids from a sample by physical, chemical, or the like methods. The isolation of high quality nucleic acids is a key step in molecular biology research. At present, nucleic acid extraction is mainly carried out by adopting a nucleic acid extraction kit. However, the conventional nucleic acid extraction kit has poor quality of extracted nucleic acid and cannot meet the actual demand.
Although the existing method for extracting nucleic acid by using magnetic beads is improved, the method still has many defects. First, a lysis solution generally used for nucleic acid extraction by the magnetic bead method usually contains proteinase K. Secondly, in the nucleic acid extraction operation in the prior art, the lysis of the sample and the combination of the magnetic beads and the DNA are often carried out step by step, and the corresponding kit often comprises independently subpackaged lysis solution and combination solution, so that the nucleic acid extraction process is complicated, the probability of nucleic acid pollution is increased to a great extent, and the subsequent detection and the detection accuracy are not facilitated.
The traditional nucleic acid extraction lysate has low extraction rate, and particularly for samples with more impurities such as feces, saliva and the like, the nucleic acid with high purity and high yield is difficult to obtain, and the dosage required by the subsequent related tumor detection is difficult to meet. Therefore, the development of the lysis solution for quickly extracting the nucleic acid and the preparation method thereof, which have the advantages of high efficiency, less pollution, simple and convenient operation, high extraction efficiency and the like, has great significance.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a bacterial nucleic acid extraction lysate so as to achieve the aim of simply and quickly extracting bacterial nucleic acid in a sample.
In order to achieve the aim, the invention provides a bacterial nucleic acid extraction lysate, which comprises the following components in parts by weight: 1-15 mM of metal ion chelating agent, 0.05-5% of anionic surfactant, 0.5-5% of nonionic surfactant, 5-30 mM of buffer solution, 0.05-0.2 mg/mL of lysozyme, 5-8M of high chaotropic salt, a pH regulator and a solvent, wherein the pH regulator regulates the pH of the lysate to be 7-8.
Further, the metal ion chelating agent is nitrilotriacetic acid and/or EDTA-2 Na.
Further, the buffer solution is 5-10 mM Tris-HCl.
Further, the anionic surfactant is 0.1-2% of polyacrylamide.
Further, the nonionic surfactant is 1-5% of PEG400 and/or 0.5% -3% of TritonX-100.
Further, the high chaotropic salt is 5-8M guanidine hydrochloride or urea.
Further, the pH regulator is phosphate buffer.
The invention also provides a preparation method of the bacterial nucleic acid extraction lysate, which comprises the following steps: preparing a mixed solution from 1mM EDTA-2Na, 1% polyacrylamide, 2% PEG400, 10mM Tris-HCL, 0.2mg/mL lysozyme, 6M guanidine hydrochloride and water, and adjusting the pH value to 7.0 by using a phosphate buffer solution to obtain the compound.
In another embodiment of the present invention, a method for preparing the bacterial nucleic acid extraction lysate is provided, which includes the following steps: preparing a mixed solution from 1mM aminotriacetic acid, 1% polyacrylamide, 1% Triton X-100, 1% PEG400, 5mM Tris-HCl, 0.1mg/mL lysozyme, 7M urea and water, and adjusting the pH value to 7.0 by using a phosphate buffer solution to obtain the finished product.
The invention also aims to provide application of the bacterial nucleic acid extraction lysate, and the lysate prepared by the method is suitable for extracting nucleic acid of biological samples such as feces, swabs and saliva.
Compared with the prior art, the invention has the beneficial effects that:
the invention has simple and convenient operation, rapidness and low cost, can provide a basis for nucleic acid amplification detection, provides support for rapidly diagnosing gastrointestinal related diseases, reduces time cost and economic cost in the process of diagnosis, has wide market prospect and larger economic and social benefits, and is suitable for large-scale popularization and application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
In the embodiment, the human excrement is used as a biological sample for nucleic acid extraction and detection.
1. Preparation of lysate: preparing 7 parts of lysis solution according to the raw material component ratio, and the following table shows:
TABLE 1 lysate 1-7 component table
2. Treating a fecal sample: for the feces samples of 7 diarrhea patients, 1.5mL of the feces samples were respectively sucked into 2mL EP tubes, centrifuged at 2500rpm for 90s at room temperature, 800. mu.L of the supernatant was sucked and mixed well, and then 200. mu.L of the supernatant was quickly dispensed into 3 new 2mL EP tubes and placed on ice.
3. Nucleic acid extraction: centrifuging the 2mL EP tube containing 200 μ L of the supernatant at 12000rpm for 2min, discarding the supernatant, adding 100 μ L of the lysis solution of the present invention into the collected precipitate, mixing well, heating in a metal bath at 37 deg.C for 20min, vortexing for 3-4 times during heating, vortexing again after heating, and boiling in a metal bath at 100 deg.C for 10 min; after slight cooling, the mixture was centrifuged at 12000rpm for 2min, and the resulting supernatant was a DNA solution which was used for nucleic acid amplification.
4. And (3) nucleic acid detection: the nucleic acid concentration detection was performed on 7 extracted stool samples of example 1, respectively, and the detection results are shown in table 2 below; the invention proves that the lysate has high efficiency and high purity for extracting nucleic acid.
TABLE 2 detection of nucleic acid concentrations in fecal samples 1-7
| Sample number | Concentration of nucleic acid | Unit of | OD260/280 |
| 1 | 42.12 | ng/μl | 1.88 |
| 2 | 15.33 | ng/μl | 1.85 |
| 3 | 37.08 | ng/μl | 1.72 |
| 4 | 18.09 | ng/μl | 1.84 |
| 5 | 38.41 | ng/μl | 1.74 |
| 6 | 20.90 | ng/μl | 1.72 |
| 7 | 19.42 | ng/μl | 1.80 |
Example 2
Taking saliva as a biological sample to carry out nucleic acid extraction and detection.
1. Sample processing and nucleic acid extraction: taking 7 parts of saliva samples, carrying out pretreatment, turning upside down and mixing uniformly, adding 7 parts of lysis solution prepared in the embodiment 1, mixing uniformly, heating in a metal bath at 37 ℃ for 20min, carrying out vortex 3-4 times in the heating process, carrying out vortex mixing again after heating, and boiling in a metal bath at 100 ℃ for 10 min; after slight cooling, the mixture was centrifuged at 12000rpm for 2min, and the resulting supernatant was a DNA solution which was used for nucleic acid amplification.
2. And (3) nucleic acid detection: the nucleic acid concentration detection was performed on 7 extracted saliva samples of example 1, respectively, and the detection results are shown in table 3 below; the invention proves that the lysate has high efficiency and high purity for extracting nucleic acid.
TABLE 3 detection of nucleic acid concentrations in saliva samples 1-7
| Sample number | Concentration of nucleic acid | Unit of | OD260/280 |
| 1 | 110.12 | ng/μl | 1.85 |
| 2 | 54.33 | ng/μl | 1.88 |
| 3 | 47.08 | ng/μl | 1.79 |
| 4 | 58.09 | ng/μl | 1.74 |
| 5 | 88.41 | ng/μl | 1.81 |
| 6 | 90.90 | ng/μl | 1.83 |
| 7 | 80.42 | ng/μl | 1.77 |
Example 3
The nasal swab is used as a biological sample for nucleic acid extraction and detection.
1. Sample processing and nucleic acid extraction: taking 7 parts of nasal swab samples, carrying out up-down inversion and uniform mixing without pretreatment, adding 7 parts of lysis solution prepared in example 1, fully and uniformly mixing, placing in a metal bath at 37 ℃, heating for 20min, carrying out vortex 3-4 times in the heating process, carrying out vortex uniform mixing again after heating, and placing in a metal bath at 100 ℃ for boiling for 10 min; after slight cooling, the mixture was centrifuged at 12000rpm for 2min, and the resulting supernatant was a DNA solution which was used for nucleic acid amplification.
2. And (3) nucleic acid detection: the nucleic acid concentration detection was performed on 7 extracted nasal swab samples of example 1, and the detection results are shown in table 4 below; the invention proves that the lysate has high efficiency and high purity for extracting nucleic acid.
TABLE 4 detection of nucleic acid concentration in nasal swab samples 1-7
Comparative example 1
The difference between the comparative example and the example 1 is that the lysis solution for extracting bacterial nucleic acid of the comparative example is prepared by replacing the equivalent amount of solvent water without adding the metal ion chelating agent EDTA-2 Na.
Comparative example 2
The difference between the comparative example and the example 1 is that in the lysis solution for extracting bacterial nucleic acid of the comparative example, no anionic surfactant polyacrylamide is added, and the same amount of solvent water is used for replacement, so that the lysis solution 2 of the comparative example is prepared.
Comparative example 3
The difference between the comparative example and the example 1 is that the lysis solution for extracting bacterial nucleic acid of the comparative example is prepared by replacing the same amount of solvent water without adding the nonionic surfactant PEG 400.
Comparative example 4
The difference between the comparative example and example 1 is that lysozyme was not added to the lysate for bacterial nucleic acid extraction in the comparative example, and an equal amount of solvent water was used instead to prepare a lysate for comparative example 4.
Comparative example 5
The difference between the comparative example and example 1 is that no high chaotropic salt is added to the lysate for bacterial nucleic acid extraction of the comparative example, and an equal amount of solvent water is used instead to prepare a lysate for comparative example 5.
Comparative example 6
The difference between the comparative example and example 1 is that a lysate 6 of the comparative example was prepared by adding a pH adjuster to the lysate for bacterial nucleic acid extraction to adjust the pH to 4.
Comparative example 7
The difference between the comparative example and example 1 is that a lysate 7 of the comparative example was prepared by adding a pH adjuster to the lysate for bacterial nucleic acid extraction to adjust the pH to 9.
Comparative example 8
The difference between the comparative example and the example 2 is that in the bacterial nucleic acid extraction lysate of the comparative example, the non-ionic surfactant is 1% of Triton X-100, and the lysate of the comparative example 8 is prepared.
A nucleic acid extraction experiment was performed on the lysates obtained in comparative examples 1-8 above, and the final nucleic acid concentration was determined.
1. Treating a fecal sample: for 8 feces samples of diarrhea patients, 1.5mL of the feces samples are respectively sucked into 2mLEP tubes, centrifuged at 2500rpm for 90s at room temperature, 800. mu.L of the supernatant is sucked and fully mixed, and then 200. mu.L of the supernatant is quickly subpackaged into 3 new 2mLEP tubes and placed on ice.
2. Nucleic acid extraction: centrifuging the 2mL EP tube containing 200 μ L of the supernatant at 12000rpm for 2min, discarding the supernatant, adding 100 μ L of the lysis solution of the present invention into the collected precipitate, mixing well, heating in a metal bath at 37 deg.C for 20min, vortexing for 3-4 times during heating, vortexing again after heating, and boiling in a metal bath at 100 deg.C for 10 min; after slight cooling, the mixture was centrifuged at 12000rpm for 2min, and the resulting supernatant was a DNA solution which was used for nucleic acid amplification.
3. And (3) nucleic acid detection: respectively carrying out nucleic acid concentration detection on the 8 extracted fecal samples, wherein the detection results are shown in the following table 5; the specific detection results are as follows.
TABLE 5 investigation of nucleic acid extraction efficiency of the lysates in comparative examples 1-8
| Comparative example | Sample number | Concentration of nucleic acid | Unit of | OD260/280 |
| Comparative example 1 | 1 | 14.13 | ng/μl | 1.03 |
| Comparative example 2 | 2 | 22.54 | ng/μl | 1.22 |
| Comparative example 3 | 3 | 20.10 | ng/μl | 0.98 |
| Comparative example 4 | 4 | 17.41 | ng/μl | 1.35 |
| Comparative example 5 | 5 | 10.17 | ng/μl | 1.21 |
| Comparative example 6 | 6 | 22.08 | ng/μl | 1.54 |
| Comparative example 7 | 7 | 35.60 | ng/μl | 1.52 |
| Comparative example 8 | 8 | 28.21 | ng/μl | 1.45 |
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (10)
1. The bacterial nucleic acid extraction lysate is characterized by comprising the following components in parts by weight:
1 to 15mM of a metal ion chelating agent;
0.05-5% of an anionic surfactant;
0.5-5% of a nonionic surfactant;
5 to 30mM of buffer solution;
0.05-0.2 mg/mL lysozyme;
5-8M of high chaotropic salt;
a pH adjuster and a solvent;
and the pH regulator regulates the pH of the lysate to 7-8.
2. The lysis solution for extracting bacterial nucleic acid according to claim 1, wherein the metal ion chelating agent is nitrilotriacetic acid and/or EDTA-2 Na.
3. The bacterial nucleic acid extraction lysate of claim 1, wherein the buffer solution is 5-10 mM Tris-HCl.
4. The bacterial nucleic acid extraction lysate according to claim 1, wherein the anionic surfactant is 0.1-2% polyacrylamide.
5. The bacterial nucleic acid extraction lysate according to claim 1, wherein the non-ionic surfactant is 1-5% PEG400 and/or 0.5% -3% Triton X-100.
6. The lysis solution for bacterial nucleic acid extraction according to claim 1, wherein the high chaotropic salt is 5-8M guanidine hydrochloride or urea.
7. The bacterial nucleic acid extraction lysate according to claim 1, wherein the pH regulator is a phosphate buffer.
8. A method of preparing a bacterial nucleic acid extraction lysate according to any one of claims 1-7, comprising the steps of:
preparing a mixed solution from 1mM EDTA-2Na, 1% polyacrylamide, 2% PEG400, 10mM Tris-HCL, 0.2mg/mL lysozyme, 6M guanidine hydrochloride and water, and adjusting the pH value to 7.0 by using a phosphate buffer solution to obtain the compound.
9. A method of preparing a bacterial nucleic acid extraction lysate according to any one of claims 1-7, comprising the steps of:
preparing a mixed solution from 1mM aminotriacetic acid, 1% polyacrylamide, 1% Triton X-100, 1% PEG400, 5mM Tris-HCl, 0.1mg/mL lysozyme, 7M urea and water, and adjusting the pH value to 7.0 by using a phosphate buffer solution to obtain the finished product.
10. The use of a bacterial nucleic acid extraction lysate according to any one of claims 1 to 7, wherein: the lysate is suitable for extracting bacterial nucleic acid of biological samples of feces, swabs and saliva.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112813062A (en) * | 2021-04-08 | 2021-05-18 | 武汉纳磁生物科技有限公司 | Normal-temperature-stored lysis binding solution for genome extraction and kit |
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| CN112813062A (en) * | 2021-04-08 | 2021-05-18 | 武汉纳磁生物科技有限公司 | Normal-temperature-stored lysis binding solution for genome extraction and kit |
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