CN114276932A - Microbial cell lysate - Google Patents
Microbial cell lysate Download PDFInfo
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- CN114276932A CN114276932A CN202210130887.0A CN202210130887A CN114276932A CN 114276932 A CN114276932 A CN 114276932A CN 202210130887 A CN202210130887 A CN 202210130887A CN 114276932 A CN114276932 A CN 114276932A
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- 230000000813 microbial effect Effects 0.000 title claims abstract description 31
- 239000013592 cell lysate Substances 0.000 title claims abstract description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 36
- 239000004094 surface-active agent Substances 0.000 claims abstract description 16
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 13
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 10
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 8
- 229960002233 benzalkonium bromide Drugs 0.000 claims description 7
- 229960000686 benzalkonium chloride Drugs 0.000 claims description 7
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims description 7
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 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
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 210000002421 cell wall Anatomy 0.000 abstract description 20
- 210000004027 cell Anatomy 0.000 abstract description 9
- 244000005700 microbiome Species 0.000 abstract description 4
- 210000000170 cell membrane Anatomy 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 19
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 239000006166 lysate Substances 0.000 description 11
- 238000001514 detection method Methods 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- 241000233866 Fungi Species 0.000 description 7
- 150000004676 glycans Polymers 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229920002101 Chitin Polymers 0.000 description 5
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 5
- 108010013639 Peptidoglycan Proteins 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 229940083575 sodium dodecyl sulfate Drugs 0.000 description 5
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 210000003296 saliva Anatomy 0.000 description 4
- 238000011895 specific detection Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229950006780 n-acetylglucosamine Drugs 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- MNLRQHMNZILYPY-MDMHTWEWSA-N N-acetyl-alpha-D-muramic acid Chemical compound OC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O)[C@@H]1NC(C)=O MNLRQHMNZILYPY-MDMHTWEWSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- -1 chitin Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 150000002337 glycosamines Chemical class 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a microbial cell lysate, which comprises 90-95% of base solution, 0.1-4% of surfactant, 0.1-2% of sodium hydroxide, 0.1-2% of sodium dodecyl sulfate and 0.1-3% of Triton x-1000.5, wherein the pH value of the microbial cell lysate is 8.5 +/-0.2. The microbial cell lysate provided by the invention can rapidly destroy cell membranes and cell walls of microorganisms, so that contents in the microbial cells are released.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a microbial cell lysate.
Background
The cell wall (cellwall) is the outer layer of the cell, and the thickness of the cell wall is different according to different tissues and functions.
The main component of bacterial cell walls is peptidoglycan (peptidoglycan), also known as mucopeptide (mucopeptide). The mechanical strength of the cell wall depends on the presence of peptidoglycan. Synthetic peptidoglycans are specific for prokaryotes. The peptidoglycan is a polysaccharide scaffold formed by connecting two amino sugars, namely n-acetylglucosamine and n-acetylcysteine, through beta-1.4 glycosidic bonds and arranging at intervals. The n-acetylmuramic acid molecule is connected with a tetrapeptide side chain, and peptide chains are connected by a peptide bridge or a peptide chain to form a mechanically strong net structure. The peptidoglycan scaffolds of various bacterial cell walls are the same, and the composition of the tetrapeptide side chains and the connection mode thereof vary with the strain.
The bacterial cell wall is tough and elastic, protects the bacteria against a low-permeability environment, bears the osmotic pressure of 5-25 atmospheres in the environment, and ensures that the cells of the bacteria are not easy to rupture in the low-permeability environment; the cell wall plays an important role in maintaining the inherent morphology of the bacteria; the water and soluble small molecules with the diameter less than 1nm can be allowed to freely pass through, and are related to material exchange; the cell wall carries various antigenic determinants which determine the antigenicity of the bacterial cells.
The main component of the fungal cell wall is chitin. The wall thickness of the fungal cell is about 100-250 nm, and the fungal cell accounts for 30% of dry cell matter. The main component of the cell wall is polysaccharide, and the second is protein and lipoid. The types of cell wall polysaccharides vary among different groups of fungi.
The fungal cell wall polysaccharide mainly comprises chitin (chitin), cellulose, glucan, mannan, etc., and these polysaccharides are polymers of monosaccharides, such as chitin, which is a polysaccharide formed by connecting N-acetylglucosamine molecules with b-1, 4 glucosidic bonds. The cell wall components of lower fungi are mainly cellulose, yeast are mainly glucan, and higher fungi are mainly chitin. The cell wall components of a fungus are not fixed and the cell wall composition is significantly different during different stages of its growth.
However, because bacteria and fungi have rapid variation and many varieties, the type of the bacteria and fungi is difficult to be identified completely and accurately by a common identification method, and the current nucleic acid detection is a means capable of accurately judging the type, the cell walls of the bacteria and fungi need to be broken sufficiently to extract enough DNA or RNA with high quality, so that the nucleic acid is released sufficiently. In view of the above, the present application provides a microbial cell lysate.
Disclosure of Invention
The invention aims to provide a microbial cell lysate which can rapidly destroy cell membranes and cell walls of microorganisms and release contents in the microbial cells.
The invention adopts the following technical scheme to solve the technical problems:
a microbial cell lysate comprising:
90-95% of base solution, 0.1-4% of surfactant, 0.1-2% of sodium hydroxide, 0.1-2% of sodium dodecyl sulfate and 0.1-3% of Triton x-1000.5, wherein the pH value of the microbial cell lysate is 8.5 +/-0.2.
Further, the surfactant comprises one or more of benzalkonium chloride, benzalkonium bromide, triton 100 and tween 20.
Further, the surfactant comprises 0-1% benzalkonium chloride, 0-1% benzalkonium bromide, 0-1% triton 100, and 0-0.5% tween 20.
Further, the concentration of the sodium hydroxide is 6-7 wt%.
Further, the base fluid comprises 30-50mM Tris-HCl pH 7.4, 160-170mM NaCl, 1-2mM PMSF, 1-3mM EDTA.
The invention has the advantages that:
the microbial cell lysate can rapidly destroy cell membranes and cell walls of microorganisms, so that contents in the microbial cells are released.
Detailed Description
The invention is further illustrated by the following examples, which are intended to illustrate, but not to limit the invention further.
Example 1
1. Preparation of lysate: and mixing the materials to prepare a solution, and filtering and sterilizing the prepared solution to obtain the lysate. The microbial cell lysate in this embodiment includes:
93% of base solution, 3% of surfactant, 1% of sodium hydroxide, 1% of sodium dodecyl sulfate and Triton x-1002%, wherein the pH value of the microbial cell lysate is 8.5 +/-0.2.
Wherein the surfactant comprises 1% benzalkonium chloride, 0.5% benzalkonium bromide, 1% triton 100, and 0.5% tween 20; the concentration of the sodium hydroxide is 6.5 wt%;
the base solution included 40mM Tris-HCl pH 7.4, 165mM NaCl, 1.5mM PMSF, 2mM EDTA.
2. The method for lysing the microorganisms specifically comprises:
(1) and adding the sample into the prepared lysate, oscillating, uniformly mixing, and carrying out boiling water bath for 10 min. After cooling, centrifuging at 6000rpm for 10min, and sucking supernatant; the DNA was precipitated by adding an equal volume of isopropanol and mixed by gently inverting it upside down. The mixture was left at room temperature for 15min, centrifuged at 8000rpm for 20min, and the supernatant liquid was discarded.
(2) Adding 1mL of 75% ethanol, slightly reversing, mixing, centrifuging at 4 deg.C and 12000rpm for 10min, removing supernatant, standing at room temperature for several minutes, air drying the residual liquid in the tube, and drying.
3. Respectively carrying out DNA concentration detection on the extracted saliva samples, wherein the specific detection method adopts a nucleic acid concentration detection kit in the prior art; the results are shown in table 1 below:
TABLE 1 sample nucleic acid concentration determination
| Sample number | Concentration of nucleic acid | Unit of | OD260/280 |
| 1 | 80.5 | ng/μl | 1.83 |
| 2 | 88.7 | ng/μl | 1.79 |
| 3 | 100.4 | ng/μl | 1.88 |
| 4 | 99.1 | ng/μl | 1.82 |
Example 2
Preparation of lysate: and mixing the materials to prepare a solution, and filtering and sterilizing the prepared solution to obtain the lysate. The microbial cell lysate in this embodiment includes:
90% of base solution, 3% of surfactant, 2% of sodium hydroxide, 2% of sodium dodecyl sulfate and Triton x-1003%, wherein the pH value of the microbial cell lysate is 8.5 +/-0.2.
Wherein the surfactant comprises 1% benzalkonium chloride, 1% benzalkonium bromide, 0.5% triton 100, and 0.5% tween 20; the concentration of the sodium hydroxide is 6 wt%;
the base solution included 30mM Tris-HCl pH 7.4, 160mM NaCl, 1mM PMSF, 1mM EDTA.
Respectively carrying out DNA concentration detection on the extracted saliva samples, wherein the specific detection method adopts a nucleic acid concentration detection kit in the prior art; the results are shown in table 2 below:
TABLE 2 sample nucleic acid concentration determination
Example 3
Preparation of lysate: and mixing the materials to prepare a solution, and filtering and sterilizing the prepared solution to obtain the lysate. The microbial cell lysate in this embodiment includes:
95% of base solution, 1% of surfactant, 1% of sodium hydroxide, 2% of sodium dodecyl sulfate and 1001% of Triton x, wherein the pH value of the microbial cell lysate is 8.5 +/-0.2.
Wherein the surfactant comprises 0.2% benzalkonium chloride, 0.1% benzalkonium bromide, 0.6% triton 100, and 0.1% tween 20; the concentration of the sodium hydroxide is 6-7 wt%;
the base solution included 50mM Tris-HCl pH 7.4, 170mM NaCl, 2mM PMSF, 3mM EDTA.
Respectively carrying out DNA concentration detection on the extracted saliva samples, wherein the specific detection method adopts a nucleic acid concentration detection kit in the prior art; the results are shown in table 3 below:
TABLE 3 sample nucleic acid concentration determination
| Sample number | Concentration of nucleic acid | Unit of | OD260/280 |
| 1 | 98.0 | ng/μl | 1.85 |
| 2 | 88.1 | ng/μl | 1.78 |
| 3 | 54.8 | ng/μl | 1.80 |
| 4 | 39.7 | ng/μl | 1.88 |
Comparative example 1
The difference from example 1 is that in the microbial cell lysate of comparative example 1, sodium hydroxide was not added, and an equal amount of solvent water was used instead, to prepare comparative example lysate 1.
Comparative example 2
The difference from example 1 is that in the microbial cell lysate of comparative example 2, the surfactant is a single surfactant, and all of the surfactants are tween 20, and comparative example lysate 2 was prepared.
Comparative example 3
The difference from example 1 is that in the microbial cell lysate of comparative example 3, no Tris-HCl was added, and an equal amount of solvent water was used instead, to prepare comparative example lysate 3.
Comparative example 4
The difference from example 1 was that the microbial cell lysate of comparative example 4 was adjusted to pH 7.
Comparative example 5
The difference from example 1 is that in the microbial cell lysate of comparative example 5, sodium dodecylsulfate was not added, and an equal amount of solvent water was used instead, thereby preparing comparative example lysate 5.
Respectively carrying out DNA concentration detection on the extracted saliva samples, wherein the specific detection method adopts a nucleic acid concentration detection kit in the prior art; the results are shown in table 4 below:
| sample number | Concentration of nucleic acid | Unit of | OD260/280 |
| Comparative example 1 | 74.3 | ng/μl | 1.35 |
| Comparative example 2 | 45.1 | ng/μl | 1.61 |
| Comparative example 3 | 66.7 | ng/μl | 1.12 |
| Comparative example 4 | 71.8 | ng/μl | 0.87 |
| Comparative example 5 | 33.9 | ng/μl | 0.51 |
Finally, it should be noted that: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; it will be understood by those skilled in the art that the present invention may be modified and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (5)
1. A microbial cell lysate comprising:
90-95% of base solution, 0.1-4% of surfactant, 0.1-2% of sodium hydroxide, 0.1-2% of sodium dodecyl sulfate and 0.1-3% of Triton x-1000.5, wherein the pH value of the microbial cell lysate is 8.5 +/-0.2.
2. The microbial cell lysate of claim 1, wherein the surfactant comprises one or more of benzalkonium chloride, benzalkonium bromide, Triton 100, and Tween 20.
3. The microbial cell lysate of claim 2, wherein the surfactant comprises 0-1% benzalkonium chloride, 0-1% benzalkonium bromide, 0-1% triton 100, 0-0.5% tween 20.
4. A microbial cell lysate according to claim 1, wherein the concentration of sodium hydroxide is 6-7 wt%.
5. A microbial cell lysate according to claim 1, wherein the base solution comprises 30-50mM Tris-HCl pH 7.4, 160-170mM NaCl, 1-2mM PMSF, 1-3mM EDTA.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210130887.0A CN114276932A (en) | 2022-02-12 | 2022-02-12 | Microbial cell lysate |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210130887.0A CN114276932A (en) | 2022-02-12 | 2022-02-12 | Microbial cell lysate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024146532A1 (en) * | 2023-01-04 | 2024-07-11 | 深圳市真迈生物科技有限公司 | Kit and nucleic acid extraction method |
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| WO2016024263A1 (en) * | 2014-08-14 | 2016-02-18 | Molecular Detection Israel Ltd. | Methods for isolating microbial dna from a blood sample |
| CN106282160A (en) * | 2016-07-14 | 2017-01-04 | 中国计量大学 | Lysate, extracting solution, cracking and extracting method, kit, application and PCR system |
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Application publication date: 20220405 |