CN114276932A

CN114276932A - Microbial cell lysate

Info

Publication number: CN114276932A
Application number: CN202210130887.0A
Authority: CN
Inventors: 马振桥; 丁海平; 李坤
Original assignee: Hefei Peakedness Biological Technology Co ltd
Current assignee: Hefei Peakedness Biological Technology Co ltd
Priority date: 2022-02-12
Filing date: 2022-02-12
Publication date: 2022-04-05

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

Microbial cell lysate

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

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

1. A microbial cell lysate comprising:

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.