CN117987506A

CN117987506A - Kit for extracting microorganism DNA with universality, method and application

Info

Publication number: CN117987506A
Application number: CN202211355342.6A
Authority: CN
Inventors: 蔡伟文; 范世睿; 黄亦杭
Original assignee: Zhongshan Kangyuan Gene Technology Technology Co ltd
Current assignee: Zhongshan Kangyuan Gene Technology Technology Co ltd
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2024-05-07
Also published as: WO2024092946A1

Abstract

The invention discloses a kit for extracting microorganism DNA with universality, a method and application thereof. The kit comprises pretreatment liquid, enzymolysis liquid, lysis liquid, magnetic bead purification liquid, eluent and protection liquid. The kit is used for extracting the microorganism DNA, and comprises the steps of pretreatment, enzymolysis, cracking, adsorption, elution, purification and the like. The kit is suitable for extracting most bacteria and fungi, has no toxic or pollution reagent, has simple extraction process, is suitable for automatic equipment extraction, has high sensitivity, and can extract effective DNA only by 10 ² bacteria concentration.

Description

Kit for extracting microorganism DNA with universality, method and application

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a kit for extracting microbial DNA with universality, a method and application thereof.

Background

In recent years, as various disease problems are spread, people have become more and more aware of the pathogen infection. Most diseases occur in close relation with bacteria and fungi, however, traditional detection methods include: the detection methods such as enrichment, separation culture, biochemical reaction, serum agglutination and the like can not meet the requirement of rapid detection in the modern society. The real-time fluorescent quantitative PCR technology has the advantages of strong specificity, good stability, high sensitivity, high detection speed and the like, and is widely applied to the field of medical clinical detection, wherein the extraction of template DNA is one of important steps, and how to extract the DNA template rapidly is the key for improving the detection sensitivity and speed of the real-time fluorescent quantitative PCR method.

At present, many methods for extracting DNA are available, mainly physical extraction, chemical extraction, filter centrifugal column method, magnetic bead method and the like. Physical extraction methods include grinding and high-temperature heating, and if the strength is too strong, DNA double strand is easily broken, and if the strength is insufficient, grinding cannot effectively destroy cell structures for bacteria, and it is difficult to ensure that the cell structures are sufficiently broken; the chemical extraction method comprises a CTAB method, an SDS method, a phenol chloroform extraction method and the like, the chemical extraction method is mature in operation, but the chemical extraction method is complex in DNA operation, needs multiple times of transfer, is long in time consumption and easy to lose, and the phenol chloroform extraction method needs to use organic toxic solvents such as chloroform in the extraction process, so that the risk of injury to human bodies is high; the filter membrane centrifugal column method has stable extraction effect and high operation speed, but needs to transfer liquid for many times, and has low extraction efficiency on fungi and small-fragment DNA with more sugar substances.

In addition, manual operation steps such as grinding, centrifugation, repeated transfer of reaction liquid, a filter membrane centrifugal column and the like are not beneficial to implementation of automatic equipment, are not easy to be suitable for an automatic extraction flow, have small extraction flux, have large operation errors among manual individuals and high labor cost, and are difficult to adapt to efficient detection work. The magnetic bead method can be used for rapidly separating and purifying DNA, the magnetic beads and the nucleic acid are combined specifically, so that the nucleic acid is high in specificity, centrifugation and filtration are not needed in the extraction process, manual operation can be realized, an automatic working platform can be also used, the extraction flux is high, the automatic flow is good, but the magnetic bead method is commonly used for conventional sampling and virus sampling specimens, the difficulty in extracting micro microorganisms is high, and the nucleic acid extraction can be performed after the treatment is particularly performed on fungi with cell walls;

At present, the conventional kit on the market has different treatment modes and methods for cell membranes and cell walls of each fungus, and often a single extraction kit corresponds to a single fungus, so that the flux of extracted fungus is small, and the extraction efficiency and sensitivity of nucleic acid extraction of other fungi are obviously reduced or even can not be extracted. For blood and sputum samples with complex components, the conventional extraction kit cannot effectively extract DNA of trace bacteria and fungi in the samples, or cannot extract DNA of all microorganisms in multi-bacteria compound infection, so false detection and omission are easy to cause.

Therefore, in order to adapt to the characteristic of large sample size of medical institutions and detection institutions, the invention provides a universal kit for extracting microorganism DNA, a method and application thereof, which has the advantages of high flux, high sensitivity, low risk and low pollution, economy, simple steps, applicability to an automatic process and great significance.

Disclosure of Invention

The invention aims at overcoming the defects and shortcomings of the prior art and providing a kit for extracting microorganism DNA with universality.

Another object of the present invention is to provide a method for extracting microbial DNA with universality, which is achieved by using the kit. The method solves the problem of damage to the DNA structure by the traditional physical extraction method by using the methods of extracting DNA by physical, chemical and enzymatic methods; solves the defects of the traditional chemical extraction method, and does not use toxic and polluted reagents; and DNA can be extracted from the ultramicro microorganisms, so that the extraction sensitivity is greatly improved; the device is simple and convenient to use, is applicable to automatic equipment, reduces labor cost, is economical and practical, and reduces human errors.

It is a further object of the present invention to provide the use of the above kit and method.

The aim of the invention is achieved by the following technical scheme:

A kit for extracting microorganism DNA with universality comprises pretreatment liquid, enzymolysis liquid, lysis liquid, magnetic bead purification liquid, eluent and protection liquid; wherein:

the pretreatment liquid comprises the following components: 0.1 to 1mg/mL of saponin, 0.2 to 1mol/L of sodium hydroxide, 0.05 to 0.1mol/L of potassium chloride and pH 11 to 13; the following are preferred: 0.5mg/mL of saponin, 0.5mol/L of sodium hydroxide, 0.05mol/L of potassium chloride and pH 13;

The enzymolysis liquid comprises the following components: tris 10-30 mmol/L, EDTA 0.2-2 mmol/L, naCl 0.01.01-0.15 mol/L, triton X-100.1-3% (v/v), polyethylene glycol 35000 1-8% (w/v), trehalose 0.1-1 mol/L, RNA enzyme A0.1-0.5 mg/mL, lysozyme 1-10 mg/mL, snailase 1-10 mg/mL, pH 6.0-8.0; the following are preferred: tris 20mmol/L, EDTA mmol/L, naCl 0.1mol/L, triton X-100% (v/v), polyethylene glycol 350005% (w/v), trehalose 0.5mol/L, RNA enzyme A0.2 mg/mL, lysozyme 5mg/mL, snailase 5mg/mL, pH7.5;

the composition of the lysate was as follows: tris 10-30 mmol/L, EDTA 0.2-2 nmol/L, triton X-100.1-3% (v/v), sodium chloride 0.5-1 mol/L, protease 10-30 mg/mL, pH 6.0-8.0; the following are preferred: tris 25nmol/L, EDTA 1nmol/L, triton X-100% (v/v), sodium chloride 0.8mol/L, protease 20mg/mL, pH7.5;

The composition of the magnetic bead purification liquid is as follows: 4-6 mg/mL of magnetic nano ferroferric oxide, wherein the solvent is isopropanol; the following are preferred: 5mg/mL of magnetic nano ferroferric oxide, and isopropanol as a solvent;

The eluent is isopropanol with the concentration of 75-85% (v/v); preferably 80% (v/v) isopropyl alcohol;

The protective solution is TE solution.

The RNase A is preferably 60U/mg RNase A.

The lysozyme is preferably 2000U/mg lysozyme.

The snailase is preferably 10mg/mL snailase.

The protease is preferably 20U/mg protease.

The ferroferric oxide particles are magnetic ferroferric oxide particles with the diameter of 30-60 nanometers; more preferred are magnetic ferroferric oxide particles having a diameter of 50 nm.

The TE solution comprises the following components: 50mM Tris-HCl, 1mM EDTA, pH8.0.

A method for extracting microbial DNA with universality, which uses the kit to extract microbial DNA, comprising the following steps:

(1) Taking 1-5 mL of sample, adding an equivalent amount of pretreatment liquid for reaction, centrifuging, pouring out the supernatant, and sucking up the residual liquid as much as possible; drying the obtained precipitate;

(2) Adding 100 mu L of enzymolysis liquid, and carrying out vortex oscillation and uniform mixing for enzymolysis reaction;

(3) Adding 250 mu L of lysate, vortex oscillating, uniformly mixing and precipitating, and carrying out a cracking reaction;

(4) Adding 350 mu L of magnetic bead purification liquid, uniformly mixing by vortex, performing adsorption reaction, and discarding the supernatant after the reaction;

(5) Washing the precipitate with 1mL of eluent by vortex shaking, and discarding the supernatant; repeatedly cleaning the precipitate, and drying the obtained precipitate;

(6) Adding 30 mu L of protective solution, mixing by vortex oscillation, sucking the supernatant, and removing the precipitate to obtain the microorganism DNA.

The reaction conditions in the step (1) are preferably 36-38 ℃ for 5-15 min; more preferably at 37℃for 10 minutes.

The centrifugation conditions in the step (1) are preferably 10000-15000 rpm for 1-5 min; more preferably 14000rpm for 2 minutes.

The drying condition in the step (1) is preferably that the drying is carried out for 3 to 6 minutes at the temperature of 65 to 75 ℃; more preferably at 65℃for 5 minutes.

The enzymolysis reaction condition in the step (2) is preferably 36-38 ℃ for 30-40 min; more preferably at 37℃for 5 minutes.

The conditions of the cracking reaction in the step (3) are preferably 68-72 ℃ for 30-40 min; more preferably at 70℃for 35 minutes.

The conditions of the adsorption reaction in the step (4) are preferably room temperature reaction for 8-12 minutes; more preferably for 10 minutes at room temperature.

The room temperature is 10-40 ℃; preferably 20 to 30 ℃; more preferably 24 to 26 ℃.

The reactions described in the steps (2) to (4) are performed in a stationary state or a rotating state.

The rotating speed of the rotation is below 50 rpm; preferably 5 to 30rpm; more preferably 10rpm. The effect of the rotation is to accelerate the reaction.

The number of repeated washings described in step (5) is preferably 2.

The drying condition in the step (5) is preferably that the drying is carried out for 3 to 6 minutes at the temperature of 65 to 75 ℃; more preferably at 70 c for 5 minutes.

The application of the kit and the method in the extraction of microorganism DNA is suitable for the extraction of ultramicro microorganism DNA.

The application comprises application for scientific research and diagnosis.

The microorganism comprises bacteria and fungi.

The fungi comprise saccharomycetes and candida.

The candida comprises candida albicans, candida glabrata, candida parapsilosis, candida vinosa and candida krusei.

The bacteria include Staphylococcus aureus, escherichia coli, klebsiella pneumoniae, legionella pneumophila, streptococcus pneumoniae, staphylococcus epidermidis, staphylococcus haemolyticus, and enterococcus faecium.

The trace amount refers to a bacterial content as low as 10 ² cells/mL.

Compared with the prior art, the invention has the following advantages and effects:

(1) The invention is applicable to the extraction of most microorganisms, including most bacterial fungi, such as: coli, klebsiella pneumoniae, legionella pneumophila, streptococcus pneumoniae, staphylococcus epidermidis, staphylococcus hemolyticus, etc.; fungi such as: candida krusei, candida parapsilosis, candida vinifera, and the like.

(2) The reagent used in the extraction process of the invention is nontoxic and pollution-free, and all the reagents are cheap biochemical reagents.

(3) The extraction process of the invention uses instruments and equipment which are all common and conventional laboratory instruments and equipment.

(4) The invention does not need complicated physical wall breaking processes such as liquid nitrogen grinding and the like, and avoids cross contamination.

(5) The method has the advantages of mild process, avoidance of DNA damage and high extraction efficiency.

(6) The invention has simple extraction steps, no complex steps and easy manual operation.

(7) The invention realizes no transfer of test solution, and extraction is completed on one test tube or centrifuge tube.

(8) Compared with the commercial extraction kit, the method provided by the invention can extract effective DNA only by the concentration of about 10 ³-10⁴ bacteria, and the extraction sensitivity is higher.

(9) The invention is applicable to automatic machine feeding, and reduces labor cost and human error.

Drawings

FIG. 1 is a standard graph of Staphylococcus aureus.

FIG. 2 is a fluorescent PCR graph of the Staphylococcus aureus DNA templates a1 and a2 extracted by the kit.

FIG. 3 is a fluorescent PCR graph of staphylococcus aureus DNA templates a3 and a4 extracted by a commercially available kit.

FIG. 4 is a fluorescent PCR profile of staphylococcus aureus DNA templates a5 and a6 extracted from a commercial kit.

Fig. 5 is a standard graph of candida glabrata.

FIG. 6 is a fluorescent PCR graph of Candida glabrata DNA templates b1 and b2 extracted by the present kit.

FIG. 7 is a fluorescent PCR profile of Candida glabrata DNA templates b3 and b4 extracted from commercial kits.

FIG. 8 is a fluorescent PCR profile of Candida glabrata DNA templates b5 and b6 extracted from commercial kits.

FIG. 9 is a fluorescent PCR graph of enterococcus faecium DNA templates D1-D7 and D1-D7 extracted according to the present invention.

FIG. 10 is a fluorescent PCR graph of enterococcus faecium DNA templates E1-E7 and E1-E7 extracted according to the invention.

FIG. 11 is a graph showing fluorescence PCR curves obtained using Candida albicans DNA template k1 obtained by the pretreatment liquid of the present invention, candida albicans DNA template k2 obtained by the replacement of the treatment liquid, and Candida albicans DNA template k3 obtained by the treatment with physiological saline.

Fig. 12 is a graph of morphology and number under an initial candida glabrata microscope.

Fig. 13 is a morphology and number map under a candida glabrata microscope after treatment.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

And (3) preparation of a reagent:

1. Preparing an enzymolysis liquid:

(1) The components and the functions of the enzymolysis liquid are as follows:

the concentration of the tris hydrochloride is 20mmol/L, and the tris hydrochloride has the function of providing a buffer environment so as to keep the pH of the solution within a certain range;

the concentration of the ethylenediamine tetraacetic acid is 1mmol/L, and the ethylenediamine tetraacetic acid is used for chelating divalent metal ions, so that the activity of endonuclease can be inhibited;

The concentration of sodium chloride is 0.1mol/L, and the sodium chloride with the concentration can reduce the DNA solubility;

the concentration of the triton X-100 is 1% (v/v), and the triton X-100 acts as a cell permeabilizer to dissolve lipids on cell membranes;

polyethylene glycol 35000 at a concentration of 5% (w/v) and functions to precipitate DNA;

The concentration of trehalose is 0.1mol/L, and the trehalose acts as a protective reagent;

RNase A (60U/mg) at a concentration of 0.5mg/mL, which acts to remove non-specifically bound RNA; the concentration of lysozyme (20000U/mg) and helicase (original concentration 10 mg/mL) were both 5mg/mL, which served to lyse various cell walls.

(2) Preparing enzymolysis liquid:

Weighing Tris (Tris, molecular weight 121.14), ethylenediamine tetraacetic acid (EDTA, molecular weight 372.24), sodium chloride (NaCl, molecular weight 58.44), triton X-100, trehalose and polyethylene glycol 35K, dissolving in a proper amount of sterile water, adjusting pH to 7.5, and sterilizing at 121deg.C under high pressure; cooling, adding lysozyme, snailase and RNase, and preserving at 2-8deg.C.

2. Preparing a cracking liquid

(1) The components and effects of the lysate are as follows:

The concentration of the tris hydrochloride is 25nmol/L, and the tris hydrochloride is used for providing a buffer environment so as to keep the pH of the solution within a certain range;

the concentration of the ethylenediamine tetraacetic acid is 1nmol/L, and the ethylenediamine tetraacetic acid is used for chelating divalent metal ions, so that the activity of endonuclease can be inhibited;

the concentration of the triton X-100 is 1 percent, and the triton X-100 acts as a cell permeabilizer to dissolve lipid on cell membranes;

The concentration of protease (20U/mg) is 20mg/mL, and the protease is used for hydrolyzing protein to produce amino acid;

The concentration of sodium chloride is 0.8mol/L, and sodium chloride at this concentration provides a high salt environment to break cell membranes and expose DNA.

(2) Preparing a lysate:

weighing tris (hydroxymethyl) aminomethane hydrochloride (molecular weight 121.14), ethylenediamine tetraacetic acid (molecular weight 372.24), sodium chloride (molecular weight 58.44) and triton X-100, dissolving in a proper amount of sterile water, adjusting pH to 7.5, and sterilizing at 121deg.C under high pressure for use; cooling, adding protease, and preserving at 2-8deg.C.

3. Preparing magnetic bead purifying liquid:

commercially available magnetic nano ferroferric oxide has a diameter of 50 nanometers. The function is to adsorb nucleic acid and purify; weighing magnetic nano ferroferric oxide, and diluting isopropanol to 5mg/mL for later use.

4. Preparing eluent

The eluent was 80% (v/v) isopropanol, which acts to elute impurities on the nucleic acid. And measuring isopropanol, diluting with sterile water, and then standing by.

5. Preparing protective liquid

The protection solution is TE solution, 50mM Tris-HCl and 1mM EDTA are prepared, the pH value is 8.0, and the protection solution is used for maintaining the pH value of the solution and the stability of DNA. Weighing tris (hydroxymethyl) aminomethane hydrochloride (molecular weight 121.14) and ethylenediamine tetraacetic acid (molecular weight 372.24), dissolving in a proper amount of sterile water, adjusting pH to 8.0, and sterilizing at 121deg.C under high pressure.

6. Preparing pretreatment liquid

The pretreatment liquid is a saponin solution under an alkaline buffer system, and the alkaline buffer system comprises sodium hydroxide and potassium chloride. The concentration of sodium hydroxide in the pretreatment liquid is 0.5mol/L, and the concentration of potassium chloride is 0.05mol/L, so that the pretreatment liquid is used for liquefying a viscous body fluid sample.

The concentration of the saponin is 0.5mg/mL, and the saponin has the function of dissolving red blood cells, preventing a large number of red blood cells from influencing detection, and simultaneously not damaging the microbial cell structure.

Weighing saponin, sodium hydroxide and potassium chloride, dissolving in sterile water, and regulating pH to 13.

And (3) strain selection:

(1) Selecting fungi:

Candida albicans (Candida albicans, standard number ATCC10231, vendor code LWCC 2005); candida glabrata (Candida glabra, standard number ATCC15126, supplier code LWCC 2008); candida parapsilosis (Candida parapsilosis, standard number ATCC22019, vendor code BNCC 263246); candida krusei (Candida krusei, standard number ATCC6258, supplier code BNCC 185429).

Selecting bacteria:

staphylococcus aureus (Staphylococcus aureus, standard number CMCC (B) 26003, vendor code NW 2003), enterococcus faecium (Enterococcus faecium, standard number ATCC35667, vendor code NW 2019).

Example 1: the method and the commercial kit are used for carrying out comparative experiments of extracting bacterial DNA

1. The kit of the invention extracts bacterial DNA

1.1 Taking 1mL of staphylococcus aureus suspension diluted to 5X 10 ⁶/mL, adding an equal amount of pretreatment liquid for reaction for 10min at 37 ℃, centrifuging for 2min at 14000 r/min by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

1.2 Adding 100 mu L of enzymolysis liquid, mixing uniformly by vortex oscillation, placing at 37 ℃ and rotating for 35 minutes at 10 r/min.

1.3 Adding 250 mu L of lysate, vortex oscillating, mixing, precipitating, standing at 70deg.C, and rotating at 10r/min for 35 min.

1.4 Adding 350. Mu.L of magnetic bead purification liquid, mixing by vortex, rotating for 10 minutes at room temperature of 10r/min, centrifuging for 5 minutes at 14000 r/min, and discarding the supernatant.

1.5 Washing the precipitate with 1mL of eluent, vortexing, discarding the supernatant, and repeating the steps for 2 times. The precipitate was dried at 70℃for 5 minutes.

1.6 Adding 30 mu L of protective solution, mixing by vortex oscillation, sucking the supernatant, removing the precipitate, placing at 70 ℃ for 5 minutes, and mixing by vortex oscillation for 10-20 seconds to obtain the DNA.

1.7 Two sets of experiments were performed to obtain DNA templates a1 and a2.

2. Bacterial DNA extraction from commercially available Tiangen kit (product number: PD 302)

2.1 Taking 1mL of staphylococcus aureus suspension cultured to 5X 10 ⁶/mL, centrifuging for 1 min at 11000 r/min by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the visible residual liquid as much as possible.

2.2 To the bacterial pellet 110. Mu.L buffer and 70. Mu.L lysozyme solution were added and treated at 37℃for 30min.

2.3 Add 20. Mu.L proteinase K solution to the tube and mix well.

2.4 220. Mu.L of buffer GB was added, shaken for 15sec, left at 70℃for 10min, and after the solution became clear, centrifuged briefly for 5 seconds.

2.5 Adding 220 mu L absolute ethanol, shaking and mixing for 15sec, and centrifuging for 5 sec.

2.6 Adding the solution obtained in the last step and flocculent precipitate into an adsorption column CB3, placing the adsorption column into a collecting pipe, centrifuging for 30sec at 12000 r/min, pouring out waste liquid, and placing the adsorption column CB3 into the collecting pipe.

2.7 To the adsorption column CB3, 500. Mu.L of the buffer solution GD was added, and the mixture was centrifuged at 12000 rpm for 30sec, and the waste liquid was poured off to place the adsorption column CB3 into a collection tube.

2.8 Adding 600. Mu.L of the rinse solution PW to the adsorption column CB3, centrifuging at 12000 rpm for 30esc, pouring out the waste liquid, and placing the adsorption column CB3 into a collection tube.

2.9 Repeating step 2.8.

2.10 Put the adsorption column CB3 back into the collection tube, centrifuge for 2min at 12000 rpm, and pour out the waste liquid. The adsorption column CB3 was left at room temperature for 8 minutes.

2.11 Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 200 mu L of elution buffer TE into the middle part of the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12000 r/min, and collecting the solution into the centrifuge tube.

2.12 Two sets of experiments were performed to obtain DNA templates a3 and a4.

3. Bacterial DNA extraction from commercial omega kit (cat# PD 3350)

3.1 1ML of Staphylococcus aureus suspension incubated to 5X 10 ⁶/mL, centrifuged at 4000 rpm for 10 minutes, and the supernatant was decanted.

3.2 Adding 100. Mu.L TE buffer, vortex shaking to suspend the pellet, and keep the temperature at 37℃for 10 minutes.

3.3 100. Mu.L of BTL buffer and 20. Mu.L of proteinase K solution were added and mixed by vortexing.

3.4 Water bath in a water bath tank at 55℃for 20 minutes, after brief vortexing, in a water bath for 20 minutes.

3.5 Adding 5. Mu.L RNase A, turning over and mixing, incubating for 5 minutes at room temperature.

3.6 Centrifuge at 10000 rpm for 2 minutes, transfer the supernatant to a new 1.5mL microcentrifuge tube.

3.7 Add 220. Mu.L BDL buffer, vortex mix and incubate at 65℃for 10 minutes.

3.8 Add 220. Mu.L ethanol and mix by vortex shaking.

3.9 The off-column was inserted into a 2mL collection tube, the whole sample was transferred to the column, centrifuged at 10000rpm for 1min, the filtrate and collection tube were discarded, and the column was inserted into a new 2mL collection tube.

3.10 Add 500. Mu.L HBC buffer, centrifuge at 10000 rpm for 1 min, discard the filtrate and reuse the collection tube.

3.11 Add 700. Mu.L of DNA wash buffer, centrifuge for 1 min at 10000 rpm, discard the filtrate and reuse the collection tube.

3.12 Repeat step 3.11.

3.13 The column and the collection tube were centrifuged at 12000 rpm and dried for 2 minutes.

3.14 The column was inserted into a new 1.5mL centrifuge tube, 100. Mu.L of elution buffer was added to the column, and the column was allowed to stand at room temperature for 5 minutes and centrifuged at 10000 rpm for 1 minute.

3.15 Repeat 3.14 steps.

3.16 Two sets of experiments were performed to obtain DNA templates a5 and a6.

4. Fluorescent PCR amplification

4.1 Apparatus: tianlong Gentier E full-automatic PCR analysis system

4.2 The amplification reaction system is: SYBR GREEN QPCR mix 12.5. Mu.L, primer F1 (0.2 mmol/L) 2. Mu.L, primer R1 (0.2 mmol/L) 2. Mu.L, DNA template 1. Mu.L, sterile water 3.5. Mu.L.

4.3 Primer information for PCR amplification with Staphylococcus aureus DNA as template (examples):

primer F1:5'-GAAGCTTGCTTCTCTGATGTT-3';

Primer R1:5'-CAGCAAGACCGTCTTTCACT-3'.

4.4PCR reaction procedure: 94 ℃ for 5min; 15s at 94 ℃, 30s at 55 ℃ and 15s at 72 ℃ for 40 cycles.

4.5 Fluorescent PCR amplification was performed with the DNA templates of a1-a6 and CT values were recorded.

4.6 The extraction efficiency was calculated from the standard curve formula y= -3.4757x+33.931 for staphylococcus aureus at each concentration CT of the standard curve.

Table 1: CT value and extraction efficiency of standard curve and contrast kit staphylococcus aureus amplification curve

5. Analysis of results:

according to the data in Table 1, the method provided by the invention has higher efficiency in extracting bacterial DNA than other kits.

Example 2: the method and the commercial kit are used for carrying out the comparative experiment of extracting the fungal DNA

1. The kit of the invention extracts the fungus DNA

1.1 Taking 1mL of candida glabrata suspension diluted to 5X 10 ⁶/mL, adding an equivalent amount of pretreatment liquid, reacting for 10min at 37 ℃, centrifuging for 2min at 14000 r/min by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

1.2 Adding 100 mu L of enzymolysis liquid, mixing uniformly by vortex oscillation, and placing at 37 ℃ for 10r/min, rotating and preserving heat for 35 minutes.

1.3 Adding 250 mu L of lysate, vortex oscillating, mixing, precipitating, standing at 70deg.C for 10r/min, and rotating for 35 min.

1.4 Adding 350 mu L of magnetic bead purification liquid, mixing by vortex, rotating for 10 minutes at room temperature of 10r/min, and discarding the supernatant.

1.6 Adding 30 mu L of protective solution, mixing by vortex oscillation, sucking the supernatant, removing the precipitate, standing at 70 ℃ for 5 minutes, and mixing by vortex oscillation for 10-20 seconds. Thus obtaining DNA.

1.7 Two sets of experiments were performed to obtain DNA templates b1 and b2.

2. Extraction of fungal DNA from a commercially available Tiangen kit (cat# PD 307)

2.1 Taking 1mL of candida glabrata suspension diluted to 5X 10 ⁶/mL, centrifuging for 1 min at 12000 r/min by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the visible residual liquid as much as possible.

2.2 To the bacterial pellet 600. Mu.L of sorbitol buffer and 50U of lycrase (lycrase) were added and thoroughly mixed. The treatment is carried out at 30 ℃ for 30min. Centrifuge at 4000 rpm for 10 min, discard supernatant and collect pellet.

2.3 Adding 200. Mu.L buffer GA to the pellet to resuspend the pellet, and mixing well.

2.4 Add 20. Mu.L proteinase K solution and mix well.

2.5 Adding 220. Mu.L buffer GB, mixing well upside down, standing at 70deg.C for 10min, and centrifuging briefly for 5 seconds after the solution becomes clear.

2.6 Adding 220 mu L absolute ethanol, fully reversing and uniformly mixing, and briefly centrifuging for 5 seconds.

2.7 Adding the solution obtained in the previous step into an adsorption column CB3, placing the adsorption column into a collecting pipe, centrifuging for 30sec at 12000 r/min, pouring out waste liquid, and placing the adsorption column CB3 into the collecting pipe.

2.9 Repeating step 2.8.

2.12 Two sets of experiments were performed to obtain DNA templates b3 and b4.

3. Extraction of fungal DNA from commercial omega kit (cat# PD 3370)

3.1 1 Taking 1mL of Candida glabrata suspension diluted to 5X 10 ⁶/mL, centrifuging at 4000 rpm for 10 minutes, and decanting the supernatant.

3.2 Adding 480 u L SE buffer, 10u L2-hydroxy ethanol and 20u L lyase solution, resuspension bacteria solution, 30 degrees C incubation for 30 minutes.

3.3 Centrifugation at 4000 rpm for 5 minutes at room temperature.

3.4 200. Mu.L of buffer YL and 50mg of glass beads (0.4-0.6 mm) were added to the sample, mixed rapidly and spun for 5 minutes, and the supernatant transferred to a new 1.5mL centrifuge tube.

3.5 25. Mu.L of proteinase K solution are added, vortexed and mixed well and incubated in an oscillating water bath at 65℃for 30 minutes.

3.6 5. Mu.L RNase A was added, mixed by tumbling and incubated at room temperature for 10 minutes.

3.6 Centrifuge at 10000 rpm for 5 minutes, transfer the supernatant to a new 1.5mL microcentrifuge tube.

3.7 Add 220. Mu.L YDL buffer and 220. Mu.L absolute ethanol and mix rapidly for 20 seconds.

3.8 The column was inserted into a 2mL collection tube, the whole sample was transferred to the column, centrifuged at 10000 rpm for 1 min, the filtrate and collection tube were discarded, and the column was inserted into a new 2mL collection tube.

3.9 Add 500. Mu.L HB buffer and centrifuge at 10000 rpm for 1 min, discard the filtrate and reuse the collection tube.

3.10 Adding 700 u L DNA washing buffer, 10000 rotation centrifugal 1 minutes, discarding the filtrate, reuse collection tube.

3.11 Repeat 3.10 steps.

3.15 Repeat 3.14 steps.

3.16 Two sets of experiments were performed to obtain DNA templates b5 and b6.

4. Fluorescent PCR amplification

4.1 Apparatus: tianlong Gentier E full-automatic PCR analysis system

4.2 The amplification reaction system is: SYBR GREEN QPCR mix 12.5. Mu.L, primer F2 (0.2 mmol/L) 2. Mu.L, primer R2 (0.2 mmol/L) 2. Mu. L, DNA template 1. Mu.L, sterile water 3.5. Mu.L.

4.3 Primer information for PCR amplification with Candida glabrata DNA as template:

primer F2:5'-CGATTTTTTCGTGTACTGGAATG-3';

Primer R2:5'-CGCCAAGCCACAAGGACTT-3'.

4.5 Fluorescent PCR amplification was performed with the DNA templates of b1-b6 and CT values were recorded.

4.6 Calculating the extraction efficiency from the standard curve formula y= -3.397x+29.992 of candida glabrata standard curve with each concentration CT value.

Table 2: CT value and extraction efficiency of standard curve and candida glabrata amplification curve of contrast kit

5. Analysis of results:

the method provided by the invention has higher efficiency for extracting the fungal DNA than other kits according to the data of Table 2.

Example 3: the method extracts bacterial DNA and makes concentration gradient test.

1. Taking 1mL of enterococcus faecium suspension diluted to 10 ²、10³、10⁴、10⁵、10⁶、10⁷、10⁸/mL, adding an equivalent amount of pretreatment liquid, reacting for 10min at 37 ℃, centrifuging for 2min at 14000 r/min by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

2. Adding 100 μl of enzymolysis solution, mixing under vortex oscillation, standing at 37deg.C, and rotating at 10r/min for 35 min.

3. Adding 250 μl of lysate, mixing with vortex vibration, precipitating, standing at 70deg.C, and rotating at 10r/min for 35 min.

4. 350. Mu.L of the bead purification solution was added, the mixture was vortexed and spun at 10r/min for 10 minutes at room temperature, and the supernatant was discarded.

5. The precipitate was washed with 1mL of eluent, vortexed and shaken, the supernatant was discarded, and the step was repeated for 2 times. The precipitate was dried at 70℃for 5 minutes.

6. Adding 30 μl of protective solution, mixing under vortex shaking, sucking supernatant, removing precipitate, standing at 70deg.C for 5min, and mixing under vortex shaking for 10-20 seconds. Thus obtaining DNA.

7. 2 Groups of experiments were performed to obtain DNA templates D1 to D7 and repeat groups D1 to D7.

8. Fluorescent PCR amplification

8.1 Apparatus: tianlong Gentier E full-automatic PCR analysis system

8.2 The amplification reaction system is: SYBR GREEN QPCR mix 12.5. Mu.L, primer F3 (0.2 mmol/L) 2. Mu.L, primer R3 (0.2 mmol/L) 2. Mu. L, DNA template 1. Mu.L, sterile water 3.5. Mu.L.

8.3 Primer information for PCR amplification with enterococcus faecium DNA as template:

Primer F3:5'-CGAACGCTTCTTTTTCCACC-3';

Primer R3:5'-CTTTCAAATCAAAACCATGCGGTT-3'.

8.4 PCR reaction procedure: 94 ℃ for 5min; 15s at 94 ℃, 30s at 55 ℃ and 15s at 72 ℃ for 40 cycles.

8.5 Fluorescent PCR amplification with DNA templates of D1-D7 and D1-D7 and recording the CT values.

TABLE 3 CT values of enterococcus faecium amplification curves at different concentrations

Name of the name

Encoding

Concentration of

CT value

Encoding

Concentration of

CT value

Enterococcus faecium

d1

10⁸

12.064

D1

10⁸

12.728

Enterococcus faecium

d2

10⁷

16.145

D2

10⁷

15.540

Enterococcus faecium

d3

10⁶

20.835

D3

10⁶

21.605

Enterococcus faecium

c4

10⁵

25.224

D4

10⁵

25.465

Enterococcus faecium

d5

10⁴

26.840

D5

10⁴

26.651

Enterococcus faecium

d6

10³

27.927

D6

10³

27.113

Enterococcus faecium

d7

10²

29.956

D7

10²

29.881

Enterococcus faecium-negative

-

9 Analysis of results

As shown in FIG. 9, the obtained CT values are shown in Table 3, and the CT values are proportionally increased along with the decrease of the concentration of the bacteria-enterococcus faecium liquid, and the amplification performance is still realized at a low concentration, namely, the method provided by the invention still has the extraction effect at a low concentration of a sample.

Example 4: the method extracts the fungus DNA and makes a concentration gradient test.

1. Taking 1mL of candida otophylla suspension diluted to 10 ²、10³、10⁴、10⁵、10⁶、10⁷、10⁸/mL, adding an equivalent amount of pretreatment liquid, reacting for 10min at 37 ℃, centrifuging for 2min at 14000 r/min by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

2. Adding 100 μl of enzymolysis solution, mixing under vortex oscillation, standing at 37deg.C for 10r/min, and maintaining the temperature for 35 min.

3. Adding 250 μl of lysate, mixing with vortex vibration, precipitating, standing at 70deg.C for 10r/min, and rotating for 35 min.

7. 2 Sets of experiments were performed to obtain DNA templates E1 to E7 and repeat sets E1 to E7.

8. Fluorescent PCR amplification

8.1 Apparatus: tianlong Gentier E full-automatic PCR analysis system

8.2 The amplification reaction system is: SYBR GREEN QPCR mix 12.5. Mu.L, primer F4 (0.2 mmol/L) 2. Mu.L, primer R4 (0.2 mmol/L) 2. Mu. L, DNA template 1. Mu.L, sterile water 3.5. Mu.L.

8.3 Primer information for PCR amplification with Candida otorhinoca DNA as template:

primer F4:5'-GTACTTCCATATCCAAGACCTTT-3';

primer R4:5'-GCCTGCTTTGAACACTCT-3'.

8.4PCR reaction procedure: 94 ℃ for 5min; 15s at 94 ℃, 30s at 55 ℃ and 15s at 72 ℃ for 40 cycles.

8.5 Fluorescent PCR amplification with DNA templates of E1-E7 and E1-E7 and recording the CT values.

Table 4: CT values of candida otophylla amplification curves with different concentrations

Name of the name

Encoding

Concentration of

CT value

Encoding

Concentration of

CT value

Candida otophylla (L.) Ohwi

e1

10⁸

13.154

E1

10⁸

12.026

Candida otophylla (L.) Ohwi

e2

10⁷

20.147

E2

10⁷

18.945

Candida otophylla (L.) Ohwi

e3

10⁶

22.556

E3

10⁶

22.354

Candida otophylla (L.) Ohwi

e4

10⁵

25.024

E4

10⁵

25.412

Candida otophylla (L.) Ohwi

e5

10⁴

26.840

E5

10⁴

26.651

Candida otophylla (L.) Ohwi

e6

10³

27.527

E6

10³

27.913

Candida otophylla (L.) Ohwi

e7

10²

28.256

E7

10²

28.181

Candida otophylla-negative

-

9. Analysis of results

As shown in FIG. 10, the obtained CT values are shown in Table 4, and the CT values are proportionally increased along with the decrease of the concentration of the fungus-candida otophylla liquid, and the amplification performance is still realized at a low concentration, namely, the method provided by the invention still has the extraction effect at a low concentration of a sample. Example 5: and (5) comparing the components of the enzymolysis liquid after increasing and decreasing.

1. Candida krusei, candida parapsilosis and candida albicans are selected, the condition that components are reduced, extraction efficiency is reduced, and no data is reflected is avoided, bacterial liquid is diluted to medium-high concentration 10 ⁸、10⁶、10⁴/mL, and 1mL of suspension is taken. Adding an equal amount of pretreatment liquid to react for 10min at 37 ℃, centrifuging for 2 min at 14000 rpm by a centrifuge, collecting thalli to the bottom of a tube, pouring out the supernatant, and sucking the residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

2.1 Adding 100. Mu.L of enzymolysis solution (containing RNase A0.2 mg/mL, lysozyme 5mg/mL and snailase 5 mg/mL), mixing by vortex oscillation, and standing at 37deg.C for 10r/min for rotation and heat preservation for 35 min.

2.2 Control 100. Mu.L of the enzymatic hydrolysate of lysozyme and RNase A (i.e.containing only 10.2mg/mL of helicase) was added, mixed by vortexing, and incubated at 37℃for 35 minutes with 10r/min rotation.

6. Adding 30 μl of the protective solution, mixing under vortex shaking, removing precipitate, standing at 70deg.C for 5 min, and mixing under vortex shaking for 10-20 seconds.

7. 2 Groups of tests are carried out to obtain candida krusei DNA templates F1-F3, repeated groups F1-F3, comparison groups F4-F6 and comparison groups repeated F4-F6; candida parapsilosis DNA templates G1-G3, repeated groups G1-G3, control groups G4-G6 and control groups repeated G4-G6; candida albicans templates I1-I3, repeated groups I1-I3, control groups I4-I6 and control groups repeated I4-I6.

8. Fluorescent PCR amplification

8.1 Apparatus: tianlong Gentier E full-automatic PCR analysis system

8.2 The amplification reaction system is: SYBR GREEN QPCR mix 12.5. Mu.L, upstream primer (0.2 mmol/L) 2. Mu.L, downstream primer (0.2 mmol/L) 2. Mu. L, DNA template 1. Mu.L, sterile water 3.5. Mu.L.

8.3 Primer information for PCR amplification with Candida krusei, candida parapsilosis, candida albicans DNA as template:

candida krusei:

primer F5:5'-GGACGGTCTACCTATGGTAA-3';

primer R5:5'-ACTCTAATTTCCTCAAAGTAATCGT-3'.

Candida parapsilosis:

primer F6:5'-CCTTCTGGCTAGCCTTTTTG-3';

primer R4:5'-GCCTGCTTTGAACACTCT-3'.

Candida albicans:

primer F7:5'-TTCCTTCTGGGTAGCCATTT-3';

primer R4:5'-GCCTGCTTTGAACACTCT-3'.

8.5 Fluorescent PCR amplification was performed with DNA templates of C.krusei, C.parapsilosis, C.albicans and CT values were recorded.

TABLE 5 comparison of CT values and extraction efficiencies of amplification curves for full fraction and snailase alone

9. Analysis of results

The results of the experiment are shown in Table 5, and the extraction efficiency is higher than that of the whole enzymatic hydrolysate only. Therefore, the enzymolysis liquid used in the method provided by the invention is prepared to be the optimal proportion, and if other reagent components in the method provided by the invention are reduced, the extraction efficiency is also affected.

Example 6: and (5) after replacement of the pretreatment liquid, comparing the experiment.

1.1 Taking 1mL of candida albicans suspension diluted to 5X 10 ⁶/mL by using blood of a healthy person, adding an equivalent amount of pretreatment liquid, reacting for 10min at 37 ℃, centrifuging for 2min at 14000 rpm by a centrifuge, collecting thalli to the bottom of the tube, pouring out the supernatant, and sucking the visible residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

1.2 Control k2 was diluted with blood to 1mL of 5X 10 ⁶/mL Candida albicans suspension, an equal amount of 0.5% (w/v) SDS+1% (v/v) Triton X-100+0.5M NaCl mixed solution was added and reacted at 37℃for 10min, and the cells were collected to the bottom of the tube by centrifugation at 14000 rpm for 2 min, and the supernatant was decanted and the residual liquid was blotted as much as possible. The precipitate was dried at 65℃for 5 minutes.

1.3 Control k3 was diluted with blood to 1mL of Candida albicans suspension of 5X 10 ⁶/mL, added with an equivalent amount of physiological saline, reacted for 10min at 37℃and centrifuged at 14000 rpm for 2 min in a centrifuge, the cells were collected to the bottom of the tube, the supernatant was decanted, and the residual liquid was blotted as dry as possible. The precipitate was dried at 65℃for 5 minutes.

4.1 Adding 350 mu L of magnetic bead purification liquid, mixing by vortex, rotating for 10 minutes at room temperature of 10r/min, and discarding the supernatant.

6. Adding 30 μl of the protective solution, mixing under vortex shaking, removing precipitate, standing at 70deg.C for 5min, and mixing under vortex shaking for 10-20 seconds. Obtain candida albicans DNA template k1, control group k2 and control group k3.

8. Fluorescent PCR amplification

8.1 Apparatus: tianlong Gentier E full-automatic PCR analysis system

8.3 Primer information for PCR amplification with Candida albicans DNA as template:

Candida albicans:

primer F7:5'-TTCCTTCTGGGTAGCCATTT-3';

primer R4:5'-GCCTGCTTTGAACACTCT-3'.

8.5 Fluorescent PCR amplification with Candida albicans DNA template and CT values were recorded.

TABLE 6 CT value comparison table for comparison experiment amplification curve after pretreatment liquid replacement

9. Analysis of results

The experimental results are shown in Table 6 and FIG. 11, and the pretreatment liquid of the present invention has significantly better effect.

Example 7: after the enzymolysis liquid and the lysate are treated, the shape and the quantity of the bacteria change

1. The bacterial liquid of candida glabrata is cultured, and after proper dilution, the original form and the number of the bacterial liquid are observed by using a microscope.

2. Taking 1mL of the bacterial suspension, adding an equivalent amount of pretreatment liquid for reaction for 10min at 37 ℃, centrifuging for 2 min at 14000 r/min by a centrifuge, collecting bacterial cells to the bottom of a tube, pouring out the supernatant, and sucking the residual liquid as much as possible. The precipitate was dried at 65℃for 5 minutes.

3. Adding 100 μl of enzymolysis solution, mixing under vortex oscillation, standing at 37deg.C, and rotating at 10r/min for 35 min.

4. Adding 250 μl of lysate, mixing with vortex vibration, precipitating, standing at 70deg.C, and rotating at 10r/min for 35 min.

5. And then taking the treated bacterial liquid, and observing the bacterial forms and the bacterial quantity by using a microscope.

6. Analysis of results

The experimental results are shown in fig. 12 and 13, and the number of candida glabrata with cell structures after treatment is obviously reduced, and the tissue structures after cell rupture are present. Therefore, the method provided by the invention can effectively break the wall and crack the microorganism, and does not influence the efficiency of subsequent extraction of nucleic acid.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A kit for extracting microbial DNA with universality, which is characterized in that: comprises pretreatment liquid, enzymolysis liquid, pyrolysis liquid, magnetic bead purifying liquid, eluent and protective liquid; wherein:

The pretreatment liquid comprises the following components: 0.1 to 1mg/mL of saponin, 0.2 to 1mol/L of sodium hydroxide, 0.05 to 0.1mol/L of potassium chloride and pH 11 to 13;

The enzymolysis liquid comprises the following components: tris 10-30 mmol/L, EDTA 0.2-2 mmol/L, naCl 0.01.01-0.15 mol/L, triton X-100.1-3% (v/v), polyethylene glycol 35000 1-8% (w/v), trehalose 0.1-1 mol/L, RNA enzyme A0.1-0.5 mg/mL, lysozyme 1-10 mg/mL, snailase 1-10 mg/mL, pH 6.0-8.0;

The composition of the lysate was as follows: tris 10-30 mmol/L, EDTA 0.2-2 nmol/L, triton X-100.1-3% (v/v), sodium chloride 0.5-1 mol/L, protease 10-30 mg/mL, pH 6.0-8.0;

the composition of the magnetic bead purification liquid is as follows: 4-6 mg/mL of magnetic nano ferroferric oxide, wherein the solvent is isopropanol;

the eluent is isopropanol with the concentration of 75-85% (v/v);

The protective solution is TE solution.

2. The kit for extracting microbial DNA having universality according to claim 1, wherein: the pretreatment liquid comprises the following components: 0.5mg/mL of saponin, 0.5mol/L of sodium hydroxide, 0.05mol/L of potassium chloride and pH 13;

The enzymolysis liquid comprises the following components: tris 20mmol/L, EDTA mmol/L, naCl 0.1mol/L, triton X-1001% (v/v), polyethylene glycol 35000 5% (w/v), trehalose 0.5mol/L, RNA enzyme A0.2 mg/mL, lysozyme 5mg/mL, snailase 5mg/mL, pH7.5;

the composition of the lysate was as follows: tris 25nmol/L, EDTA 1nmol/L, triton X-100% (v/v), sodium chloride 0.8mol/L, protease 20mg/mL, pH7.5;

The composition of the magnetic bead purification liquid is as follows: 5mg/mL of magnetic nano ferroferric oxide, and isopropanol as a solvent;

The eluent was 80% (v/v) isopropyl alcohol.

3. Kit for the extraction of microbial DNA with universality according to claim 1 or 2, characterized in that:

the RNase A is 60U/mg RNase A;

the lysozyme is 2000U/mg lysozyme;

the snailase is 10mg/mL snailase;

the protease is 20U/mg protease;

the ferroferric oxide particles are magnetic ferroferric oxide particles with the diameter of 30-60 nanometers.

4. A method for extracting microbial DNA with universality, which is characterized by comprising the following steps:

5. The method according to claim 4, wherein:

the reaction condition in the step (1) is 36-38 ℃ for 5-15 min;

The centrifugation condition in the step (1) is 10000-15000 rpm for 1-5 min;

The drying condition in the step (1) is that the drying is carried out for 3 to 6 minutes at the temperature of 65 to 75 ℃;

The enzymolysis reaction condition in the step (2) is 36-38 ℃ for 30-40 min;

The condition of the cracking reaction in the step (3) is 68-72 ℃ for 30-40 min;

the conditions of the adsorption reaction in the step (4) are room temperature reaction for 8-12 minutes.

6. The method according to claim 5, wherein:

the reaction conditions in the step (1) are that the reaction is carried out for 10 minutes at 37 ℃;

The centrifugation conditions in step (1) were 14000rpm for 2 minutes;

the enzymolysis reaction condition in the step (2) is that the reaction is carried out for 5 minutes at 37 ℃;

the conditions of the cracking reaction in the step (3) are 70 ℃ for 35 minutes;

the conditions for the adsorption reaction described in step (4) were room temperature reaction for 10 minutes.

7. The method according to claim 4, wherein:

The drying condition in the step (5) is that the drying is carried out for 3 to 6 minutes at the temperature of 65 to 75 ℃.

8. Use of a kit according to any one of claims 1 to 3 or a method according to any one of claims 4 to 7 in the extraction of microbial DNA.

9. The use according to claim 8, characterized in that: the microorganisms include bacteria and fungi.