A mixed microbial gene extraction reagent combination and kit for magnetic bead method

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a mixed microbial gene extraction reagent combination and a kit for a magnetic bead method. The present invention first provides a mixed microbial gene extraction reagent combination for a magnetic bead method, wherein the reagent combination includes a wall-breaking reagent, a wall-breaking enzyme reagent, a protease reagent, and a cleavage reagent. The reagent combination includes a variety of functional reagents, and each functional reagent cooperates to achieve efficient and accurate extraction of samples containing unknown mixed microorganisms without the need for particularly complex magnetic bead modification. Therefore, it has good universality and is applicable to a large number of magnetic bead types.

Description

[0001] Divisional application This application is a divisional application based on the Chinese invention patent application with application number CN2024114995061, application date October 25, 2024, and invention name “A magnetic bead microbial genome extraction kit and microbial genome extraction method”. Technical Field

[0002] The invention belongs to the technical field of biomedicine, and in particular relates to a mixed microbial gene extraction reagent combination and a kit for a magnetic bead method. Background Art

[0003] Magnetic bead-based nucleic acid extraction is a method that uses nanotechnology to improve and modify the surface of superparamagnetic nanoparticles, and then specifically recognize and efficiently bind the magnetic nanoparticles to nucleic acid molecules. Under the action of an external magnetic field, nucleic acids are separated from samples such as blood, animal tissues, food, and pathogenic microorganisms. It is widely used in clinical disease diagnosis, environmental microbial testing, and food safety testing.

[0004] Nucleic acid testing is currently an important means of disease diagnosis and screening. In the process of extracting nucleic acids using magnetic beads, it is often necessary to ensure that the nucleic acid is fully extracted and the integrity of the primary structure of the nucleic acid is guaranteed, while also avoiding contamination by biological macromolecules such as proteins, lipids, and polysaccharides. The current extraction methods and extraction reagents must simultaneously achieve the above technical objectives, and the extraction process is often more complicated; or the above technical objectives cannot be achieved simultaneously when the extraction process is simplified.

[0005] In addition, the extraction of bacteria and fungi in commonly used commercial genome extraction kits are set up separately. It is necessary to first estimate the types of pathogenic microorganisms in the sample and then select the appropriate extraction reagent for extraction. For samples where the types of pathogenic microorganisms are not clear and samples with mixed types of pathogenic microorganisms (such as mixed samples of bacteria and fungi), it is often necessary to try different extraction reagents separately, and multiple tests are required before successful extraction. Therefore, there is a waste of sample processing time and the reagents used.

[0006] The patent with publication number CN105821034A and invention name "A method for extracting nucleic acids using magnetic beads to improve purification effect" discloses a technical solution for recovering microbial genomes using magnetic beads. However, the patent method requires special modification of magnetic beads to achieve the technical purpose of improving the quality of nucleic acid purification. However, the scope of application of the technical solution of special modification of magnetic beads is slightly limited.

[0007] The patent with publication number CN112941066A and invention name “A lysis binding solution for bacterial nucleic acid extraction, its kit and application” discloses a method for extracting bacterial nucleic acid, including treating the sample with lysozyme solution and proteinase K solution, adding lysis binding solution, releasing nucleic acid from the biological sample, adding magnetic beads, obtaining magnetic bead-nucleic acid complex under the action of an external magnetic field, collecting and washing the magnetic bead-nucleic acid complex, and obtaining extracted nucleic acid. However, the bacterial nucleic acid extracted by the patented method needs to be amplified by PCR before the result can be detected, and PCR amplification has an amplifying effect on the original nucleic acid. It is speculated that this may be because the amount of original nucleic acid extracted by this method is not large. In addition, the patented method cannot process mixed microbial samples containing multiple species.

[0008] In summary, a more convenient and efficient tool for extracting microbial nucleic acids is urgently needed. Summary of the invention

[0009] In view of this, the object of the present invention is to provide a mixed microbial gene extraction reagent combination and a kit for the magnetic bead method, which can partially solve or alleviate the above-mentioned deficiencies in the prior art. The specific technical solution is as follows.

[0010] A mixed microbial gene extraction reagent combination for a magnetic bead method, the reagent combination comprising a wall-breaking reagent, a wall-breaking enzyme reagent, a protease reagent and a cleavage reagent; the volume ratio of the wall-breaking reagent, the wall-breaking enzyme reagent, the protease reagent and the cleavage reagent is 16:1:1:6 (80μL:5μL:5μL:30μL); The wall-breaking reagent is composed of 5-10 mM dithiothreitol, 10-20 mM tris(hydroxymethyl)aminomethane hydrochloride, 1-5 mM ethylenediaminetetraacetic acid and 0.1%-0.5% polyethylene glycol octylphenyl ether; the pH value of the wall-breaking reagent is 7.5-8.5; The wall-breaking enzyme reagent is a wall-breaking enzyme composition, which consists of lytic enzyme, lysozyme and lysostaphin; the specific activity of the lytic enzyme is ≥20000U / mg, the specific activity of the lysozyme is ≥20000U / mg, and the specific activity of the lysostaphin is ≥1200U / mg; The protease reagent is proteinase K, and the specific activity is ≥30 units / mg; The lysis reagent consists of 200-400 mM tris(hydroxymethyl)aminomethane hydrochloride, 20-50 mM ethylenediaminetetraacetic acid, 1%-5% polyethylene glycol octylphenyl ether, 500-1000 mM sodium chloride, 0.1-0.5 mM sodium methyl paraben and 1%-5% Tween; the pH value of the lysis reagent is 7.5-8.5.

[0011] It is understood that the wall-breaking reagent is used to provide a weakly alkaline reducing environment for the wall-breaking enzyme composition to ensure enzyme activity. For example, the wall-breaking reagent contains the reducing agent dithiothreitol, and the wall-breaking reagent may contain more than one reducing agent.

[0012] Proteinase K was chosen because it is a powerful proteolytic enzyme with strong activity in decomposing proteins; secondly, it is highly stable and maintains activity over a wide pH range (4-12.5) and high temperature (50-70°C), so it can be combined with other reagents to exert a synergistic effect.

[0013] Furthermore, the microorganisms include one or more selected from fungi, Gram-positive bacteria and / or Gram-negative bacteria.

[0014] Preferably, the cell wall breaking reagent consists of 5 mM dithiothreitol, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 2 mM ethylenediaminetetraacetic acid and 0.1% polyethylene glycol octylphenyl ether; and the pH value of the cell wall breaking reagent is 8.

[0015] Preferably, the lysis reagent consists of 400 mM tris(hydroxymethyl)aminomethane hydrochloride, 40 mM ethylenediaminetetraacetic acid, 5% polyethylene glycol octylphenyl ether, 800 mM sodium chloride, 0.1 mM sodium methyl paraben and 5% Tween; the pH value of the lysis reagent is 8.

[0016] Furthermore, the reagent combination also includes a high-salt magnetic bead binding reagent for magnetic bead binding and a low-salt magnetic bead elution reagent for magnetic bead elution; the high-salt magnetic bead binding reagent is composed of 30-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 1-4M sodium chloride and 5% polyethylene glycol 8000; the low-salt magnetic bead elution reagent is nuclease-free water.

[0017] The present invention further provides the use of the above-mentioned mixed microbial gene extraction reagent combination in the preparation of a magnetic bead method microbial genome extraction kit.

[0018] A magnetic bead method microbial genome extraction kit, characterized in that it comprises a microbial wall breaking unit and a microbial lysis unit; the microbial wall breaking unit comprises a wall breaking reagent and a wall breaking enzyme composition reagent; the microbial lysis unit comprises a protease reagent and a lysis reagent; wherein, The wall-breaking reagent is composed of 5-10 mM dithiothreitol, 10-20 mM tris(hydroxymethyl)aminomethane hydrochloride, 1-5 mM ethylenediaminetetraacetic acid and 0.1%-0.5% polyethylene glycol octylphenyl ether; the pH value of the wall-breaking reagent is 7.5-8.5; The wall-breaking enzyme composition reagent is composed of lytic enzyme, lysozyme and lysostaphin; the specific activity of the lytic enzyme is ≥20000U / mg, the specific activity of the lysozyme is ≥20000U / mg, and the specific activity of the lysostaphin is ≥1200U / mg; The protease reagent is set to proteinase K, and the specific activity is ≥30 units / mg; The lysis reagent is composed of 200-400 mM tris(hydroxymethyl)aminomethane hydrochloride, 20-50 mM ethylenediaminetetraacetic acid, 1%-5% polyethylene glycol octylphenyl ether, 500-1000 mM sodium chloride, 0.1-0.5 mM sodium methyl paraben and 1%-5% Tween; the pH value of the lysis reagent is 7.5-8.5; The magnetic bead method microbial genome extraction kit is also provided with magnetic beads whose surfaces are modified with carboxyl and / or hydroxyl groups; a high-salt magnetic bead binding reagent and a low-salt magnetic bead elution reagent; the high-salt magnetic bead binding reagent is composed of 30-50mM 4-hydroxyethylpiperazineethanesulfonic acid, 1-4M sodium chloride and 5% polyethylene glycol 8000; the low-salt magnetic bead elution reagent is nuclease-free water.

[0019] It can be understood that the "high salt" and "low salt" described in the present invention are a set of relative concepts in the setting of this kit.

[0020] Furthermore, the concentration ratio of the lytic enzyme, lysozyme and lysostaphin is 40:100:1.

[0021] Furthermore, the concentration of the proteinase K is 20-50 mg / mL.

[0022] Furthermore, the magnetic beads include silica hydroxy magnetic beads with a particle size of 1 μm, silica carboxyl magnetic beads with a particle size of 300 nm and / or silica carboxyl magnetic beads with a particle size of 600 nm.

[0023] As a preference, the unit dosage of each set of cell wall breaking reagents provided in the magnetic bead method mixed microbial genome extraction kit is not less than 80 µL.

[0024] Preferably, the unit dosage of each set of wall-breaking enzyme reagents provided in the magnetic bead mixed microbial genome extraction kit is not less than 5 µL.

[0025] Preferably, the unit dosage of each group of protease reagents in the magnetic bead method mixed microbial genome extraction kit is not less than 5 µL, and the unit dosage of each group of lysis reagents is not less than 30 µL.

[0026] In some preferred embodiments, the magnetic bead method mixed microbial genome extraction kit is also provided with a reagent to assist magnetic bead sedimentation.

[0027] In some preferred embodiments, the enzyme activity ratio of the lysozyme, lysozyme and lysostaphin is 100:100:3 (20000:20000:1200).

[0028] In some preferred embodiments, the magnetic bead method mixed microbial genome extraction kit can be provided with multiple sets of reagents with the same functions.

[0029] Another aspect of the present invention provides a method for extracting mixed microbial genomes based on the above kit.

[0030] The method for extracting microbial genomes based on the above-mentioned magnetic bead method mixed microbial genome extraction kit comprises the following steps: S01: Collecting a sample containing pathogenic microorganisms, wherein the sample includes a body fluid sample or a urine sample; the microorganisms include one or more of fungi, Gram-positive bacteria and / or Gram-negative bacteria; S02: adding the cell wall breaking reagent and cell wall breaking enzyme combination reagent in the kit to the sample containing pathogenic microorganisms, respectively, and reacting them for a short time under the first temperature condition; S03: continue to add the protease reagent and the cleavage reagent in the kit to the sample containing pathogenic microorganisms, respectively, and react for a short time under a second temperature condition; the second temperature is higher than the first temperature and the second temperature inactivates the wall-breaking enzyme composition; S04: Continue to add the magnetic beads and magnetic bead binding reagent in the kit to the sample containing the pathogenic microorganisms after the reaction, vortex and mix, and adsorb and extract the nucleic acid of the pathogenic microorganisms; S05: Use the magnetic bead elution reagent in the kit to elute the magnetic beads adsorbed with nucleic acid to release the nucleic acid sample.

[0031] Furthermore, the body fluid sample includes a blood sample.

[0032] Furthermore, the first temperature condition is 30-40°C; and the second temperature condition is 70-80°C.

[0033] Furthermore, the short-time reaction means that the reaction time does not exceed 30 minutes.

[0034] Beneficial technical effects: The present invention first provides a mixed microbial gene extraction reagent combination for magnetic bead method, wherein the reagent combination contains a plurality of functional reagents, and the functional reagents cooperate with each other to achieve efficient and accurate extraction of samples containing unknown mixed microorganisms without the need for particularly complex magnetic bead modification. Therefore, the reagent combination provided by the present invention has good universality, is applicable to a large number of magnetic bead types, and has good industrialization prospects.

[0035] Secondly, the present invention provides a microbial genome extraction kit based on the above reagent combination, which can realize rapid genome (DNA+RNA) extraction of samples containing multiple unknown microorganisms without distinguishing the types of microorganisms. Specifically, the kit can perform one-time genome extraction of samples containing fungi (Candida albicans), Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium), and Gram-negative bacteria (Escherichia coli).

[0036] The kit provided by the present invention can achieve sufficient release of nucleic acids even under mild reaction conditions through the optimized configuration and coordinated cooperation of various functional reagents. For example, the present invention provides a composite enzyme composition containing low enzyme activity and high concentration of lysostaphin to achieve mild but sufficient cell wall breaking release of microbial nucleic acids. Since the overall reaction conditions of the kit of the present invention are mild when used, the nucleic acid structure will not be destroyed, so the total amount of genes finally extracted is large and of high quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0038] Figure 1 The figure is a graph of agarose gel electrophoresis results of extracting genome in one of the embodiments; Figure 2 This is a graph showing the agarose gel electrophoresis result of extracting genomic PCR products in one of the embodiments; Figure 3 This is a schematic diagram of a mixed microbial genome extraction kit using a magnetic bead method according to the present invention; Figure 4 The present invention is a schematic diagram of the process of extracting samples using the magnetic bead method mixed microbial genome extraction kit of the present invention.

[0039] Summary of reference numerals: mixed microbial genome extraction kit 10, biological wall breaking unit 20, biological lysis unit 30, others 40, wall breaking reagent 21, wall breaking enzyme composition reagent 22, protease reagent 31, lysis reagent 32, magnetic beads 41, magnetic bead binding reagent 42, magnetic bead elution reagent 43. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0042] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0043] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0044] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0045] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0046] Definition of noun: The "genome performance" described in the present invention includes genome concentration and genome quality; it should be noted that the kit of the present invention involves the co-extraction of DNA and RNA. The genome quality refers to A 260 / A 280 The detection value of .

[0047] A 260 / A 280 Judgment index reference: A 260 / A 280 A ratio range of around 2.0 indicates good genome quality; a ratio greater than 2.2 indicates partial RNA degradation; a ratio less than 1.8 indicates contamination by proteins (aromatic) or phenolic substances and poor genome quality.

[0048] Example 1 This embodiment provides an example of a mixed microbial genome extraction kit using a magnetic bead method (referred to as a mixed microbial genome extraction kit). The schematic diagram of the mixed microbial genome extraction kit using a magnetic bead method is shown in Figure 3 .

[0049] The mixed microbial genome extraction kit 10 provided in this embodiment includes a biological wall breaking unit 20, a biological lysis unit 30 and others 40; specifically, the biological wall breaking unit 20 includes a wall breaking reagent 21 and a wall breaking enzyme composition reagent 22; the biological lysis unit 30 includes a protease reagent 31 and a lysis reagent 32; the others 40 may include magnetic beads 41, magnetic bead binding reagents 42 and magnetic bead elution reagents 43.

[0050] Others 40 may also include nucleic acid precipitation aids, washing solutions, etc., which are described in detail below.

[0051] (1) The cell wall breaking reagent includes 5 mM dithiothreitol (DTT), 20 mM Tris-HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.1% Triton X-100. The unit dosage of the cell wall breaking reagent is set to 80 µL.

[0052] (2) The wall-breaking enzyme combination reagent includes lytic enzyme, lysozyme and lysostaphin; wherein the lytic enzyme has a specific activity ≥ 20,000 U / mg and a concentration of 20 mg / mL; the lysozyme has a specific activity ≥ 20,000 U / mg and a concentration of 50 mg / mL; the lysostaphin has a specific activity ≥ 1,200 U / mg and a concentration of 0.5 mg / mL; and the unit dosage of the wall-breaking enzyme combination reagent is set to 5 µL.

[0053] (3) The proteinase K specific activity is ≥30 units / mg protein, and the concentration is 30 mg / mL; the unit dosage is set to 5 µL.

[0054] (4) The lysis reagent comprises 400 mM Tris-HCl (pH 8.0), 40 mM ethylenediaminetetraacetic acid (EDTA), 5% Triton X-100, 800 mM sodium chloride (NaCl), 0.1 mM sodium methyl paraben and 5% Tween 20; the unit dosage of the lysis reagent is set to 30 µL.

[0055] (5) The nucleic acid precipitation aid is anhydrous ethanol; the volume ratio of anhydrous ethanol to sample is set to 1.5:1, and the dosage is 180 µL.

[0056] (6) The magnetic bead binding reagent includes 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 4 M sodium chloride and 5% polyethylene glycol PEG8000; the volume ratio of the magnetic bead binding reagent to the sample is set to 1:5, and the amount used is 75 µL.

[0057] (7) The washing solution is composed of 80% ethanol; the unit dosage is set to 500 µL. The washing solution is used to clean impurities on the magnetic beads.

[0058] (8) The magnetic bead elution reagent comprises nuclease-free water.

[0059] Furthermore, this embodiment also discloses a method for preparing each reagent in the above-mentioned microbial genome extraction kit.

[0060] (1) Preparation of the cell wall breaking reagent: Take 10 mg Triton X-100 in a 15 mL centrifuge tube, then add 200 µL 1 M Tris-HCl (pH 8.0), 40 µL 0.5 M EDTA, and add sterile water to make up to 10 mL.

[0061] (2-1) Preparation of the lytic enzyme: 40 mg / mL of lytic enzyme was prepared using 1 / 15 phosphate buffer, and then an equal volume of 100% glycerol was added to prepare a 20 mg / mL lytic enzyme storage solution, which was stored at -20°C.

[0062] (2-2) Preparation of lysozyme: 100 mg / mL lysozyme was prepared using 10 mM Tris-HCl (pH 8.0), and then an equal volume of 100% glycerol was added to prepare a 50 mg / mL lysozyme storage solution, which was stored at -20°C.

[0063] (2-3) Preparation of the lysostaphin: 6000 U / mL of lysostaphin was prepared using 50 mM Tris-HCl (pH 8.0), and then an equal proportion of 100% glycerol was added to prepare a 3000 U / mL lysostaphin storage solution, which was stored at -20°C and diluted to 600 U / mL when used.

[0064] (3) Preparation of the proteinase K: Use 10 mM Tris-HCl (pH 8.0) to prepare 60 mg / mL of proteinase K, then add an equal volume of 100% glycerol to prepare a 30 mg / mL proteinase K storage solution, and store it at -20°C.

[0065] (4) Preparation of the lysis reagent: Take 400 mg Triton X-100, 400 mg Tween 20, and 468 mg NaCl in a 15 mL centrifuge tube, then add 4 mL 1 M Tris-HCl (pH8.0), 800 µL 0.5 M EDTA, and 10 µL 100 mM sodium methyl paraben, and add sterile water to make up to 10 mL.

[0066] (6) Preparation of the magnetic bead binding reagent: Take 500 mg PEG8000 in a 15 mL centrifuge tube, then add 8 mL 5 M NaCl, 500 µL 1 M HEPES, and add sterile water to make up to 10 mL.

[0067] Example 2 This embodiment provides a method for extracting microbial genomes based on the magnetic bead method mixed microbial genome extraction kit of embodiment 1. The extraction method flow chart is shown in Figure 4It should be noted that the method of this embodiment is mainly used for scientific research and clinical trials, rather than for diagnostic purposes. The specific steps are as follows.

[0068] S01, place the sample containing unknown pathogenic microorganisms (referred to as the test sample) in a centrifuge tube. The test sample generally contains 1×10 5 -1×10 8 The sample source includes a body fluid sample, a urine sample and / or a blood sample; the sample to be tested is centrifuged in a centrifuge at 12000 rpm for 2 minutes, the supernatant is discarded, and the precipitate is collected; the precipitate is the bacterial cell.

[0069] S02, add 80 µL of the cell wall breaking reagent in the microbial genome extraction kit to the bacteria, vortex mix, then add 5 µL of the cell wall breaking enzyme combination reagent, vortex mix, and incubate at 37°C and 400 rpm for 30 minutes to fully break the cell wall.

[0070] S03, continue to add 5 µL of proteinase K and 30 µL of lysis reagent to the reacted bacteria, and then heat at 70°C and 400 rpm for 20 minutes to fully lyse the bacteria.

[0071] S04, continue to add 20 µL magnetic beads, 180 µL nucleic acid precipitation aid and 75 µL magnetic bead binding reagent to the lysed bacteria and vortex to mix.

[0072] S05, continue to add 30-200 µL of magnetic bead elution reagent to the sample to be tested containing magnetic beads, vortex to mix, and let stand for 5 minutes to obtain the gene to be tested.

[0073] Furthermore, the specific steps of S04-S05 include: 1) Add 20 µL of magnetic beads to the sample to be tested after S04 treatment, vortex to mix, and let stand at room temperature for 5 minutes. The types of magnetic beads available include silica hydroxy magnetic beads with a particle size of 1 µm; silica carboxyl magnetic beads with a particle size of 300 nm; silica carboxyl magnetic beads with a particle size of 600 nm, and silica hydroxy magnetic beads with a particle size of 500 nm.

[0074] 2) Place the sample to be tested after adding the magnetic beads on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant.

[0075] 3) Add 200 µL of washing solution to the sample containing the magnetic beads, rotate and wash on a magnetic rack, let it stand, and aspirate the supernatant as much as possible.

[0076] 4) Repeat the previous step.

[0077] 5) Remove the sample from the magnetic rack and let it stand for 5-10 minutes to allow the residual liquid to evaporate completely.

[0078] 6) Continue to add 30-200 µL of magnetic bead elution reagent to the sample containing magnetic beads, vortex to mix, and let stand for 5 minutes.

[0079] 7) Place the sample to be tested containing the magnetic beads on a magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant to obtain the purified genome.

[0080] The adsorption of nucleic acids by magnetic beads requires certain conditions, such as a high-salt, low-pH solution, which is then replaced with a low-salt solution to elute and release the nucleic acids from the magnetic beads.

[0081] Example 3 This example compares the performance of genomes extracted by different kits. Samples to be extracted: (i) Candida albicans sample; (ii) Escherichia coli sample; (iii) Staphylococcus aureus sample; (iv) a mixed bacterial solution sample of Candida albicans, Escherichia coli, Staphylococcus aureus, and Enterococcus faecium.

[0082] Extraction reagents: the mixed microbial genome extraction kit provided in Example 1, the Tiangen bacterial genome DNA extraction kit (control 1), and the Takara high-efficiency extraction and refined yeast genome DNA kit (control 2).

[0083] (I) The steps for extraction using different kits are as follows.

[0084] 1. Extract using the magnetic bead mixed microbial genome extraction kit provided in Example 1.

[0085] 1) Take 1 mL of a Candida albicans sample with a concentration of 2 OD / mL as test sample 1; 1 mL of a Escherichia coli sample with a concentration of 1 OD / mL as test sample 2; 1 mL of a Staphylococcus aureus sample with a concentration of 1 OD / mL as test sample 3; 1 mL of a mixed bacterial solution containing 0.5 OD / mL Candida albicans, 0.25 OD / mL Escherichia coli, 0.25 OD / mL Staphylococcus aureus and 0.25 OD / mL Enterococcus faecium as test sample 4.

[0086] 2) Treat the above sample using the method of Example 2 2. Extract using the Tiangen Bacteria Genomic DNA Extraction Kit (Control 1).

[0087] (1) Extraction of Escherichia coli genome 1) Take 1 mL of Escherichia coli sample with a concentration of 1 OD / mL as test sample 5.

[0088] 2) Centrifuge at 10,000 rpm for 1 minute, and remove the supernatant as much as possible; add 200 µL buffer GA to the bacteria, vortex to mix until the bacteria are completely suspended; add 20 µL proteinase K solution, vortex to mix; add 220 µL buffer GB, shake for 15 seconds, and react at 70°C for 10 minutes; add 220 µL anhydrous ethanol, vortex to mix for 15 seconds, and centrifuge instantly; add all the solutions to an adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube; add 500 µL buffer GD to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube; add 600 µL buffer PW to the adsorption column, centrifuge at 12,000 Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube; repeat the previous step; centrifuge at 12000 rpm for 2 minutes, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the rinsing liquid remaining in the adsorption material; transfer the adsorption column to a new 1.5 mL centrifuge tube, add 80 µL of enzyme-free water to the middle position of the adsorption membrane, let it stand at room temperature for 5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

[0089] (2) Extraction of Staphylococcus aureus genome 1) Take 1 mL of Staphylococcus aureus sample with a concentration of 1 OD / mL as test sample 6.

[0090] 2) Centrifuge at 10,000 rpm for 1 minute and aspirate the supernatant as much as possible; add 110 µL G +Buffer (20 mMTris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton) and 70 µL lysozyme solution, treated at 37℃ for 30 min; added 20 µL proteinase K solution and vortexed; added 220 µL buffer GB, oscillated for 15 s, reacted at 70℃ for 10 min; added 220 µL anhydrous ethanol, vortexed for 15 s, and centrifuged instantly; added all the solutions to an adsorption column CB3, centrifuged at 12000 rpm for 30 s, discarded the waste liquid, and placed the adsorption column CB3 in a collection tube; added 500 µL buffer GD to the adsorption column, centrifuged at 12000 rpm for 30 s, discarded the waste liquid, and placed the adsorption column CB3 in a collection tube; added 600 µL buffer PW to the adsorption column, centrifuged at 12000 rpm for 30 s, discarded the waste liquid, and placed the adsorption column CB3 in a collection tube; repeated the previous step; 12000 rpm for 2 minutes, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinsing liquid in the adsorption material; transfer the adsorption column to a new 1.5 mL centrifuge tube, add 80 µL of elution reagent to the middle position of the adsorption membrane in mid-air, let it stand at room temperature for 5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

[0091] 3. Extract using Takara high-efficiency extraction and purification yeast genomic DNA kit (control 2).

[0092] 1) Take 1 mL of Candida albicans sample with a concentration of 1.5 OD / mL as test sample 7.

[0093] 2) Centrifuge at 12,000 rpm for 1 minute; Remove the supernatant with a micropipette (try to remove as much liquid as possible); Add 500 µL of GenTLE Yeast Solution A to the precipitate, vortex gently to fully suspend the precipitate, and incubate at 37°C for 1 hour, gently vortexing the centrifuge tube several times; Add 100 µL of GenTLE Yeast Solution B, vortex gently to mix, and heat at 70°C for 10 minutes; Add 200 µL of GenTLE Yeast Solution C, vortex gently to mix, and cool on ice for 5 minutes; Centrifuge at 12,000 rpm, 4°C for 5 minutes; Carefully transfer the supernatant to a new microtube, add 400 µL of isopropanol, and invert the centrifuge tube to mix thoroughly; Centrifuge at 12,000 rpm, 4°C for 5 minutes, and discard the supernatant; Add 500 µL of pre-cooled 70% ethanol to the precipitate, gently invert the precipitate to wash, and incubate at 12,000 rpm for 5 minutes. rpm, centrifuge at 4℃ for 5 minutes; discard the supernatant with a micropipette, and dry the DNA precipitate naturally at room temperature until there is no ethanol smell; add 80 μL of elution reagent to dissolve the genome.

[0094] (B) Experimental results of genome extraction using different kits.

[0095] (1) Genome concentration detection: Take 1 µL of the genome of different microbial samples extracted by different kits, add 199 µL Qubit working solution, vortex mix, centrifuge briefly, and use Qubit4.0 to detect the genome concentration and total genome amount. The results are shown in Tables 1 and 2.

[0096] (2) Genome quality detection: 2 μL of the genome extracted from each of the test samples 1-7 was used for genome quality detection using Nanodrop. The results are shown in Tables 1 and 2.

[0097] Table 1 Microbiological sample detection grouping Table 2 Genome detection results The above results show that, under the same bacterial amount, the genome concentration of the microbiome extracted by the kit of Example 1 (i.e., the kit of the present invention) is higher, the total amount is larger, and the genome quality is better (A 260 / A 280 The value of is basically around 2). In addition, only the kit of Example 1 can extract the unknown mixed bacterial solution sample. Further, the extracted genome of the above sample was verified by agarose gel electrophoresis: 100 ng of the genome was taken and electrophoresed on 1% agarose gel at 140 V for 25 minutes. The results are as follows Figure 1 shown.

[0098] Lane 1 shows the Candida albicans genome extracted by this kit; Lane 2 shows the Escherichia coli genome extracted by this kit; Lane 3 shows the Staphylococcus aureus genome extracted by this kit; Lane 4 shows the mixed bacteria genome extracted by this kit; Lane 5 shows the Candida albicans genome extracted by the Takara kit; Lane 6 shows the Escherichia coli genome extracted by the Tiangen kit; Lane 7 shows the Staphylococcus aureus genome extracted by the Tiangen kit.

[0099] The results showed that compared with the genome extracted by this kit, the genome extracted by the Tiangen kit showed more obvious diffusion phenomenon in agarose gel electrophoresis, that is, the degree of degradation was higher, which also proved that the genome extracted by this kit was of better quality than the genome extracted by the Tiangen kit.

[0100] (C) PCR verification of genomes extracted using different kits.

[0101] 1) Primer design: Use the NCBI database to search for the 16S region sequences of Enterococcus faecium and Staphylococcus aureus and design primers; cite the Escherichia coli primers in patent CN110499378A (a method for detecting Escherichia coli pathogens from clinical blood); and the Candida albicans primers in CN102417931A (a Candida albicans PCR fluorescence detection kit and detection method thereof). The primer sequences are shown in Table 3.

[0102] Table 3 PCR validation primers 2) PCR reaction: The extracted genome was verified according to the following system, wherein each bacteria was subjected to PCR using its corresponding primers, and the mixed bacteria were subjected to PCR using four primers: Candida albicans primers, Staphylococcus aureus primers, Enterococcus faecium primers, and Escherichia coli primers.

[0103] Table 4 PCR reaction system Table 5 PCR reaction procedure 3) Agarose gel electrophoresis verification: Take 10 µL of PCR product and run electrophoresis on 3.5% agarose gel at 140 V for 30 minutes. Figure 2 shown.

[0104] Lane 1 is the PCR product of Escherichia coli genome extracted by this kit; Lane 2 is the PCR product of Candida albicans genome extracted by this kit; Lane 3 is the PCR product of Enterococcus faecium genome extracted by this kit; Lane 4 is the PCR product of Staphylococcus aureus genome extracted by this kit; Lane 5 is the PCR product of Escherichia coli primers of mixed bacteria genome extracted by this kit; Lane 6 is the PCR product of Candida albicans primers of mixed bacteria genome extracted by this kit; Lane 7 is the PCR product of Enterococcus faecium primers of mixed bacteria genome extracted by this kit; Lane 8 is the PCR product of Staphylococcus aureus primers of mixed bacteria genome extracted by this kit; Lane 9 is the PCR product of Escherichia coli genome extracted by Tiangen kit; Lane 10 is the PCR product of Candida albicans genome extracted by Takara kit; Lane 11 is the PCR product of Staphylococcus aureus genome extracted by Tiangen kit.

[0105] The results showed that the kit of the present invention can normally extract single bacteria and mixed bacteria. There are single bright bands at the corresponding primer fragments, and the mixed bacteria genome uses primers of four bacteria for PCR. There are single bright bands at the corresponding primer fragment size, which proves that the kit of the present invention can perform one-time genome extraction for samples containing fungi (Candida albicans), Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium), and Gram-negative bacteria (Escherichia coli).

[0106] Example 4 This example compares the performance of extracting genomes based on the magnetic bead mixed microbial genome extraction kit provided in Example 1.

[0107] (A) Genome extraction.

[0108] 1. Extract the genome using the mixed microbial genome extraction kit provided in Example 1.

[0109] 1) Take 1 mL of Candida albicans with a concentration of 2 OD / mL as sample A; take 1 mL of Staphylococcus aureus with a concentration of 1 OD / mL as sample B.

[0110] 2) The above sample was treated with the method of Example 2 to obtain a purified genome.

[0111] 2. The cell wall breaking methods were changed to Tiangen and Takara kits respectively, and the genome was extracted using other reagents of the mixed microbial genome extraction kit provided in Example 1.

[0112] 1) Take 1 mL of Candida albicans with a concentration of 2 OD / mL as sample C; take 1 mL of Staphylococcus aureus with a concentration of 1 OD / mL as sample D.

[0113] 2) Centrifuge the microbial sample at 12,000 rpm for 2 minutes, discard the supernatant, and collect the precipitate. Add 85 µL GenTLE Yeast Solution A to the Candida albicans cells, vortex to mix, and incubate at 37°C, 400 rpm for 60 minutes; add 52 µL G + Buffer (20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton) and 70 µL lysozyme were vortexed and incubated at 37°C, 400 rpm for 30 min.

[0114] 3) Add 5 µL proteinase K and 30 µL cell lysis reagent to the cell wall-broken sample, and then heat at 70°C and 400 rpm for 20 minutes; add 180 µL nucleic acid precipitation agent and 75 µL magnetic bead binding reagent to the lysed sample, vortex to mix; add 20 µL magnetic beads, vortex to mix, and let stand at room temperature for 5 minutes; place the sample on a magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; add 200 µL washing solution to the sample, rotate and wash on the magnetic rack, let stand for 1 minute, and aspirate the supernatant as much as possible; repeat the previous step; remove the sample, let stand for 5-10 minutes, and wait for the residual liquid to evaporate completely; add 80 µL magnetic bead elution reagent to the sample, vortex to mix, and let stand for 5 minutes; place the sample on a magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant to obtain the purified genome.

[0115] 3. The lysis methods of the Tiangen and Takara kits were changed respectively, and the genome was extracted using other reagents of the mixed microbial genome extraction kit provided in Example 1.

[0116] 1) Take 1 mL of Candida albicans with a concentration of 2 OD / mL as sample E; take 1 mL of Staphylococcus aureus with a concentration of 1 OD / mL as sample F.

[0117] 2) Centrifuge the microbial sample at 12,000 rpm for 2 minutes, discard the supernatant, and collect the precipitate; add 80 µL of cell wall breaking reagent to the bacteria, vortex to mix, then add 5 µL of cell wall breaking enzyme combination reagent, vortex to mix, and incubate at 37°C and 400 rpm for 30 minutes.

[0118] 3) Add 35 µL GenTLE Yeast Solution B to the disrupted sample E, add 5 µL proteinase K solution and 30 µL buffer GB to the disrupted sample F, vortex to mix, and then heat at 70℃, 400 rpm for 20 minutes; add 180 µL nucleic acid precipitation agent and 75 µL magnetic bead binding reagent to the lysed sample, vortex to mix; add 20 µL magnetic beads, vortex to mix, and let stand at room temperature for 5 minutes; place the sample on a magnetic rack, wait for the magnetic beads to completely aggregate, and then aspirate the supernatant; add 200 µL washing solution to the sample, rotate and wash on the magnetic rack, let stand for 1 minute, and aspirate the supernatant as much as possible; repeat the previous step; remove the sample, let stand for 5-10 minutes, and wait for the residual liquid to completely evaporate; add 80 µL magnetic bead elution reagent to the sample, vortex to mix, and let stand for 5 minutes; place the sample on a magnetic rack, wait for the magnetic beads to completely aggregate, and then aspirate the supernatant to obtain the purified genome.

[0119] Table 6 Reagent components and contents of different extraction methods used in this kit to extract genome (ii) Genome concentration and quality testing.

[0120] (1) Genome concentration detection: Take 1 µL of the extracted genome, add 199 µL of Qubit working solution, vortex to mix, centrifuge briefly, and then use Qubit4.0 to detect the genome concentration. The results are shown in Table 7.

[0121] (2) Genome quality detection: Take 2 µL of genome and use Nanodrop to perform genome quality detection. The results are shown in Table 7.

[0122] Table 7 Kit performance test results The above results show that the genome extraction efficiency is significantly reduced after the cell wall breaking method of the kit in Example 1 is replaced with the Tiangen and Takara cell wall breaking methods, which is manifested in a smaller total amount of extracted genomes and a worse quality of the extracted genomes.

[0123] After the lysis method of the kit in Example 1 was replaced with the Tiangen and Takara lysis methods, the genome extraction efficiency was also significantly reduced, which was manifested in a smaller total amount of extracted genomes and a worse quality of the extracted genomes.

[0124] Example 5 This example provides a verification of the cell wall breaking effect of a lysozyme composition.

[0125] The main component of bacterial cell wall is peptidoglycan, also known as mucopetide.

[0126] According to the different structures and chemical compositions of bacterial cell walls, they can be divided into Gram-positive bacteria (Gram-positive bacteria) and Gram-negative bacteria (Gram-negative bacteria). Gram-positive bacteria have thicker cell walls (20-80 nm), but their chemical composition is relatively simple, containing only 90% peptidoglycan and 10% teichoic acid; Gram-positive bacteria have thinner cell walls (10-15 nm), but have multi-layer structures (peptidoglycan and lipopolysaccharide layers, etc.), and their chemical composition contains not only peptidoglycan, but also certain amounts of lipids and proteins. Yeast cell walls are rich in a variety of bioactive substances such as β-glucan and mannan (MOS).

[0127] It is difficult for a single lysozyme to break the cell wall of a mixed microbial sample. Composite enzymes can achieve this, but the combination is diverse and the cost is high.

[0128] Lysostaphin is a Zn-containing 2+The metalloproteinase has endopeptidase activity and can specifically hydrolyze the five glycine peptide bond bridges (peptide bonds formed by the 2nd and 3rd Gly) of bacterial cell wall peptidoglycan, thereby quickly dissolving the bacterial cell wall and producing a wall-breaking and bacteriolytic effect. The present invention achieves gentle treatment of multi-microorganism mixed samples by adding a suitable proportion of lysostaphin.

[0129] Table 8 Verification of gene quantity extracted after lysis by different complex enzymes The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A mixed microbial gene extraction reagent combination for magnetic bead method, characterized in that: The reagent combination includes a wall-breaking reagent, a wall-breaking enzyme reagent, a protease reagent and a cleavage reagent; the volume ratio of the wall-breaking reagent, the wall-breaking enzyme reagent, the protease reagent and the cleavage reagent is 16:1:1:6; The wall-breaking reagent is composed of 5-10 mM dithiothreitol, 10-20 mM tris(hydroxymethyl)aminomethane hydrochloride, 1-5 mM ethylenediaminetetraacetic acid and 0.1%-0.5% polyethylene glycol octylphenyl ether; the pH value of the wall-breaking reagent is 7.5-8.5; The wall-breaking enzyme reagent is a wall-breaking enzyme composition, which consists of lytic enzyme, lysozyme and lysostaphin; the specific activity of the lytic enzyme is ≥20000U / mg, the specific activity of the lysozyme is ≥20000U / mg, and the specific activity of the lysostaphin is ≥1200U / mg; The protease reagent is proteinase K, and the specific activity is ≥30 units / mg; The lysis reagent consists of 200-400 mM tris(hydroxymethyl)aminomethane hydrochloride, 20-50 mM ethylenediaminetetraacetic acid, 1%-5% polyethylene glycol octylphenyl ether, 500-1000 mM sodium chloride, 0.1-0.5 mM sodium methyl paraben and 1%-5% Tween; the pH value of the lysis reagent is 7.5-8.

5.

2. The mixed microbial gene extraction reagent combination according to claim 1, characterized in that: The microorganisms include one or more selected from fungi, Gram-positive bacteria and / or Gram-negative bacteria.

3. The mixed microbial gene extraction reagent combination according to claim 1, characterized in that: The wall-breaking reagent consists of 5 mM dithiothreitol, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 2 mM ethylenediaminetetraacetic acid and 0.1% polyethylene glycol octylphenyl ether; the pH value of the wall-breaking reagent is 8.

4. The mixed microbial gene extraction reagent combination according to claim 1, characterized in that: The lysis reagent consists of 400 mM tris(hydroxymethyl)aminomethane hydrochloride, 40 mM ethylenediaminetetraacetic acid, 5% polyethylene glycol octylphenyl ether, 800 mM sodium chloride, 0.1 mM sodium methyl p-hydroxybenzoate and 5% Tween; the pH value of the lysis reagent is 8.

5. The mixed microbial gene extraction reagent combination according to claim 1, characterized in that: The reagent combination also includes a high-salt magnetic bead binding reagent for magnetic bead binding and a low-salt magnetic bead elution reagent for magnetic bead elution; the high-salt magnetic bead binding reagent is composed of 30-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 1-4 M sodium chloride and 5% polyethylene glycol 8000; the low-salt magnetic bead elution reagent is nuclease-free water.

6. Use of the mixed microbial gene extraction reagent combination according to any one of claims 1 to 5 in the preparation of a magnetic bead method microbial genome extraction kit.

7. A magnetic bead method microbial genome extraction kit, characterized in that: It includes a microbial wall breaking unit and a microbial lysis unit; the microbial wall breaking unit includes a wall breaking reagent and a wall breaking enzyme composition reagent; the microbial lysis unit includes a protease reagent and a lysis reagent; wherein, The wall-breaking reagent is composed of 5-10 mM dithiothreitol, 10-20 mM tris(hydroxymethyl)aminomethane hydrochloride, 1-5 mM ethylenediaminetetraacetic acid and 0.1%-0.5% polyethylene glycol octylphenyl ether; the pH value of the wall-breaking reagent is 7.5-8.5; The wall-breaking enzyme composition reagent is composed of lytic enzyme, lysozyme and lysostaphin; the specific activity of the lytic enzyme is ≥20000U / mg, the specific activity of the lysozyme is ≥20000U / mg, and the specific activity of the lysostaphin is ≥1200U / mg; The protease reagent is set to proteinase K, and the specific activity is ≥30 units / mg; The lysis reagent is composed of 200-400 mM tris(hydroxymethyl)aminomethane hydrochloride, 20-50 mM ethylenediaminetetraacetic acid, 1%-5% polyethylene glycol octylphenyl ether, 500-1000 mM sodium chloride, 0.1-0.5 mM sodium methyl paraben and 1%-5% Tween; the pH value of the lysis reagent is 7.5-8.5; The magnetic bead method microbial genome extraction kit is also provided with magnetic beads whose surfaces are modified with carboxyl and / or hydroxyl groups; a high-salt magnetic bead binding reagent and a low-salt magnetic bead elution reagent; the high-salt magnetic bead binding reagent is composed of 30-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 1-4 M sodium chloride and 5% polyethylene glycol 8000; the low-salt magnetic bead elution reagent is nuclease-free water.

8. The magnetic bead method microbial genome extraction kit as claimed in claim 7, characterized in that: The concentration ratio of the lytic enzyme, lysozyme and lysostaphin is 40:100:

1.

9. The magnetic bead method microbial genome extraction kit according to claim 7, characterized in that: The concentration of the proteinase K is 20-50 mg / mL.

10. The magnetic bead method microbial genome extraction kit according to claim 7, characterized in that: The magnetic beads include silica hydroxy magnetic beads with a particle size of 1 μm, silica carboxyl magnetic beads with a particle size of 300 nm and / or silica carboxyl magnetic beads with a particle size of 600 nm.

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