Method and kit for removing host nucleic acid from biological sample

Abstract

There is provided a method for removing host nucleic acid from a biological sample, the method comprising the steps of: a) contacting the sample with an excess amount of a saponin-containing formulation so as to sufficiently react the host in the sample with the saponin-containing formulation; b) adding at least one saponin inhibitor in order to inhibit the activity of excess saponin; and c) adding at least one nucleic acid digestive enzyme to allow removal of the host nucleic acid. Also provided are methods for detecting microorganisms in a biological sample, kits for host nucleic acid removal of a biological sample or kits for detecting microorganisms in a biological sample, and uses thereof. The provided method does not need to calculate or consider the concentration of host cells in a sample, can reduce the loss of microorganisms, and ensures the effect of removing the host genome.

Description

Method and kit for removing host nucleic acid from biological samples

[0001] Technology

[0002] The present invention relates to the field of biological detection, and specifically relates to a method and a kit for removing host nucleic acid from biological samples. Background Art

[0003] Pathogenic microorganisms refer to microorganisms that can invade hosts such as the human body and cause infection or even infectious diseases, or pathogens. Among pathogens, bacteria and viruses are the most harmful. Pathogenic microorganisms refer to prions, fungi, bacteria, spirochetes, mycoplasmas, rickettsiae, chlamydiae, viruses, and parasites (protozoa, worms, and medical insects). Infectious and infectious diseases caused by pathogenic microorganisms are one of the most common diseases faced in clinical practice, accounting for 50% of human diseases. When pathogenic microorganisms infect the human body, it is very likely to cause the infected person to develop corresponding infection symptoms. Moreover, with the changes in population structure, environmental pollution, drug abuse and other factors, as well as the high variability of the pathogenic microorganisms themselves, pathogenic microorganisms are showing a trend of diversification and complexity, seriously threatening the health of the infected person. In addition, infectious diseases caused by pathogenic microorganisms often develop rapidly and there are unknown pathogens, which brings great difficulties to diagnosis and detection.

[0004] Metagenomic sequencing (mNGS), as a new technology that does not require culture, can quickly and deeply identify infectious pathogens. Compared with traditional culture methods, it is more sensitive and consistent with the concept of "precision diagnosis and treatment". It has the advantages of short cycle, wide coverage, and simple operation for the detection of clinical microbial pathogens, providing valuable data for clinical diagnosis and treatment. At the same time, metagenomic sequencing also faces a huge challenge, that is, clinical samples contain a large amount of host nucleic acids such as DNA. In the context of high human DNA, it is very difficult to detect low-abundance pathogen sequences, not to mention more in-depth analysis such as typing of pathogenic microorganisms and drug-resistant genes through genome sequencing.

[0005] Therefore, in the process of detecting pathogenic microorganisms, it is necessary to remove host nucleic acids. In the prior art, saponin has been selected as a reagent for removing the host, and then nuclease is used to degrade and remove the nucleic acids released in the host cells. Saponin is composed of amphoteric hydrophilic sapogenin and one or more hydrophilic sugars, has a high degree of structural diversity, and its physiological and pharmacological activities include antibacterial, anticancer, immunomodulatory, etc. However, this method requires an appropriate amount of saponin, otherwise it will affect the effect of removing the host nucleic acids, thereby affecting the efficiency of pathogen detection. Since the prior art does not disclose the reasons for this phenomenon, there is no corresponding solution.

[0006] Summary of the Invention

[0007] The inventors have found that when an excess of saponin is used to ensure sufficient destruction of the host cells and then nuclease is added to digest the nucleic acid, the activity of the nuclease will be affected, which reduces the effect of removing the host nucleic acid. In order to solve this problem, it is necessary to remove the inhibition of saponin on the nucleic acid digestion enzyme. The common methods currently used to solve this problem are to optimize the experimental conditions, use other types of nucleases, reduce the concentration of saponin, and find non-inhibitory compounds with similar structures to saponins. However, these methods have poor universality and are not suitable for all application scenarios. Therefore, in actual applications, they need to be adjusted and optimized according to specific circumstances. In response to the above problems, the present application provides a method for removing host nucleic acids from biological samples that can be widely applied to a variety of situations.

[0008] A first aspect of the present invention provides a method for removing host nucleic acid from a biological sample, the method comprising the following steps:

[0009] a) contacting the sample with an excess saponin-containing preparation so that the host in the sample fully reacts with the saponin-containing preparation;

[0010] b) adding at least one saponin inhibitor to inhibit the activity of excess saponin; and

[0011] c) adding at least one nucleic acid digesting enzyme to allow for the removal of host nucleic acids.

[0012] After the sample is contacted with an excess of saponin, the saponin ruptures the host cell membrane by lysing the host cell, thereby releasing the host nucleic acid, which includes mitochondrial nucleic acid and non-mitochondrial nucleic acid. The released host nucleic acid (especially non-mitochondrial nucleic acid) will be digested by the nucleic acid digestion enzyme and removed. Excess saponin can ensure the full lysis of the host cell. However, the inventors have found that excessive saponin inhibits the activity of the nucleic acid digestion enzyme, thereby affecting the removal of the host nucleic acid. Therefore, after adding a saponin inhibitor, the inhibition of the nucleic acid digestion enzyme by the excess saponin can be lifted, so that the nucleic acid digestion enzyme can function normally to remove the host nucleic acid. In this process, a variety of methods can be used to determine whether the host has fully reacted with the saponin-containing preparation, such as by observing whether the cell membrane of the host cell is completely ruptured, or by detecting whether the non-mitochondrial genes of the host cell are completely eliminated by qPCR.

[0013] In some embodiments, steps a), b), and c) are performed in a manner selected from the group consisting of: (1) steps a), b), and c) are performed simultaneously; (2) steps a) and b) are performed simultaneously, and then step c) is performed; (3) step a) is performed first, and then steps b) and c) are performed simultaneously; or (4) steps a), b), and c) are performed sequentially.

[0014] Here, "concurrently performed" means that multiple steps are performed simultaneously or within a short period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, or 1, 2, or 3 minutes). For example, steps a), b), and c), or a) and b), or b) and c) are performed simultaneously or within a short period of time. For example, "steps a), b), and c) are performed simultaneously" can mean that the sample is contacted with an excess saponin-containing preparation, at least one saponin inhibitor, and at least one nucleic acid-digesting enzyme simultaneously and within a short period of time, or that the excess saponin-containing preparation, at least one saponin inhibitor, and at least one nucleic acid-digesting enzyme are mixed with the sample simultaneously or within a short period of time.

[0015] In some embodiments, the saponin-containing formulation is a formulation containing saponin, such as a solution having a saponin concentration of 10% to 20%.

[0016] When executed simultaneously, the reaction operation is simpler, and the reaction system is in a dynamic equilibrium state, resulting in a better host removal effect.

[0017] “Performed sequentially” means performing step a) for a period of time (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 minutes) and then performing step b), and then performing step c) after a period of time (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 minutes).

[0018] In some embodiments, the sum of the reaction times of steps a), b), and c) is 5-60 min, 10-50 min, 10-40 min, 10-30 min, 15-50 min, 15-40 min, 15-30 min, or 20-30 min. In some embodiments, the sum of the reaction times of steps a), b), and c) is 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 min.

[0019] In some embodiments, the reaction time of step a) is 5-20 min, the reaction time of step b) is 5-20 min, and the reaction time of step c) is 10-30 min.

[0020] In some embodiments, no separation step is included before step c), preferably no centrifugation and / or washing steps. In the prior art, multiple centrifugation and washing steps are required to obtain a host-free sample. During the centrifugation and washing process, some supernatant must be removed, which inevitably results in the loss of some microorganisms, making it impossible to fully detect all microorganisms. Using a nuclease without separation can prevent the loss of microorganisms while ensuring the effectiveness of host genome removal.

[0021] In some embodiments, the at least one saponin inhibitor is an agent for inhibiting excess saponin from inhibiting the activity of nucleic acid digesting enzymes, and the saponin inhibitor is selected from a saponin neutralizer or a saponin adsorbent.

[0022] In some embodiments, the saponin neutralizer is a preparation capable of reacting with saponin. Preferably, the saponin neutralizer is selected from one or more of cholesterol, liposomes containing cholesterol, cholesterol red blood cells or their analogs. More preferably, the saponin neutralizer is selected from one or more of water-insoluble cholesterol, water-soluble cholesterol or liposomes containing cholesterol. Most preferably, the saponin neutralizer is selected from water-soluble cholesterol and / or liposomes containing cholesterol.

[0023] In some embodiments, the saponin adsorbent is a preparation capable of adsorbing saponins. Preferably, the saponin adsorbent is a preparation capable of adsorbing saponins; preferably, the saponin adsorbent is selected from one or more of reversed-phase chromatography fillers, ion exchange chromatography fillers, protein adsorbents, sugar adsorbents, polydopamine, or adsorbents based on molecular imprinting technology. More preferably, the reversed-phase chromatography filler is selected from octylsilane bonded silica gel (ODS) or phenylsilane bonded silica gel; the ion exchange chromatography filler is selected from sulfonic acid type cation exchange resin and quaternary ammonium type anion exchange resin; the protein adsorbent is selected from albumin or serum protein; and the sugar adsorbent is selected from dextran or cellulose.

[0024] The addition of saponin inhibitors can remove or inhibit saponins through chemical reactions or adsorption, thereby preventing the inhibition of nucleic acid-digesting enzymes. Saponin inhibitors, such as cholesterol, act as saponin substrates and react with excess saponins to form insoluble molecular complexes, thereby removing excess saponins. Cholesterol, as a component of cell membranes, does not adversely affect the reaction system.

[0025] In some embodiments, the at least one nucleic acid digesting enzyme is a nuclease having DNase and / or RNase activity; preferably, the at least one nucleic acid digesting enzyme is selected from one or more of Nuclease M-SAN, Nuclease HL-SAN, Serratia nuclease Benzonase, Universal Nuclease, Super Nuclease, and High Salt-Tolerant Enzyme. In some embodiments, the nucleic acid digesting enzyme is used in an amount of 1 U / ml to 5000 U / ml, preferably 250 U / ml to 2500 U / ml.

[0026] In some embodiments, step a) is performed in the presence of a host removal reagent. Preferably, the host removal reagent comprises: 0-500mM NaCl, 4-15mM MgCl2, and 15-50mM Tris-HCl, pH 7-9 or 7.2-8.7. Alternatively, the host removal reagent comprises: NaCl: 0-500mM, 10-450mM, 50-300mM, 100-250mM, 125-200mM, or 130-160mM; MgCl2: 4-15mM or 8-12mM; Tris-HCl: 15-50mM or 20-30mM, and the pH of the nuclease buffer is 7-9 or 7.2-8.7. The inclusion or non-inclusion of the host removal reagent in the reaction system, or the selection of the components of the host removal reagent, can be determined based on the type of nucleic acid digestion enzyme.

[0027] In some embodiments, in the reaction system for removing host nucleic acids, the mass concentration of the saponin is 0.01%-5%, preferably 0.02%-2.5%, more preferably 0.025%-1.25%. In some embodiments, in the reaction system for removing host nucleic acids, the mass concentration of the saponin is 0.2%-2.5% or 0.25%-2.5%. In some embodiments, in the reaction system for removing host nucleic acids, the mass concentration of the saponin is 0.01%, 0.02%, 0.025%, 0.1%, 0.2%, 0.25%, 0.3125%, 0.5%, 0.625%, 1%, 1.25%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0028] In some embodiments, the saponin is a Quillaja saponin, and the saponin contains a saponin active ingredient, such as saponin. Preferably, in the reaction system for removing host nucleic acid, the concentration of the saponin active ingredient is 0.002-1.25%, preferably 0.004%-0.63%, and more preferably 0.005%-0.32%.

[0029] In some embodiments, in the reaction system for removing host nucleic acid, the concentration of cholesterol is no greater than 1 mg / mL, preferably 0.02-0.5 mg / mL, more preferably 0.04-0.4 mg / mL, and most preferably 0.1-0.2 mg / mL.

[0030] In some embodiments, the concentration of host cells in the sample is 10 3 -10 11 cells / mL, more preferably 10 3 -10 8 cells / mL, and the most preferred is 10 4 -10 7cells / mL.

[0031] In this reaction system, the concentration of host cells, saponins, and saponin inhibitors do not affect the activity of nucleic acid digestion enzymes. Therefore, there is no need to calculate or consider the concentration of host cells in the sample, which can reduce microbial loss and ensure the effectiveness of host genome removal.

[0032] In some embodiments, the host cell can be a mammalian cell, for example, a human cell, a dog cell, a monkey cell, a mouse cell, or a cat cell, preferably a human cell.

[0033] In some embodiments, the sample is selected from one or more of blood, sputum, bronchoalveolar lavage fluid, cerebrospinal fluid, tissue, and pleural and ascites fluid.

[0034] In some embodiments, a method for removing host nucleic acid from a biological sample comprises the following steps:

[0035] a) reacting a sample, an optional host-removing reaction reagent, and an excess saponin-containing preparation for 5-20 minutes, so that the host in the sample fully reacts with the saponin-containing preparation;

[0036] b) adding cholesterol or liposomes and reacting for 5-20 minutes to neutralize excess saponin; and

[0037] c) Add nuclease and incubate for 10-30 minutes to allow removal of host nucleic acids.

[0038] In some embodiments, a method for removing host nucleic acid from a biological sample comprises the following steps:

[0039] Add the optional host-removal reagent, excess saponin-containing preparation, cholesterol or liposomes, and nuclease to the sample and incubate for 10-30 minutes.

[0040] In some embodiments, a method for removing host nucleic acid from a biological sample comprises the following steps:

[0041] The optional host-free reaction reagent, excess saponin-containing preparation, and cholesterol or liposomes are added to the sample at the same time and reacted for 5-20 minutes, and then nuclease is added and incubated for 10-30 minutes.

[0042] In some embodiments, a method for removing host nucleic acid from a biological sample comprises the following steps:

[0043] Add the optional host-free reaction reagent and excess saponin-containing preparation to the sample and react for 5-20 minutes, and then incubate with cholesterol or liposomes and nuclease for 10-30 minutes.

[0044] A second aspect of the present invention provides a method for detecting microorganisms in a biological sample, the method comprising:

[0045] Perform the method for removing host nucleic acid according to the first aspect of the present invention; and

[0046] Nucleic acids are extracted and metagenomic sequencing is performed on the nucleic acids to determine the detection results of the microorganisms.

[0047] In some embodiments, the sequencing includes PCR amplification-based next-generation sequencing, or nanopore-based single-molecule sequencing, optionally, nanopore-based single-molecule sequencing.

[0048] In some embodiments, the microorganisms include bacteria, fungi and / or viruses, and the viruses include DNA viruses and / or RNA viruses.

[0049] In some embodiments, the bacteria include at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella aerogenes, Klebsiella oxytoca, Streptococcus pneumoniae, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus capitis, and Streptococcus agalactiae.

[0050] In some embodiments, the fungus comprises at least one of Candida albicans, Cryptococcus gattii, Aspergillus nidulans, Pseudomonas glabrata / Candida glabrata, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Cryptococcus neoformans / Cryptococcus neoformans, and Aspergillus fumigatus.

[0051] In some embodiments, the DNA virus comprises at least one of a DNA phage, an Epstein-Barr virus, a mumps virus, and an adenovirus.

[0052] In some embodiments, the RNA virus comprises at least one of an RNA bacteriophage, influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, and coronavirus.

[0053] The method of the present application helps to fully lyse the isolated host cells and maintain the activity of microbial nucleic acids that may be present in the isolated host cell sample, thereby increasing the amount of microbial nucleic acids that can be effectively detected in the sample, thereby improving the detection rate and improving the detection sensitivity.

[0054] A third aspect of the present invention provides a kit for removing host nucleic acid from a biological sample or a kit for detecting microorganisms in a biological sample, the kit comprising

[0055] a. Preparations containing saponins,

[0056] b. at least one saponin inhibitor, the at least one saponin inhibitor is an agent used to inhibit the activity of nucleic acid digesting enzymes inhibited by excess saponin; preferably, the saponin inhibitor is selected from a saponin neutralizer or a saponin adsorbent, the saponin neutralizer is an agent capable of reacting with saponin, and the saponin adsorbent is an agent capable of adsorbing saponin; more preferably, the saponin neutralizer is selected from one or more of liposomes, cholesterol, red blood cells or their analogs, preferably, the at least one saponin neutralizer is selected from one or more of water-insoluble cholesterol, water-soluble cholesterol or liposomes, more preferably, the at least one saponin neutralizer is selected from water-soluble cholesterol and / or liposomes;

[0057] c. at least one nucleic acid digesting enzyme, wherein the at least one nucleic acid digesting enzyme is a nuclease having DNase and / or RNase activity; preferably, the at least one nucleic acid digesting enzyme is selected from one or more of nuclease M-SAN, nuclease HL-SAN, Serratia nuclease Benzonase, universal nuclease, super nuclease, and high salt tolerant enzyme;

[0058] d. Optionally, a description for use in the method according to the present invention, preferably, the description describes the method according to the first aspect or the second aspect of the present application; and

[0059] e. Optionally, a host-removal reaction reagent comprising: 0-500 mM NaCl, 4-15 mM MgCl2, and 15-50 mM Tris-HCl, pH 7-9. Optionally, the host-removal reaction reagent comprises: NaCl: 0-500 mM, 10-450 mM, 50-300 mM, 100-250 mM, 125-200 mM, or 130-160 mM; MgCl2: 4-15 mM or 8-12 mM; Tris-HCl: 15-50 mM or 20-30 mM, and the pH of the nuclease buffer is 7-9 or 7.2-8.7.

[0060] In some embodiments, the instructions describe the method for removing host nucleic acid from a biological sample according to the first aspect of the present invention or the method for detecting microorganisms in a biological sample according to the second aspect of the present invention.

[0061] Preferably, the description states:

[0062] The mass concentration of the saponin is 0.01%-5%, preferably 0.02%-2.5%, more preferably 0.025%-1.25%; further preferably, the saponin is Quillaja saponin, and the saponin contains a saponin active ingredient, such as saponin. Preferably, in the reaction system, the concentration of the saponin active ingredient is 0.002%-1.25%, preferably 0.004%-0.63%, more preferably 0.005%-0.32%. And / or

[0063] The concentration of cholesterol is no greater than 1 mg / mL, preferably 0.02-0.5 mg / mL, more preferably 0.04-0.4 mg / mL, most preferably 0.1-0.2 mg / mL; and / or

[0064] The amount of nucleic acid digestion enzyme used is 1U / ml-5000U / ml, preferably 250U / ml-2500U / ml;

[0065] The above mass concentrations and concentrations are calculated relative to the entire reaction system.

[0066] The fourth aspect of the present invention provides the use of the method according to the first or second aspect of the present invention or the kit according to the third aspect of the present invention in pathogenic microorganism metagenomic sequencing, microbial typing or drug resistance gene analysis, or in the preparation of products for pathogenic microorganism metagenomic sequencing, microbial typing or drug resistance gene analysis.

[0067] This application has at least the following beneficial effects:

[0068] The method of the present invention adds a saponin inhibitor that inhibits the activity of excessive saponin, thereby preventing the inhibitory effect of excessive saponin on nucleic acid digestion enzymes.

[0069] The method of the present invention uses nuclease without separation, which can reduce the loss of microorganisms and ensure the effect of removing host nucleic acid.

[0070] In the methods of the present invention, the concentration of host cells, saponins, and saponin inhibitors do not affect the activity of nucleic acid digesting enzymes. Therefore, there is no need to calculate or consider the concentration of host cells in the sample.

[0071] The method of the present invention is simple to operate and can be applied to various situations, such as various nucleic acid digestion enzymes, various biological samples, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

[0073] FIG1 shows the qPCR detection results of the experimental operation in Table 3, wherein MT represents the mitochondrial gene in human host cells, rpoA represents the non-mitochondrial gene in human host cells, and T1 represents the phage gene.

[0074] FIG2 shows the qPCR detection results of the experimental operation of Table 6, wherein rpoA represents a non-mitochondrial gene in human host cells.

[0075] Figure 3 shows the qPCR test results of the experimental operation in Table 8, where MT represents the mitochondrial gene in the human host cell, rpoA represents the non-mitochondrial gene in the human host cell, and KLE represents the gene of the target bacteria Klebsiella pneumoniae spiked into the simulated sample. The missing part has a CT value greater than 40, which means the test is negative.

[0076] Figure 4 shows the nucleic acid length distribution of the experimental operation in Table 8, where L represents marker, the black band at the position of the pink line is the upper internal standard (Upper Marker) with a length of 10 kb, and the black band at the position of the green line is the lower internal standard (Lower Marker) with a length of 15 bp, which is not the extracted nucleic acid; the black band at the position marked with a triangle is the extracted nucleic acid.

[0077] FIG5 shows the qPCR detection results of the experimental operation of Table 11, wherein rpoA represents a non-mitochondrial gene in human host cells.

[0078] FIG6 shows the qPCR detection results of the experimental operation of Table 12, wherein rpoA represents a non-mitochondrial gene in human host cells.

[0079] FIG7 shows the qPCR detection results of the experimental operation of Table 14, wherein rpoA represents a non-mitochondrial gene in human host cells. DETAILED DESCRIPTION

[0080] In order to make the application purpose, technical solution and beneficial technical effects of this application clearer, the application is further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are only for explaining this application and are not intended to limit this application.

[0081] For simplicity, this application only explicitly discloses certain numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and similarly, any upper limit can be combined with any other upper limit to form an unspecified range. In addition, although not explicitly stated, each point or individual value between the endpoints of a range is included in the range. Thus, each point or individual value can serve as its own lower limit or upper limit and be combined with any other point or individual value, or with other lower limits or upper limits, to form an unspecified range.

[0082] In the description of the present application, it should be noted that, unless otherwise specified, the “multiple” in “one or more” means two or more.

[0083] In the description of this application, the mass concentration of saponin refers to the mass-to-volume ratio (m / v).

[0084] The above disclosure of the present application is not intended to describe every disclosed embodiment or every implementation in the present application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided through a series of examples, which can be used in various combinations. In each example, the enumeration is intended only as a representative group and should not be construed as exhaustive.

[0085] Example

[0086] The present invention is further illustrated by way of examples, but the invention is not limited to the scope of the examples. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions, or selected according to the product specifications. The experimental reagents and instruments used in the following examples are all commercially available reagents or instruments, and the sequences used were synthesized by a biotechnology company.

[0087] The experimental procedures used in the following examples are as follows:

[0088] (1) Host neutralization experimental treatment

[0089] 1. Cholesterol-neutralizing saponins:

[0090] Take 300 μL of sample, 50 μL of 10× host-free reaction reagent (20 mM Tris-HCl, 6 mM MgCl2, pH 8.5), 100 μL of 10% saponin (abbreviated as SA, Sigma, product number S4521, from Quillaja saponaria bark, containing the active ingredient Sapogenin 20-25%), react at room temperature for 5 minutes, add 50 μL of 2 mg / mL cholesterol (Sigma, product number C3045) and react at room temperature for 5 minutes, 10 μL of Benzonase (Vazyme, RM1022-02), treat at 37°C 1000 rpm for 15 minutes, add 20 μL of 0.5 M EDTA, shake to mix, and incubate at room temperature for 5 minutes.

[0091] 2. Liposome neutralization of saponins:

[0092] Take 300 μL of sample, 50 μL of 10× host-free reaction reagent (20 mM Tris-HCl, PH8.5, 2 mM MgCl2, 20 mM NaCl), 100 μL of 5% saponin, mix well, add 50 μL of liposomes (Yisheng Bio, 40338ES08, the main components are phospholipids and cholesterol, the concentration of cholesterol is 2 mg / mL), react at room temperature for 15 minutes, 10 μL of Yisheng all-purpose enzyme (Yisheng Bio, 20157ES25), treat at 37°C 1000 rpm for 20 minutes, add 20 μL of 0.5 M EDTA, shake to mix, and incubate at room temperature for 5 minutes.

[0093] (2) Pathogen DNA / RNA co-extraction

[0094] 1. Add 40 μl Proteinase K, 200 μl Lysis Buffer 2, and 200 μl Binding Buffer 2 to Lysis Tube 2 in sequence and vortex to mix.

[0095] 2. Place Lysis Tube 2 on a vortex mixer and vortex at maximum speed for 10 minutes, or place it in an oscillating disruptor to lyse the cells.

[0096] 3. Place Lysis Tube 2 in a 56°C water bath for 10 min, centrifuge at 12,000 rpm (13,800 × g) for 3 min to eliminate foam, and transfer 600 μl of supernatant to a new 2 ml Nuclease-free Tube.

[0097] 4. Add 350 μl of isopropanol to the centrifuge tube in step 3 and vortex to mix.

[0098] 5. Add 20 μl of Magnetic Beads to the centrifuge tube in step 4, vortex mix, place the centrifuge tube in a vortex mixer, vortex mix at 1500 rpm for 3 minutes, let it stand for 1 minute, centrifuge for 1-3 seconds, place the centrifuge tube on a magnetic stand, and let it stand for 1 minute.

[0099] 6. Discard the supernatant, remove the centrifuge tube from the magnetic stand, add 900 μl Wash Buffer A (with anhydrous ethanol added) along the tube wall, vortex mix for 1 minute, let it stand for 1 minute, centrifuge for 1-3 seconds, place the centrifuge tube on the magnetic stand, and let it stand for 1 minute.

[0100] 7. Repeat step 6 once.

[0101] 8. Discard the supernatant, remove the centrifuge tube from the magnetic stand, add 900 μl Wash Buffer B (with anhydrous ethanol added) along the wall of the tube, vortex mix for 1 minute, let it stand for 1 minute, centrifuge for 1-3 seconds, place the centrifuge tube on the magnetic stand, and let it stand for 1 minute.

[0102] 9. Repeat step 8 once.

[0103] 10. Discard the supernatant, open the lid and let it dry for 1 minute, add 25μl Nuclease-free ddH2O, vortex mix for 1 minute, centrifuge for 1-3 seconds, place the centrifuge tube on a magnetic rack, and let it stand for 3 minutes.

[0104] 11. Pipette 25 μl of eluted product into a new 1.5 ml Nuclease-free Tube. The eluted product can be used directly in downstream experiments or stored at -80°C for a long time.

[0105] (III) MDA amplification of metagenomes

[0106] 1. Prepare Buffer D1 and Stop Solution Mix. (The volumes of Buffer D1 and Stop Solution Mix given in the table below are sufficient for 12 reactions. Any unused volume in one experiment can be stored at -30 to -15°C for no more than 3 months.)

[0107] Prepare Buffer D1

[0108] Prepare Stop Solution Mix

[0109] 2. Add 2.5 μl of DNA sample to the PCR tube. If the sample volume is less than 2.5 μl, use Nuclease-free ddH2O or TE to make up to 2.5 μl.

[0110] 3. Add 2.5 μl of Buffer D1, gently tap the tube to mix, and centrifuge briefly.

[0111] 4. Incubate at room temperature for 3 minutes.

[0112] 5. Add 5 μl of Stop Solution Mix, gently tap the tube to mix, and centrifuge briefly. Keep samples on ice until ready for the next reaction.

[0113] 6. Prepare the reaction mixture.

[0114] 7. Immediately add 40 μl of the reaction mixture to the prepared 10 μl DNA sample (step 5), gently tap the tube wall to mix, and briefly centrifuge to collect.

[0115] 8. Incubate at 30°C for 1 hour (when the sample amplification efficiency is low, the reaction time can be adjusted to 2 hours).

[0116] 9. Incubate at 65°C for 5 minutes to inactivate Discover-sc WGA Enzyme Mix.

[0117] (IV) Digestion of MDA products

[0118] Directly take 1-2ug of MDA product for digestion

[0119] Incubate at 37°C for 15 min, add 1.5 μL of 0.25 M EDTA to terminate the enzyme digestion reaction, and purify with 0.8x DNA beads.

[0120] 1. Add 0.8x balanced Mix the DNA Clean Beads and incubate at room temperature for 5 minutes; place the PCR tube on a magnetic rack and remove the supernatant after the solution is clear.

[0121] 2. Add 200 μL of 80% anhydrous ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat once

[0122] 3. Keep the PCR tube on the magnetic rack and use a 10μL pipette to remove the ethanol remaining at the bottom of the tube. Dry at room temperature until no ethanol remains (beads appear frosted).

[0123] 4. Remove the PCR tube, add 41 μL of Nuclease-free water, mix the magnetic beads thoroughly, and incubate at room temperature for 2 minutes.

[0124] 5. Place the PCR tube back on the magnetic rack and wait for the solution to clarify before aspirating 40 μL for subsequent experiments.

[0125] 6. Purify the product with Qubit TM 4 Fluorometer and Quantify using 1× dsDNA HS Assay Kit and record.

[0126] (V) End repair and ligation of digestion products

[0127] Take 1ug of digestion product for end repair

[0128] 30℃30min, 65℃30min, cover temperature 75℃. End repair products were treated with 0.8x DNA Clean Beads purification was performed as above, eluted with 67 μL of Nuclease-free water, and 66 μL was recovered for ligation.

[0129] Ligation of end-repair products

[0130] React at room temperature for 10 min, add 0.4x DNA Clean Beads purification.

[0131] 1. Add 40 μL (1:0.4 ratio) of magnetic beads equilibrated at room temperature to the ligation reaction system, gently tap the tube wall to mix, and let it stand at room temperature for 5 minutes.

[0132] 2. Place the centrifuge tube on the magnetic stand and remove the supernatant after the magnetic beads are completely adsorbed.

[0133] 3. Remove the centrifuge tube from the magnetic stand, add 200 μL of room temperature equilibrated washing buffer LWB, gently tap the tube wall to resuspend the magnetic beads, then place the centrifuge tube on the magnetic stand again. After the magnetic beads are completely adsorbed, remove the supernatant.

[0134] 4. Repeat the above steps once.

[0135] 5. Aspirate all the remaining liquid in the centrifuge tube as much as possible, then resuspend the magnetic beads with 15 μL elution buffer AEB and let it stand at room temperature for 5 minutes.

[0136] 6. Place the centrifuge tube back on the magnetic stand, wait until the magnetic beads are completely adsorbed, and then transfer the supernatant to a new 1.5 mL centrifuge tube.

[0137] 7. Using Qubit TM 4 Fluorometer and The concentration of the library after magnetic bead purification was detected using the 1× dsDNA HS Assay Kit.

[0138] (6) Nanopore sequencing

[0139] The library in the above steps was subjected to nanopore sequencing on the sequencer Qnome3841 of Qitan Technology according to the sequencing kit QSK-V1.1.1 of Qitan Technology.

[0140] Example 1: Effect of cholesterol on nuclease

[0141] (1) Effect on nuclease Benzonase

[0142] Take 1ug of cell (293T) genomic nucleic acid DNA (5μL), add PBS to 300μL as a sample, add 10× host-free reaction reagent, and perform the cholesterol neutralization saponin experiment in step 1 of the experimental operation and the pathogen DNA / RNA co-extraction in step 2 according to the scheme in Table 1. The extracted nucleic acid is tested for Qubit nucleic acid concentration.

[0143] Table 1. Experimental protocol for the effect of cholesterol on nuclease Benzonase Note: Low refers to values ​​below the instrument sensitivity or detection limit, such as below 0.05 ng / ul. Generally, nucleic acids larger than 5 bp are included in the calculation.

[0144] According to the extraction concentrations in Table 1, in the presence of only host nucleic acid, conditions 1 and 3 show that cholesterol does not inhibit the digestion activity of the nuclease Benzonase, and conditions 1 and 2 show that the presence of 2.5% saponin does not affect the digestion activity of the nuclease Benzonase.

[0145] (2) Effect on nuclease M-SAN

[0146] Take 10 μL of 1 μg cell (293T) genomic nucleic acid DNA and add 5 μL of 10× host removal reaction reagent (i.e., the host removal reaction reagent in step 1). According to the experimental scheme in Table 2, add the following reagents (a total of 50 μL system), incubate at 37°C, 1000 rpm, and incubate for 20 minutes. Detect the nucleic acid concentration every 5 minutes:

[0147] Nuclease M-SAN (ArcticZymes, 70950-202) 10 μL, PBS buffer 25 μL;

[0148] 10 μL of cholesterol (2 mg / mL, the stock solution was diluted 10-fold with 10× host removal reaction reagent (i.e., the host removal reaction reagent in step 1)), 10 μL of nuclease M-SAN, and 15 μL of PBS;

[0149] 10 μL of 10% saponin (final concentration of 2%), 10 μL of cholesterol (2 mg / mL), 5 μL of PBS, react at room temperature for 5 min, and then add 10 μL of nuclease M-SAN;

[0150] 10 μL of 1% saponin (10% saponin diluted 10-fold with water) to a final concentration of 0.2%, 10 μL of cholesterol (2 mg / mL), 5 μL of PBS, react at room temperature for 5 min, and then add 10 μL of nuclease M-SAN;

[0151] 10 μL of 1% saponin (10% saponin diluted 10-fold with water) to a final concentration of 0.2%, 10 μL of cholesterol (0.2 mg / mL, 2 mg / mL cholesterol diluted 10-fold with 10× host removal reaction reagent (i.e., the host removal reaction reagent in step 1)), 5 μL of PBS, react at room temperature for 5 minutes, and then add 10 μL of nuclease M-SAN.

[0152] Table 2. Experimental protocol for the effect of cholesterol on nuclease M-SAN

[0153] According to the nucleic acid concentrations in Table 2, the control group, which only added the nuclease M-SAN, completely digested the nucleic acids after 15 minutes. However, in the experimental group added with cholesterol, more than 100 ng of nucleic acids still remained after 15 minutes, indicating that cholesterol inhibits the activity of the nuclease M-SAN. In the experimental group added with cholesterol and saponin, when the cholesterol concentration was 0.4 mg / mL, the inhibitory effect of 2% saponin on the M-SAN enzyme was stronger than that of 0.2% saponin.

[0154] Example 2: Cholesterol concentration test

[0155] Take 293T cells and prepare 10 5 cell / mL, 10 6A 300 μL mock sample of bacteriophage T1 (copies / mL) was prepared and, according to the protocol in Table 3, the cholesterol neutralization saponin assay in step 1 and the pathogen DNA / RNA co-extraction in step 2 were performed. The extracted nucleic acids were then analyzed by qPCR. The term "copy / ml" is commonly used to describe the concentration of a substance or solution. "Copy" represents the number of a molecule or substance, typically gene or viral copies. "ml" represents the volume, i.e., milliliters. "Copy / ml" indicates the number of copies per milliliter of solution. The above protocol was repeated in two replicates.

[0156] Table 3. Experimental protocol 1 for cholesterol concentration test

[0157] The qPCR test results for the experimental procedures in Table 3 are shown in Figure 1. Based on the extraction concentration, abnormalities during extraction, and the qPCR results in Figure 1, it can be seen that when the input cholesterol concentration is higher than 1 mg / mL, it affects the adsorption of magnetic beads during extraction, and the qPCR results are all negative, indicating that impurities in the nucleic acid affect downstream amplification. At the same cholesterol input concentration (0.2 mg / mL), there is no significant difference in the expression of the human gene rpoA between cholesterol incubation times of 10 minutes and 5 minutes, indicating that increasing the incubation time does not improve the efficiency of host removal.

[0158] Example 3: Saponin neutralization experiment on different samples

[0159] (1) Take 293T cells and prepare the concentration of 10 5 cell / mL human background, 10 3 300 μL of simulated samples of copies / mL phage T1, 100 copies / mL Klebsiella pneumoniae, 100 copies / mL Enterococcus faecalis, and 100 copies / mL Candida albicans were prepared. According to the experimental scheme in Table 4, the host removal (with saponin but no cholesterol) and non-host removal (without saponin and cholesterol) processes, as well as saponin neutralization and host removal (with saponin and cholesterol) processes were performed in step 1 of the experimental operation step 1, and the pathogen DNA / RNA was co-extracted in step 2. The extracted nucleic acids were simultaneously tested by qPCR and the metagenomic MDA amplification was performed according to step 3. Then, steps 4 to 6 were performed for sequencing.

[0160] Table 4. Saponin neutralization test on simulated samples

[0161] The nucleic acid sequencing results of the experimental operation in Table 4 are shown in Table 5.

[0162] Table 5. Sequencing results of saponin neutralization experiment on simulated samples

[0163] According to the nucleic acid extraction concentration and sequencing results, the saponin neutralization and host removal process is superior to the saponin host removal process in terms of human origin ratio, mitochondrial ratio and target bacteria detection.

[0164] (2) Take a blood sample and prepare a solution with a concentration of 10 3 300 μL of simulated samples of copies / mL phage T1, 100 copies / mL Klebsiella pneumoniae, 100 copies / mL Enterococcus faecalis, and 100 copies / mL Candida albicans were prepared according to the experimental scheme in Table 6. The host was removed (with saponin but no cholesterol) and not removed (without saponin and cholesterol), and the saponin neutralization and host removal (with saponin and cholesterol) processes were performed in step 1 of the experimental operation step 1. The pathogen DNA / RNA was co-extracted in step 2. The extracted nucleic acids were simultaneously tested by qPCR. The metagenomic MDA amplification was performed according to step 3, and then sequencing was performed in steps 4 to 6.

[0165] Table 6. Cholesterol Neutralization and Detoxification Tests in Blood Samples

[0166] The nucleic acid sequencing results of the experimental operation in Table 6 are shown in Table 7, and the qPCR detection results are shown in Figure 2.

[0167] Table 7. Cholesterol neutralization and host-free sequencing results

[0168] According to the nucleic acid extraction concentration and the sequencing results in Table 7, the saponin neutralization and host removal process and the saponin neutralization and host removal process have similar results in terms of human origin proportion, mitochondrial proportion and target bacteria detection, with no significant differences, indicating that the cholesterol neutralization experiment is suitable for blood samples.

[0169] According to the qPCR results in Figure 2, for the human gene rpoA, the lower the CT value, the higher the human origin. The CT of ropA in the saponin neutralization and host removal process is 32, and the CT of the saponin host removal process is 31. There is little difference in the CT values ​​of ropA between the two processes, indicating that there is no significant difference in the host removal effect.

[0170] Example 4: Comparison of different nucleases

[0171] Take 293T cells and prepare 10 5 cell / mL, add 10 4CFU / mL Klebsiella pneumoniae was used to simulate BALF (bronchoalveolar lavage fluid) samples. Following the protocol in Table 8, the experimental procedures in step 1 (1) were performed, including host removal (with saponin but no cholesterol), host removal (without saponin and cholesterol), and saponin neutralization and host removal (with saponin and cholesterol). In step 2, pathogen DNA / RNA was co-extracted. The extracted nucleic acids were simultaneously analyzed by qPCR and nucleic acid length distribution analysis. These procedures were repeated in two parallel experiments.

[0172] Table 8. Tests of different nucleases

[0173] The qPCR test results of the experimental operation in Table 8 are shown in Figure 3, and the nucleic acid length distribution is shown in Figure 4.

[0174] According to the results of nucleic acid concentration and nucleic acid length distribution, the nucleic acid fragments of the saponin host removal process are below 200bp, and the nucleic acid fragments of the saponin neutralization host removal process are below 100bp. The enzyme digestion ability in the saponin neutralization host removal process is better; the nucleic acid fragment distribution of the high-salt-tolerant enzyme is diffuse, and its digestion ability is worse than that of the all-round enzyme.

[0175] Compared with the host-free sample (sample 1) under different test conditions, the greater the difference in CT values ​​for RPOA, the better the host removal effect. The insignificant difference in CT values ​​between MT and KLE indicates no loss of target bacteria. In process 5-2 of saponin neutralization and host removal using Yisheng All-in-One enzyme, the qPCR result for rpoA was ΔCT = CT value (sample 5-2) - CT value (sample 1) = 6.62. For other enzymes, ΔCT was < 6. The higher the ΔCT, the lower the starting amount of target nucleic acid, that is, the better the enzyme digestion effect, and Yisheng All-in-One enzyme performed the best.

[0176] Example 5. Liposome reaction condition test

[0177] (1) Take 293T cells and prepare the concentration of 10 4 cell / mL background, 10 3 300 μL of simulated samples of copies / mL phage T1, 100 copies / mL Klebsiella pneumoniae, 100 copies / mL Enterococcus faecalis, and 100 copies / mL Candida albicans were prepared according to the scheme in Table 9. The host removal (with saponin but no liposomes) and saponin neutralization and host removal (with saponin and liposomes) processes were performed according to step 1 of the experimental operation step 1, and the pathogen DNA / RNA was co-extracted in step 2. The extracted nucleic acids were simultaneously tested by qPCR and the metagenomic MDA amplification was performed according to step 3. Then, steps 4 to 6 were performed for sequencing.

[0178] Table 9. Liposome reaction conditions tested

[0179] The nucleic acid sequencing results of the experimental operation in Table 9 are shown in Table 10.

[0180] Table 10. Sequencing results of liposome reaction conditions

[0181] Sequencing results show that at the same liposome concentration, as the saponin concentration decreases, the proportion of human origin decreases accordingly. 4T3 had the lowest proportion of human origin at 54.96% and the highest proportion of target bacteria at 32.7%, achieving the best results compared to the other tested conditions.

[0182] (2) Take negative alveolar lavage fluid and prepare a final concentration of 10 5 For the simulated samples of 100 copies / mL phage T1 and 100 copies / mL Klebsiella pneumoniae, according to the scheme in Table 11, the experimental operation step 1 (2) was performed to remove the host, not remove the host, and saponin neutralization and remove the host, respectively, and the pathogen DNA / RNA was co-extracted in step 2. The extracted nucleic acids were simultaneously tested by qPCR and the metagenomic MDA amplification was performed according to step 3. Then, steps 4 to 6 were performed for sequencing.

[0183] Table 11. Liposome neutralization saponin reaction conditions test

[0184] The qPCR detection results of the experimental operation in Table 11 are shown in Figure 5.

[0185] According to the qPCR results, the CT of the BALF sample without host rpoA was 28.38, the control group T1 (saponin diluted 100 times) had CT = 38.91, the control group T8 (1.25% SA & liposomes) had CT = 37.27, the experimental group T5 (0.25% SA + high salt-tolerant enzyme) had CT = 41.86, and the CT of the other experimental groups was between 38-40. Therefore, T5 was selected as the most optimal reaction condition.

[0186] (3) Take 293T cells and prepare a final concentration of 10 4 copies / mL human background, 10 3300 μL of simulated samples of bacteriophage T1 (100 copies / mL), Klebsiella pneumoniae (100 copies / mL), Enterococcus faecalis (100 copies / mL), and Candida albicans (100 copies / mL) were prepared according to the protocol in Table 12. Step 1 (2) included host removal, no host removal, and saponin neutralization and host removal, respectively. The pathogen DNA / RNA was co-extracted in Step 2. The extracted nucleic acids were simultaneously analyzed by qPCR and metagenomic DNA amplification was performed according to Step 3. Sequencing was then performed in Steps 4 through 6. These procedures were repeated twice.

[0187] Table 12 Liposome reaction conditions test

[0188] The qPCR detection results of the experimental operation in Table 12 are shown in Figure 6, and the nucleic acid sequencing results are shown in Table 13.

[0189] According to qPCR results, 10 4 Under the human background of copies / mL, the CT values ​​of ropA in the three experimental groups were close to 40, with no significant difference.

[0190] Table 13. Sequencing results of liposome reaction conditions

[0191] Sequencing results showed that the mitochondrial proportion in the experimental group T2 reached 99%, and the proportion of target bacteria was close to that in T1. Considering the time cost of the entire reaction, T2 was ultimately selected as the final reaction condition.

[0192] Example 6. Stability test of saponin neutralized by liposomes

[0193] (1) Take 293T cells and prepare the concentration of 10 5 cell / mL, 10 6 cell / mL,10 3 copies / mL phage T1, 100 copies / mL Klebsiella pneumoniae, 100 copies / mL Enterococcus faecalis, 100 copies / mL Candida albicans, 10 3 copies / mL Aspergillus fumigatus, 10 3 A 300 μL simulated sample of Cryptococcus neoformans containing 100 copies / mL was prepared and, according to the protocol in Table 14, the experimental procedures were followed in step 1 (2) for host removal, non-host removal, and treatment, respectively. The pathogen DNA / RNA was co-extracted in step 2. The extracted nucleic acids were then subjected to qPCR analysis and metagenomic MDA amplification according to step 3. Sequencing was then performed in steps 4 through 6. These procedures were repeated three times. In Table 14 below, T1 indicates sequential addition of the components, while T2 indicates simultaneous addition of the components.

[0194] Table 14. Liposome neutralization saponin stability test

[0195] The qPCR detection results of the experimental operation in Table 14 are shown in Figure 7, and the sequencing results of the liposome reaction conditions are shown in Tables 15-16.

[0196] The nucleic acid concentration and qPCR results showed that there was no significant difference in the CT value of ropA under the two reaction conditions in the liposome neutralization and host removal processes, indicating that there was no significant difference in the host removal effects between the two processes.

[0197] Table 15. Sequencing results of liposome neutralization saponin stability test (10 5 cell / mL background)

[0198] Table 16. Sequencing results of liposome neutralization saponin stability test (10 6 cell / mL background)

[0199] The sequencing results show that under low human background (10 5 cell / mL), in terms of mitochondrial proportion, the liposome neutralization saponin process was 99.9%; in terms of target bacteria proportion, the liposome neutralization saponin process T1 (45min) was 0.7%-1%, and the liposome neutralization saponin process T2 was 5.4%-8.2%, among which the proportion of Candida albicans and Aspergillus fumigatus increased by nearly 10 times. Overall, it can be seen that under low human background, the liposome neutralization experiment T2 process improves the host removal effect; under high human background (10 6 cell / mL), the proportion of mitochondria in the saponin-host removal process and the liposome-neutralized saponin process were both above 99.9%, the target bacteria were below 1%, the T2 process accounted for 0.3%, and the other processes accounted for less than 1%, indicating that the liposome-neutralized saponin process is feasible.

[0200] (2) Take clinical samples of alveolar lavage fluid (human background is 10 4 cell / mL, 10 5 cell / mL, 10 6A total of 3 samples (100 cells / mL) were collected. Following the liposome neutralization and host removal process, the host removal and treatment of step 1, and the co-extraction of pathogen DNA / RNA in step 2 were performed. Each sample was run in triplicate. The extracted nucleic acids were simultaneously tested by qPCR and metagenomic MDA amplification according to step 3. Sequencing was then performed in steps 4 to 6. The above procedures were repeated three times. The sequencing results are shown in Table 17.

[0201] Table 17. Sequencing results of liposome neutralization experiment on alveolar lavage fluid

[0202] According to the test results, the three parallel groups of three samples at different cell concentrations had similar results in terms of human origin ratio and mitochondrial ratio, and the results between parallel groups were stable.

[0203] Note: The percentage of saponin in the table is the final concentration of saponin in the reaction system;

[0204] The cholesterol concentration in the table is the final cholesterol concentration in the reaction system;

[0205] The liposome concentration in the table is the final concentration of cholesterol contained in the liposomes in the reaction system. Liposomes are artificial membranes containing cholesterol. During the experiment, an appropriate amount of liposomes was added to the reaction system to achieve the cholesterol concentration in the table.

[0206] The above-described embodiments and test examples merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person of ordinary skill in the art would be able to make various modifications and improvements without departing from the technical concept of the present application, and these modifications and improvements are all within the scope of protection of the present application.

Claims

1. A method for removing host nucleic acid from a biological sample, the method comprising the following steps: a) contacting the sample with an excess saponin-containing preparation so that the host in the sample fully reacts with the saponin-containing preparation; b) adding at least one saponin inhibitor to inhibit the activity of excess saponin; and c) adding at least one nucleic acid digesting enzyme to allow for the removal of host nucleic acids.

2. The method according to claim 1, wherein steps a), b), and c) are performed in a manner selected from the group consisting of: (1) steps a), b), and c) are performed simultaneously; (2) steps a) and b) are performed simultaneously, and then step c) is performed; (3) step a) is performed first, and then steps b) and c) are performed simultaneously; or (4) steps a), b), and c) are performed sequentially. Preferably, the sum of the reaction times of steps a), b) and c) is 5-60 min, preferably 10-30 min.

3. The method according to claim 2, wherein no separation step is included before step c), preferably no centrifugation and / or washing step is included.

4. The method according to any one of claims 1 to 3, wherein the at least one saponin inhibitor is an agent for inhibiting the activity of nucleic acid digesting enzymes inhibited by excess saponin, and the saponin inhibitor is selected from a saponin neutralizer or a saponin adsorbent; The saponin neutralizing agent is a preparation capable of reacting with saponin. Preferably, the saponin neutralizing agent is selected from one or more of cholesterol, liposomes containing cholesterol, red blood cells containing cholesterol, or analogs thereof. Preferably, the saponin neutralizing agent is selected from one or more of water-insoluble cholesterol, water-soluble cholesterol, or liposomes containing cholesterol. More preferably, the saponin neutralizing agent is selected from water-soluble cholesterol and / or liposomes containing cholesterol. The saponin adsorbent is a preparation capable of adsorbing saponin; preferably, the saponin adsorbent is selected from reverse phase chromatography fillers, ion exchange chromatography fillers, protein adsorbents, sugar adsorbents, polydopamine or adsorbents based on molecular imprinting technology.

5. The method according to any one of claims 1 to 4, wherein the at least one nucleic acid digesting enzyme is a nuclease having DNase and / or RNase activity; preferably, the at least one nucleic acid digesting enzyme is selected from one or more of nuclease M-SAN, nuclease HL-SAN, Serratia nuclease Benzonase, universal nuclease, super nuclease and high salt tolerant enzyme.

6. The method according to any one of claims 1 to 5, wherein in the reaction system for removing host nucleic acid, The mass concentration of the saponin is 0.01%-5%, preferably 0.02%-2.5%; and / or The concentration of cholesterol is no greater than 1 mg / mL, preferably 0.02-0.5 mg / mL, more preferably 0.04-0.4 mg / mL.

7. The method according to any one of claims 1 to 6, wherein step a) is performed in the presence of a host removal reaction reagent, preferably, the host removal reaction reagent comprises: 0-500 mM NaCl, 4-15 mM MgCl2 and 15-50 mM Tris-HCl, pH 7-9.

8. The method according to any one of claims 1 to 7, wherein the sample is selected from one or more of blood, sputum, bronchoalveolar lavage fluid, cerebrospinal fluid, tissue, and pleural and ascites fluid; Preferably, the concentration of host cells in the sample is 10 3 -10 11 cells / mL, more preferably 10 3 -10 8 cells / mL, and the most preferred is 10 4 -10 7 cells / mL.

9. A method for detecting microorganisms in a biological sample, the method comprising: Perform the method for removing host nucleic acid according to any one of claims 1 to 8; as well as Nucleic acids are extracted and metagenomic sequencing is performed on the nucleic acids to determine the detection results of the microorganisms.

10. The method according to claim 9, wherein the microorganisms include bacteria, fungi and / or viruses, and the viruses include DNA viruses and / or RNA viruses; Optionally, the bacteria include at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella aerogenes, Klebsiella oxytoca, Streptococcus pneumoniae, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus hominis, Staphylococcus hemolyticus, Staphylococcus capitis, and Streptococcus agalactiae. Optionally, the fungus includes at least one of Candida albicans, Cryptococcus gattii, Aspergillus nidulans, Candida glabrata / Candida glabrata, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Cryptococcus neoformans / Cryptococcus neoformans, and Aspergillus fumigatus, Optionally, the DNA virus includes at least one of DNA phage, Epstein-Barr virus, mumps virus, and adenovirus. Optionally, the RNA virus comprises at least one of RNA bacteriophage, influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus and coronavirus.

11. A kit for removing host nucleic acid from a biological sample or a kit for detecting microorganisms in a biological sample, the kit comprising a. Preparations containing saponins, b. At least one saponin inhibitor, the at least one saponin inhibitor is a preparation for inhibiting the activity of nucleic acid digesting enzymes inhibited by excessive saponins, the saponin inhibitor is selected from a saponin neutralizer or a saponin adsorbent, the saponin neutralizer is a preparation capable of reacting with saponin, the saponin adsorbent is a preparation capable of adsorbing saponin, the saponin neutralizer is selected from one or more selected from liposomes, cholesterol, red blood cells or their analogs, preferably, the at least one saponin The neutralizing agent is selected from one or more of water-insoluble cholesterol, water-soluble cholesterol or liposomes, more preferably, the at least one saponin neutralizing agent is selected from water-soluble cholesterol and / or liposomes; c. at least one nucleic acid digesting enzyme, wherein the at least one nucleic acid digesting enzyme is a nuclease having DNase and / or RNase activity; preferably, the at least one nucleic acid digesting enzyme is selected from one or more of nuclease M-SAN, nuclease HL-SAN, Serratia nuclease Benzonase, universal nuclease, super nuclease, and high salt tolerant enzyme; d. Optionally, a description for use in the method according to the present invention, preferably, the description describes the method according to any one of claims 1 to 10; and e. Optionally, a host-free reaction reagent, wherein the host-free reaction reagent comprises: 0-500 mM NaCl, 4-15 mM MgCl2 and 15-50 mM Tris-HCl, PH 7-9.

12. Use of the method according to any one of claims 1 to 10 or the kit according to claim 11 in pathogenic microorganism metagenomic sequencing, microbial typing or drug resistance gene analysis, or in the preparation of a product for pathogenic microorganism metagenomic sequencing, microbial typing or drug resistance gene analysis.

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