Preparation method for sample for microorganism detection, sample, detection method, kit, and use

Abstract

The present application provides a preparation method for a sample for microorganism detection, a sample, a detection method, a kit, and a use. The method comprises: a treatment step: taking an in-vitro host cell sample, and using a host cell lysis reagent and a nuclease to carry out treatment to degrade host nucleic acid, the treatment being carried out in a liquid having a salt concentration of 10-600 mM; a nuclease inactivation step: providing conditions to inactivate the nuclease; and a microorganism nucleic acid acquisition step: extracting total nucleic acid to obtain a sample for microorganism detection. The method of the present application can effectively reduce the interference of a host cell genome on the sensitivity of sample detection in metagenome sequencing, and improve effective sequencing data of microorganisms in a detection result, thereby increasing the positive detection rate of the microorganisms and improving the sensitivity of microorganism detection.

Description

Sample preparation method, sample, detection method, kit and application for microbial detection

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310869270.5, filed on July 14, 2023, entitled “Sample preparation method, sample, detection method, kit and application for microbial detection,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the technical field of biological sample preparation, and specifically relates to a sample preparation method, sample, detection method, kit and application for microbial detection. Background Art

[0004] Microorganisms are widely present in nature, and usually include viruses, bacteria, fungi, protozoa, and certain algae. Rapid detection of microorganisms is of great significance for related testing.

[0005] Compared with traditional methods of culturing and preparing samples to be tested, in traditional samples, for example, in a background containing high human DNA, the samples contain a large amount of human cell DNA, which makes it difficult to detect low-abundance pathogenic microorganism sequences, often leading to missed detections or low detection accuracy. How to improve the detection accuracy of samples to be tested is a key issue to be considered at this stage.

[0006] Summary of the Invention

[0007] In view of this, the present application provides a sample preparation method, sample, detection method, kit and application for microbial detection, aiming to provide a method for preparing samples that is convenient for metagenomic sequencing and has high detection accuracy; the samples, detection methods, kits and applications obtained based on this sample preparation method also have corresponding advantages.

[0008] In a first aspect, embodiments of the present application provide a method for preparing a sample for detecting microorganisms, comprising:

[0009] a treatment step, taking an in vitro host cell sample and treating it with a host cell lysis reagent and a nuclease to degrade host nucleic acid, wherein the treatment is performed in a liquid having a salt concentration of 10-600 mM;

[0010] a nuclease inactivation step, providing conditions to inactivate the nuclease;

[0011] The step of obtaining microbial nucleic acid is to extract total nucleic acid to obtain the sample for detecting microorganisms.

[0012] In some optional embodiments, the ex vivo host cell sample is from blood, sputum, throat swab, alveolar lavage fluid, pleural effusion, cerebrospinal fluid, tissue, tissue pus, or culture fluid.

[0013] In some optional embodiments, the method satisfies at least one of the following conditions:

[0014] 1) In the treatment step, the nuclease is capable of degrading the host nucleic acid to a first nucleic acid length that is smaller than the second nucleic acid length of the microorganism obtained by extracting the total nucleic acid in the step of obtaining microbial nucleic acid, thereby forming a first length difference;

[0015] 2) In the treatment step, the length of the nucleic acid fragment after the host nucleic acid degradation is more than 35 bp,

[0016] 3) In the treatment step, the length of the nucleic acid fragments after the degradation of the host nucleic acid is less than 900 bp, less than 800 bp, less than 700 bp, less than 600 bp, less than 500 bp, less than 400 bp, less than 300 bp, less than 250 bp, less than 200 bp, less than 150 bp, or less than 100 bp; optionally, 35-300 bp, 50-275 bp, 75-250 bp, or 100-200 bp;

[0017] 4) In the step of obtaining microbial nucleic acid, the length of the nucleic acid fragment of the microorganism in the sample for detecting the microorganism is ≥ 1 kb, and can be 1-10 kb or 1 kb-5 kb,

[0018] 5) The nuclease is capable of degrading DNA and / or RNA, and the nuclease is M-SAN nuclease, Benzonase nuclease and / or high-salt-tolerant nuclease, and can further be M-SAN nuclease.

[0019] In some optional embodiments, the method satisfies at least one of the following conditions:

[0020] 1) In the treatment step, the salt concentration is 15-590 mM or 19-565 mM, the salt includes MgCl2 and Tris-HCl, and the pH value of the liquid is 7-9 or 7.2-8.7; optionally, the salt also includes a neutral salt, and the concentration of the neutral salt in the liquid is 0-500 mM, 10-450 mM, 50-300 mM, 125-250 mM, or 130-160 mM;

[0021] 2) in the treatment step, the treatment with a host cell lysis reagent and a nuclease comprises incubating the isolated host cell sample with a host cell lysis reagent, a nuclease, and a nuclease buffer to degrade host nucleic acids;

[0022] Optionally, the components and their concentrations in the nuclease buffer are as follows: 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;

[0023] 3) The host cell lysis reagent is a host cell membrane lysis reagent, which can be further selected from saponin, further selected from Quillaja saponin, and more optionally, the active ingredient in the saponin is used at a concentration of 0.3-0.7%, optionally 0.32-0.6%, and further optionally 0.34-0.54%.

[0024] In some optional embodiments, in the step of inactivating the nuclease, any one or more of heat inactivation, tris(2-carboxyethyl)phosphine, proteinase K, or EDTA is used to inactivate the nuclease.

[0025] The step of obtaining microbial nucleic acid does not include centrifugation and / or washing steps before extracting nucleic acid.

[0026] The extracted nucleic acid in the step of obtaining microbial nucleic acid includes DNA and / or RNA.

[0027] In some optional embodiments, in the step of obtaining microbial nucleic acid, after extracting total nucleic acid, the method further comprises: using the first length difference to further enrich or separate the nucleic acid of the microorganism obtained by extracting the total nucleic acid in the step of obtaining microbial nucleic acid, so as to increase the proportion of the nucleic acid of the microorganism in the sample for detecting the microorganism; and / or,

[0028] The first length difference is maintained in the method for extracting nucleic acid.

[0029] In some optional embodiments, the step of obtaining microbial nucleic acid further comprises, after extracting the total nucleic acid, a step of degrading the host mitochondrial DNA and / or the host ribosomal RNA.

[0030] Optionally, the length of the fragments after degradation of the host mitochondrial DNA and / or host ribosomal RNA is less than the length of the nucleic acid of the microorganism obtained by extracting the total nucleic acid in the step of obtaining microbial nucleic acid, forming a second length difference.

[0031] In some optional embodiments, the second length difference can be used to further enrich or separate the nucleic acid of the microorganism obtained by extracting nucleic acid in the step of obtaining microbial nucleic acid, so as to increase the proportion of the nucleic acid of the microorganism in the sample for detecting microorganisms;

[0032] Optionally, the method for degrading host mitochondrial DNA and / or host ribosomal RNA is specific degradation, which can further be CRISPR, and further can be CRISPR / Cas9.

[0033] In some optional embodiments, the second length difference is less than or equal to the first length difference.

[0034] In some optional embodiments, in the step of obtaining microbial nucleic acid, after the nucleic acid is extracted, a step of nucleic acid enrichment and / or amplification using method a or method b is included to increase the amount of nucleic acid in the sample for detecting microorganisms.

[0035] Method a represents randomly shearing the nucleic acid and then adding a universal amplification linker, and then performing PCR amplification. Optionally, the nucleic acid is randomly sheared and then adding a universal amplification linker using Tn5 enzyme;

[0036] b Method represents enrichment and / or amplification of nucleic acid of the microorganism, further optionally, using a multiple displacement amplification (MDA) method.

[0037] In some optional embodiments, the microorganisms include bacteria, fungi and / or viruses, and the viruses include DNA viruses and / or RNA viruses.

[0038] In some optional 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 (Group A Streptococcus), Staphylococcus hominis, Staphylococcus hemolyticus, Staphylococcus capitis, and Streptococcus agalactiae (Group B Streptococcus).

[0039] In some optional embodiments, optionally, the fungus includes 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.

[0040] In some optional embodiments, the DNA virus includes at least one of DNA phage, Epstein-Barr virus, mumps virus, and adenovirus.

[0041] In some optional 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.

[0042] In a second aspect, an embodiment of the present application provides a sample for detecting microorganisms, which is prepared by the method of the first aspect.

[0043] In a third aspect, an embodiment of the present application provides a method for detecting microorganisms, comprising: obtaining a sample for detecting microorganisms obtained by the preparation method of the first aspect, or a sample for detecting microorganisms of the second aspect;

[0044] performing metagenomic sequencing on the sample to determine the detection results of the microorganisms;

[0045] The sequencing includes second-generation sequencing based on PCR amplification, or single-molecule sequencing based on nanopore, optionally, single-molecule sequencing based on nanopore.

[0046] In some optional embodiments, the upper limit of the ratio of the number of the host cells to the number of the microorganisms in the in vitro host cell sample is: (10 4 -10 7 ):1;

[0047] Optionally, the microorganism is a bacterium, and the upper limit of the number ratio is 10 7 :1;

[0048] Optionally, the microorganism is a virus, and the upper limit of the number ratio is (10 4 -10 7 ):1;

[0049] Optionally, the microorganism is a fungus, and the upper limit of the number ratio is 10 7 :1;

[0050] Optionally, the concentration of the host cells in the isolated host cell sample is 1×10 4 -1×10 7 cells / mL;

[0051] The concentration of the microorganism in the isolated host cell sample is 1-1×10 7 CFU / mL.

[0052] In a fourth aspect, the present invention provides a kit for detecting a sample of a microorganism, comprising: a host cell lysis reagent, a nuclease, and a nuclease buffer,

[0053] Optionally, the salt in the nuclease buffer is used at a concentration of 10-600 mM, 15-590 mM or 19-565 mM,

[0054] Optionally, the salt includes MgCl2, Tris-HCl and neutral salt,

[0055] Optionally, the components and their concentrations in the nuclease buffer are as follows: 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;

[0056] Optionally, the host cell lysis reagent includes a host cell membrane lysis reagent, and optionally, the host cell membrane lysis reagent is a saponin, and further optionally, a Quillaja saponin, and more optionally, the active ingredient in the saponin is used at a concentration of 0.3-0.7%, optionally 0.32-0.6%, and further optionally 0.34-0.54%.

[0057] Optionally, the nuclease is capable of degrading DNA and / or RNA, and may be M-SAN.

[0058] In a fifth aspect, the present invention provides a method for constructing a sequencing library for pathogenic microorganism detection, comprising:

[0059] R1. Providing a sample to be tested, wherein the length of the host nucleic acid fragment in the sample to be tested is less than 200 bp, and the length of the nucleic acid fragment of the pathogenic microorganism that may be present in the sample to be tested is ≥ 1 kb, which can be 1-10 kb or 1 kb-5 kb;

[0060] R2. performing nucleic acid multiple displacement amplification on the sample to be tested to increase the proportion of nucleic acid fragments of pathogenic microorganisms that may be present in the sample to be tested, thereby obtaining multiple displacement amplification products;

[0061] R3. Construct a library for the multiple displacement amplification products to obtain a sequencing library.

[0062] In some optional embodiments, the sample to be tested is prepared by any method of the first aspect or the sample of the second aspect.

[0063] Application of the method of the first aspect, the sample of the second aspect, the method of the third aspect, the kit of the fourth aspect, or the method of the fifth aspect in pathogenic microorganism metagenomic sequencing, or in preparing a product for pathogenic microorganism metagenomic sequencing.

[0064] Compared with the prior art, this application has at least the following beneficial effects:

[0065] The present application provides a sample preparation method for microbial detection, which removes host nucleic acids from in vitro host cell samples that may contain microorganisms and purifies microbial nucleic acids that may contain microorganisms as much as possible. On the one hand, the sensitivity of sequencing is improved by reducing the damage to pathogens and increasing the proportion of pathogenic nucleic acids. On the other hand, host cell lysis reagents and nuclease treatment are used to degrade host nucleic acids. The treatment is carried out in a liquid with a salt concentration of 10-600mM, maintaining the distinction between long fragments and short fragments, and enriching long fragments to increase their proportion and improve the accuracy of sequencing results. The interference of the host cell genome on the sensitivity of sample detection can be effectively reduced in metagenomic sequencing, and the effective sequencing data of microorganisms in the test results can be increased, thereby improving the positive detection rate of microorganisms and the sensitivity of detecting microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] 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.

[0067] FIG1 shows a flow chart of a method for preparing a sample for detecting microorganisms according to Example 1 of the present application;

[0068] FIG2 shows a human cell (10 6 / ml) and Escherichia coli (10 6 / ml) Fluorescence quantitative qPCR results;

[0069] FIG3 shows the qPCR results of human genes, DNA phage T1, and RNA phage M in the examples of the present application;

[0070] FIG4 shows the quantitative results of LOD of viral cells in the embodiment of the present application;

[0071] FIG5 shows the coverage of the adenovirus genome according to an embodiment of the present application;

[0072] FIG6 shows the length of the host nucleic acid after the host is removed in an embodiment of the present application;

[0073] FIG7 shows the effect of short fragments on metagenome comparison analysis in an embodiment of the present application;

[0074] FIG8 shows that the host nucleic acid becomes shorter after being treated with different nucleases in the examples of the present application;

[0075] FIG9 shows a distribution diagram of long and short fragments of bacterial cells according to an embodiment of the present application;

[0076] FIG10 shows the effect of using MDA to amplify long fragments on a simulated sample according to an embodiment of the present application. DETAILED DESCRIPTION

[0077] 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.

[0078] 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.

[0079] In the description of this application, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number, and “a variety” in “one or more” means two or more.

[0080] 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.

[0081] Traditional microbial culture and identification methods offer advantages in metagenomic sequencing, such as short turnaround times, wide coverage, and ease of use. However, metagenomic sequencing also faces a significant challenge: samples contain large amounts of host DNA. For example, when the host is human, detecting low-abundance pathogen sequences against a backdrop of high human DNA is extremely challenging, not to mention the ability to perform more in-depth analyses such as microbial typing and drug resistance gene analysis through metagenomic sequencing.

[0082] How to efficiently remove the human DNA background and maximize the retention of microbial content in the sample, thereby improving the sensitivity of metagenomic sequencing and increasing the positive detection of microorganisms, is a problem that must be solved.

[0083] In the related art, methods for removing host nucleic acids include: (1) removal based on the use of DNA / RNA probes targeting host DNA, which usually requires denaturation of the sample nucleic acid before hybridization incubation. The hybridization process is time-consuming and costly; (2) removal based on the principle that methylated CpG binding domain proteins (such as MBD1, MBD2, MBD3, and MBD4 proteins) can bind to methylated CpG islands that are widely present in eukaryotic DNA. Usually, the removal efficiency is low, the effect is not ideal, and some non-host eukaryotic organisms such as fungi or parasites will be lost. (3) Host cells are removed based on the size difference or other physical differences (such as sedimentation rate) between host cells and microorganisms. The removal efficiency is usually very low, and free host DNA cannot be removed. In addition, nucleic acids of fungi, parasites and some intracellular microorganisms are lost. (4) If the host cells are human cells, since human cells are more fragile than the shell of microorganisms, hypotonic solutions, mild detergents or other enzymes are used to destroy only the host cells. Then, propidium iodide azide (PMA), which is impermeable to the cell membrane, is used to modify the host DNA exposed to the solution. PMA is a photoreactive dye with high affinity for DNA. When exposed to strong visible light, the dye is embedded in double-stranded DNA to form a covalent bond, forming chemically modified DNA. At this time, it can no longer be amplified and complete the subsequent sequencing library construction process. This method has a high removal efficiency but also has the disadvantages of complicated steps and the removal of dead bacterial DNA at the same time; (5) Professor Grady of the United Kingdom established a host DNA removal technology route based on nanopore sequencing technology. This technology route is to first centrifuge the sample solution to obtain a PBS suspension, then destroy the host cell membrane to free the host DNA, degrade the host DNA through HL-SAN nuclease, and finally centrifuge to collect microbial pathogens mainly composed of bacteria. The above technology route is complicated to operate, and multiple centrifugation and PBS washing require the removal of part of the supernatant, which will inevitably lose some microorganisms. Only microorganisms mainly composed of bacteria can be collected, and all microorganisms cannot be fully detected.

[0084] Based on the difficulty of removing host DNA in metagenomic applications, the present application provides a method for preparing samples for detecting microorganisms, which can effectively remove host nucleic acids and use recombinant proteins that degrade related nucleic acids to reduce or remove host genome mitochondrial DNA. This method can effectively reduce host genome DNA in metagenomic sequencing and improve the effective sequencing data of microorganisms in the sample.

[0085] Method for preparing samples for detecting microorganisms

[0086] In a first aspect, an embodiment of the present application provides a method for preparing a sample for detecting microorganisms, as shown in FIG1 , comprising:

[0087] a treatment step, taking an in vitro host cell sample and treating it with a host cell lysis reagent and a nuclease to degrade host nucleic acid, wherein the treatment is performed in a liquid having a salt concentration of 10-600 mM;

[0088] a nuclease inactivation step, providing conditions to inactivate the nuclease;

[0089] The step of obtaining microbial nucleic acid is to extract total nucleic acid to obtain the sample for detecting microorganisms.

[0090] The inventors discovered that in order to minimize damage to microorganisms during the host removal process, a one-step host removal system was used. This resulted in the working environment of the nuclease not being a pure ion reaction system, but being interfered with by a large amount of cell debris, proteins released by cell lysis, and other substances. This also resulted in the host nucleic acid fragments after nuclease degradation being not a few bp in length, but around 100 bp or 200 bp. Second-generation sequencing analysis showed that these fragments were evenly distributed on human chromosomes, proving that there was no similarity between short host fragments of around 100 bp after host removal.

[0091] Furthermore, the inventors have found through testing that if this 100bp or 200bp long host nucleic acid fragment is not properly processed and used directly for sequencing, it will interfere with the accuracy of the pathogen identification results during sequencing. This application pays attention to and classifies its proportion before sequencing to reduce the proportion of host nucleic acid, thereby improving the accuracy of sequencing.

[0092] In order to preserve the nucleic acids of pathogenic microorganisms as much as possible, a one-step host removal system with a salt concentration of 10-600mM and conventional means of extracting total nucleic acid were adopted to make the length of the degradation fragments of microbial and host nucleic acids significantly different, so as to reduce the proportion of the latter according to the length difference. Special attention should be paid to the steps of extracting total nucleic acid and the differential enrichment of nucleic acid after extraction, so as to ultimately improve the sensitivity and accuracy of metagenomic sequencing.

[0093] The method of the present application, on the one hand, increases the proportion of pathogen nucleic acids by reducing damage to pathogens to improve the sensitivity of sequencing, and on the other hand, uses host cell lysis reagents and nuclease treatment to degrade host nucleic acids. The treatment is carried out in a liquid with a salt concentration of 10-600mM, maintaining the distinction between long fragments and short fragments, and enriching long fragments to increase their proportion and improve the accuracy of sequencing results.

[0094] According to the embodiments of the present application, the nuclease of the present application is a type of nucleic acid degrading enzyme that can catalyze the degradation of nucleic acids (DNA and RNA). The following are some common types of nucleic acid degrading enzymes: Nuclease A (RNase A): It is a nuclease that specifically degrades single-stranded RNA and can cut at the phosphodiester bond in the RNA chain to generate short fragments. Nuclease T1 (RNase T1): It is a nuclease that specifically degrades single-stranded RNA with guanosine phosphate (G) and can cut the phosphodiester bond on the 3' side of G. Nuclease T2 (RNase T2): It is a nuclease that non-specifically degrades RNA and can cut the phosphodiester bond at various nucleotides on the RNA chain. Nuclease U2 (RNase U2): It is a nuclease that specifically degrades tRNA and can cut at a specific position on the tRNA chain. Nuclease P1 (RNase P1): It is a nuclease that specifically degrades RNA with guanosine phosphate (G) and can cut the phosphodiester bond on the 5' side of G. Alkaline Phosphatase: Although it is not a dedicated nucleic acid degrading enzyme, alkaline phosphatase can reduce the phosphorylation level of phosphate residues on DNA and RNA chains, thereby reducing the stability of the chain and the rate of degradation.

[0095] According to the present application embodiment, host cell lysis reagent is a reagent used to lyse (dissolve) host cells to release target proteins or other cellular components. These reagents generally contain a series of components designed to destroy the cell membrane, nuclear membrane and organelle membranes, thereby releasing organelles, proteins and nucleic acids in the cell.

[0096] Common host cell lysis reagents include at least one of the following: osmotic agents, cationic / anionic detergents, protease inhibitors, nuclease inhibitors, and buffers. As an example, osmotic agents, such as mannitol and glucose, are used to change the osmotic pressure difference between the inside and outside of the cell and destroy the cell membrane. As an example, cationic / anionic detergents, such as Triton X-100, Tween 20, and SDS (sodium dodecyl sulfate), are used to destroy the cell membrane, nuclear membrane, and organelle membrane to release the cell contents. As an example, protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), are used to protect proteins in the cell from degradation. As an example, nuclease inhibitors, such as ribonuclease inhibitors (RNase inhibitors), are used to protect nucleic acids in the cell from degradation. As an example, buffers include PBS buffer. Providing appropriate pH and ion concentration to maintain suitable reaction conditions.

[0097] When degrading host nucleic acids, the commonly used neutral salts are as follows: Sodium chloride (NaCl): Sodium chloride is one of the most commonly used neutral salts. It can provide appropriate ionic strength to maintain the osmotic pressure and stability of the reaction system. Sodium dihydrogen phosphate (NaH2PO4) / disodium phosphate (Na2HPO4): Sodium dihydrogen phosphate and disodium phosphate can be used to adjust the pH value of the reaction system and the preparation of buffer solutions. They can be used as neutral salts and can also provide a certain buffering capacity. Potassium chloride (KCl): Potassium chloride is also one of the commonly used neutral salts, similar to sodium chloride, used to provide appropriate ionic strength and stability. The present application provides neutral salts of a specific concentration to provide appropriate ionic strength, maintain enzyme activity and optimize its catalytic efficiency to achieve degradation of host nucleic acids.

[0098] In some embodiments, during the processing step, the host cells can be mammalian cells, for example, dog cells, monkey cells, mouse cells, cat cells, and can be human cells, further can be human cerebrospinal fluid cells or human thoracic and abdominal cells.

[0099] In some embodiments, during the treatment step, the microorganism may be a pathogenic microorganism that parasitizes a host cell, and may be one or more of bacteria, fungi, and viruses.

[0100] In some optional embodiments, the nuclease in the treatment step can degrade the host nucleic acid to a first nucleic acid length that is smaller than the second nucleic acid length of the microorganism obtained by extracting the nucleic acid in the step of obtaining microbial nucleic acid, thereby forming a first length difference.

[0101] According to an embodiment of the present application, the first length difference may range from 100 bp to 9.6 kb.

[0102] In some optional embodiments, in the processing step, the length of the nucleic acid fragment after the host nucleic acid is degraded is greater than 35 bp.

[0103] In some optional embodiments, in the processing step, the length of the nucleic acid fragment after degradation of the host nucleic acid is less than 900bp, less than 800bp, less than 700bp, less than 600bp, less than 500bp, less than 400bp, less than 300bp, less than 250bp, less than 200bp, less than 150bp, or less than 100bp; optionally, 35-300bp, 50-275bp, 75-250bp or 100-200bp.

[0104] Optionally, the length of the nucleic acid fragment after degradation of the host nucleic acid can be 35bp, 40bp, 45bp, 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 105bp, 110bp, 115bp, 120bp, 125bp, 130bp, 135bp, 140bp, 145bp, 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp , 200bp, 205bp, 210bp, 215bp, 220bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp , 285bp, 290bp, 295bp, 300bp, 305bp, 310bp, 315bp, 320bp, 325bp, 330bp, 335bp, 340bp, 345bp, 350bp, 355bp, 360bp, 365bp, 370bp, 375bp, 380bp, 385bp, 390bp, 395bp, 400bp, 405bp, 410bp, 415bp, 420bp, 425bp, 430bp, 435b p, 440bp, 445bp, 450bp, 455bp, 460bp, 465bp, 470bp, 475bp, 480bp, 485bp, 490bp, 495bp, 500bp, 505 bp, 510bp, 515bp, 520bp, 525bp, 530bp, 535bp, 540bp, 545bp, 550bp, 555bp, 560bp, 565bp, 570bp, 575bp, 580bp, 585bp, 590bp, 595bp, 600bp, 650bp, 700bp, 750bp, 800bp, 850bp, and 900bp, or a range consisting of these values.

[0105] According to the embodiments of the present application, the length of the fragments after host nucleic acid degradation can include the above lengths, which is related to the type and amount of enzyme used in step 1, and also to the reaction system and concentration. The reaction system of the present application is a liquid with a salt concentration of 10-600mM.

[0106] In some optional embodiments, in the step of obtaining microbial nucleic acid, the length of the nucleic acid fragment of the microorganism in the sample for detecting the microorganism is ≥1 kb, optionally 1-10 kb or 1 kb-5 kb,

[0107] Alternatively, the length of the nucleic acid fragment of the microorganism in the sample used to detect the microorganism can be 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2. 9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, 5kb, 5.1kb, 5.2kb, 5.3kb, 5. 4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6kb, 6.1kb, 6.2kb, 6.3kb, 6.4kb, 6.5kb, 6.6k b, 6.7kb, 6.8kb, 6.9kb, 7kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, Any value among 7.9kb, 8kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kbkb, 8.8kbkb, 8.9kb, 9kb, 9.1kb, 9.2kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, and 10kb, or a range consisting of these values.

[0108] In related technologies, the length of second-generation sequencing is approximately 35-600 bp, while nanopore sequencing has no length restrictions and can be either long or short. In the step of obtaining microbial nucleic acid, the presence of relatively long fragments in the microbial nucleic acid fragments helps improve the accuracy of sequencing comparisons. The length of the microbial nucleic acid fragments used in the step of obtaining microbial nucleic acid is related to the extraction and / or enrichment methods. MDA is a preferred enrichment method.

[0109] In some optional embodiments, the nuclease is capable of degrading DNA and / or RNA, and the nuclease is M-SAN nuclease and / or Benzonase nuclease and / or high-salt-tolerant nuclease, and can further be M-SAN nuclease.

[0110] Through research, it was found that different nucleases were used to fully degrade the host nucleic acid, and the degradation fragments obtained were of different sizes. The degradation fragments obtained by Benzonase nuclease were about 200bp, M-SAN nuclease was about 100bp, and the degradation fragments obtained by high-salt-tolerant universal nuclease and HL-SAN were about 100-200bp. The nucleic acid fragments obtained by M-SAN nuclease are relatively lower, which is more beneficial for metagenomic detection. For example, compared with microbial nucleic acids, they are shorter, there are more or easier methods to enrich microbial nucleic acids, there are less useless data that cannot be matched to the genome during sequencing, the detection efficiency is higher, and the detection results are more accurate. Based on this, the present application provides a sample preparation method for detecting microorganisms when the host nucleic acid cannot be degraded to less than 35bp.

[0111] In some optional embodiments, the method satisfies at least one of the following conditions:

[0112] 1) In the treatment step, the treatment is performed in a liquid having a salt concentration of 10-600 mM, optionally 19-565 mM;

[0113] 2) using a host cell lysis reagent and nuclease treatment, including incubating the isolated host cell sample with a host cell lysis reagent, a nuclease, and a nuclease buffer to degrade host nucleic acids;

[0114] Optionally, the components and their concentrations in the nuclease buffer are as follows: 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;

[0115] The host cell lysis reagent is a host cell membrane lysis reagent, which can be further selected from saponin, and further selected from Quillaja sylvestris bark saponin. The active ingredient in the saponin is used at a concentration of 0.01-10%. More preferably, the active ingredient in the saponin is used at a concentration of 0.3-0.7%, optionally 0.32-0.6%, and further optionally 0.34-0.54%.

[0116] It is understood that the mass concentration of saponin in the in vitro host cell mixed system is within the above range.

[0117] According to the embodiments of the present application, the use of the above-mentioned concentrations of in vitro host cells, microorganisms that may be present in the in vitro host cell sample, and saponin concentrations helps to fully lyse the in vitro host cells and maintain the activity of microbial nucleic acids that may be present in the in vitro 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.

[0118] In some optional embodiments, the method includes: rupturing the cell membrane of the in vitro host cell sample and maintaining the activity of the nucleic acids of the microorganisms that may be present in the in vitro host cell sample by adding a saponin solution, or by adding a saponin solution and a PBS buffer, or by adding a saponin solution, a PBS buffer, NaCl and MgCl2, or by adding a cell lysis solution.

[0119] In some optional embodiments, in the step of inactivating the nuclease, any one or more of heat inactivation, tris(2-carboxyethyl)phosphine, proteinase K, or EDTA is used to inactivate the nuclease.

[0120] The step of obtaining microbial nucleic acid does not include centrifugation and / or washing steps before extracting nucleic acid.

[0121] The extracted nucleic acid in the step of obtaining microbial nucleic acid includes DNA and / or RNA.

[0122] Tris(2-carboxyethyl)phosphine (TCEP) is a reducing agent that can denature multimeric protein structures, such as large protein complexes or aggregates. By disrupting non-covalent interactions between proteins, TCEP can disaggregate multimers into individual subunits. It can also inactivate nucleases.

[0123] According to the embodiments of the present application, while saponin is used for cell lysis, nuclease is added to degrade the host nucleic acid exposed to the solution, TCEP is directly added to inactivate the nuclease, and the centrifugation and / or washing steps are not included before the nucleic acid extraction in the step of obtaining microbial nucleic acid, thereby minimizing the loss of pathogen samples, especially pathogens such as viruses.

[0124] Proteinase K breaks down proteases, effectively increasing the utilization of protease activity and enhancing the body's immunity and immune response. Proteinase K is a highly active proteolytic enzyme isolated from Candida albicans. It is used to separate plasmids or genomic DNA and can directly enhance the body's absorption and utilization of proteins.

[0125] Ethylenediaminetetraacetic acid (EDTA) is an organic compound with the chemical formula C 10 H 16N2O8 is a white powder at room temperature and pressure, which can inactivate nucleases.

[0126] In some optional embodiments, in the step of obtaining microbial nucleic acid, after extracting total nucleic acid, the method further comprises: using the first length difference to further enrich or separate the nucleic acid of the microorganism obtained by extracting nucleic acid in the step of obtaining microbial nucleic acid, so as to increase the proportion of nucleic acid of the microorganism in the sample for detecting microorganisms; and / or,

[0127] The first length difference is maintained in the method for extracting nucleic acid.

[0128] This can be understood as follows: without affecting the length of the nucleic acid of the microorganism to be detected, the host nucleic acid (which can be understood here as including nucleic acid fragments of the host's common genome) can be made to be of a shorter length. The first length difference between the length of the microorganism nucleic acid and the host nucleic acid is maintained for subsequent detection.

[0129] According to the embodiments of the present application, a long-fragment nucleic acid extraction kit can be used. The main principle or difference from the ordinary kit is that it avoids column extraction and severe mechanical grinding methods, and keeps the fragments long.

[0130] In some optional embodiments, the step of obtaining microbial nucleic acid further comprises, after extracting the nucleic acid, a step of degrading the host mitochondrial DNA and / or the host ribosomal RNA.

[0131] Optionally, the length of the fragments after degradation of the host mitochondrial DNA and / or host ribosomal RNA is less than the length of the nucleic acid of the microorganism obtained by extracting the nucleic acid in the step of obtaining microbial nucleic acid, forming a second length difference.

[0132] In some optional embodiments, the second length difference can be used to further enrich or separate the nucleic acid of the microorganism obtained by extracting nucleic acid in the step of obtaining microbial nucleic acid, so as to increase the proportion of the nucleic acid of the microorganism in the sample for detecting microorganisms;

[0133] This can be understood as shortening the host's mitochondrial nucleic acid without affecting the length of the microbial nucleic acid being tested. A second length difference between the microbial nucleic acid and the host's mitochondrial nucleic acid is maintained for subsequent testing. Optionally, the method for degrading the host's mitochondrial DNA and / or ribosomal RNA is specific degradation, further optionally using CRISPR, and further optionally using CRISPR / Cas9.

[0134] In some optional embodiments, the second length difference is less than or equal to the first length difference, so that the same method can be used to separate or enrich the microbial nucleic acid once.

[0135] In some optional embodiments, in the step of obtaining microbial nucleic acid, after the nucleic acid is extracted, a step of nucleic acid enrichment and / or amplification using method a and / or method b is included to increase the amount of nucleic acid in the sample for detecting microorganisms.

[0136] Method a represents randomly shearing the nucleic acid and then adding a universal amplification linker, and then performing PCR amplification. Optionally, the nucleic acid is randomly sheared and then adding a universal amplification linker using Tn5 enzyme; or

[0137] b Method represents enrichment and / or amplification of nucleic acid of the microorganism, further optionally, using a multiple displacement amplification (MDA) method.

[0138] Research has found that compared with method a, using method b for amplification followed by nanopore sequencing yields a larger amount of data. The larger amount of data obtained is beneficial for improving detection accuracy. Due to the varying lengths of amplified product fragments, the number of unclassified reads is significantly reduced. Since the host-depleted product undergoes a selective amplification step, the MDA-amplified product has a lower human-derived fraction and a higher pathogenic fraction after sequencing. In the examples of this application, the nucleic acids of the microorganisms remain long fragments relative to the host nucleic acid fragments after de-hosting.

[0139] MDA amplification, also known as Multiple Displacement Amplification (MDA), is an isothermal amplification method that amplifies DNA at multiple sites on DNA. It is a specialized DNA amplification technique that uses phi29 DNA polymerase to amplify DNA under isothermal conditions. By leveraging MDA's preferential amplification of long fragments and its less pronounced effect on short fragments, it can selectively amplify nucleic acids of the microorganism being detected based on either a secondary or primary length difference.

[0140] In some optional embodiments, the microorganisms include bacteria, fungi and / or viruses, and the viruses include DNA viruses and / or RNA viruses.

[0141] In some optional 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 (Group A Streptococcus), Staphylococcus hominis, Staphylococcus hemolyticus, Staphylococcus capitis, and Streptococcus agalactiae (Group B Streptococcus).

[0142] In some optional embodiments, the fungus includes 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.

[0143] In some optional embodiments, the DNA virus includes at least one of DNA phage, Epstein-Barr virus, mumps virus, and adenovirus.

[0144] In some optional 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.

[0145] In some embodiments, the method comprises adding a cell lysis solution, a nuclease, and a nuclease buffer, incubating at 37°C for 15 minutes, and then adding a nucleic acid inactivator for inactivation. The method does not require PBS washing and centrifugation, and nucleic acid extraction can be performed directly. The extracted nucleic acid further reduces the proportion of host nucleic acid by degrading mitochondrial DNA. The cell lysis solution is Quillaja saponin, and the mass concentration of the saponin in the isolated host cell mixture is 1.5%-3%. The nuclease is M-SAN, and the final concentration in the mixture is 700-900 U / ml.

[0146] The components in the nuclease buffer and their usage concentrations are as follows: 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.

[0147] The nuclease buffer, when added to the mixed system, has the following final concentrations: NaCl at 125mM–250mM, MgCl2 at 4–15mM, and Tris-HCl at pH 7.2–8.7 at a concentration of 25–50mM. The nucleic acid inactivator is TCEP, with a final concentration of 10mM in the mixed system. The method for degrading mitochondrial DNA is CRISPR-Cas9.

[0148] According to the embodiments of the present application, the product name of the nuclease M-SAN can be M-SAN HQ (Bioprocessing grade), Triton FREE, 25-25k, 70950-202, can be from the brand ArcticZymes, and can be stored at -20°C.

[0149] According to the examples of the present application, the preparation method of the present application allows for rapid release and degradation of host DNA, eliminating the time-consuming, costly, inefficient, extraction-biased, and partial microbial loss. During downstream DNA library processing, this further specifically reduces host DNA sequencing reads, effectively lowering host DNA background and increasing the available sequencing data for the remaining microorganisms.

[0150] sample

[0151] In a second aspect, an embodiment of the present application provides a sample for detecting microorganisms, which is prepared by the method of the first aspect.

[0152] According to the embodiments of the present application, the amount of microbial nucleic acid that may be detected in the sample, or the absence of the microorganisms that may be present, is determined. Metagenomic sequencing of the sample is performed with higher accuracy.

[0153] Detection methods for microorganisms

[0154] In a third aspect, the present invention provides a method for detecting microorganisms, comprising:

[0155] Obtaining a sample for detecting microorganisms obtained by the preparation method of the first aspect, or a sample for detecting microorganisms of the second aspect;

[0156] performing metagenomic sequencing on the sample to determine the detection results of the microorganisms;

[0157] The sequencing includes second-generation sequencing based on PCR amplification, or single-molecule sequencing based on nanopore, optionally, single-molecule sequencing based on nanopore.

[0158] According to the embodiments of the present application, after effectively removing the host genomic DNA from the sample, the effective sequencing data of the remaining microorganisms can be improved in the metagenomic sequencing, making the detection results more accurate and the detection more sensitive.

[0159] In some optional embodiments, the upper limit of the ratio of the number of the host cells to the number of the microorganisms in the in vitro host cell sample is: (10 4 -10 7 ):1;

[0160] Optionally, the microorganism is a bacterium, and the upper limit of the number ratio is 10 7 :1;

[0161] Optionally, the microorganism is a virus, and the upper limit of the number ratio is 10 4 -10 7 :1;

[0162] Optionally, the microorganism is a fungus, and the upper limit of the number ratio is 10 7 :1; Optionally, the concentration of the host cells in the isolated host cell sample is 1×10 4 , 1×10 5 , 1×10 6 or 1×10 7 cells / mL;

[0163] The concentration of the microorganism in the isolated host cell sample is 1, 10, 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 or 1×10 7 CFU / mL.

[0164] In some embodiments, the method further comprises:

[0165] Performing nucleic acid adapter ligation on the sample, and physically and / or chemically enriching the nucleic acid fragments ligated by the nucleic acid adapter to obtain a sample to be sequenced;

[0166] Perform metagenomic sequencing on the samples to be sequenced to determine the detection results of the microorganisms.

[0167] In some embodiments, the method further comprises:

[0168] Identify the type of microorganism,

[0169] Perform metagenomic sequencing on the sample to obtain a sequencing sequence;

[0170] According to the type of the microorganism and the sequencing sequence, it is determined whether the type of the microorganism is contained.

[0171] According to the embodiments of the present application, the relevant testing personnel have clear suspicions about the types of microorganisms in the sample and have roughly determined the types of microorganisms. They can use software or other tools to compare and analyze the sequencing sequence with the sequence of the type of microorganism, and thus determine whether the sample contains microorganisms related to the previously suspected type based on the comparison results.

[0172] In some embodiments, the method further comprises:

[0173] Perform metagenomic sequencing on the sample to obtain a sequencing sequence;

[0174] The sequencing sequence is analyzed to obtain an analysis result, so as to determine whether the sample contains microorganisms based on the analysis result.

[0175] According to the embodiments of the present application, if the tester is unsure of the type of microorganism in the sample, the tester can use software to analyze the sequence to obtain the analysis results. The tester can then use manual comparison or other software to compare and combine other information to determine whether the sample contains microorganisms.

[0176] In some embodiments, the method further comprises:

[0177] Perform metagenomic sequencing on the sample to obtain a sequencing sequence;

[0178] Analyzing the sequencing sequence to obtain an analysis result;

[0179] In response to the analysis result, a detection result of the microorganism in the sample is output.

[0180] According to the embodiments of the present application, if the tester is unsure of the type of microorganism in the sample, the tester can use software to analyze the sequence to obtain the analysis result. The software is used to compare and determine whether the sample contains microorganisms and output the test result.

[0181] In some embodiments, the method further comprises:

[0182] In response to the analysis result, the detection result of the microorganisms in the sample is output and the types of the microorganisms are displayed.

[0183] According to the embodiments of the present application, if the tester is unsure of the type of microorganism in the sample, they can use software to analyze the sequence to obtain an analysis result. Using relevant software for comparison, the software determines and outputs the test result of whether the sample contains microorganisms. If microorganisms are present, the type of microorganism is also displayed.

[0184] Kit for detecting microorganisms in samples

[0185] In a fourth aspect, the present invention provides a kit for detecting a sample of a microorganism, comprising: a host cell lysis reagent, a nuclease, and a nuclease buffer,

[0186] Optionally, the salt in the nuclease buffer is used at a concentration of 10-600 mM, 15-590 mM or 19-565 mM,

[0187] Optionally, the salt includes MgCl2, Tris-HCl and neutral salt,

[0188] Optionally, the components in the nuclease buffer and their usage concentrations are as follows: 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.

[0189] Optionally, the host cell lysis reagent includes a host cell membrane lysis reagent, and optionally, the host cell membrane lysis reagent is a saponin, and further optionally, a Quillaja saponin, and more optionally, the active ingredient in the saponin is used at a concentration of 0.3-0.7%, optionally 0.32-0.6%, and further optionally 0.34-0.54%.

[0190] Optionally, the nuclease is capable of degrading DNA and / or RNA, and may be M-SAN.

[0191] Method for constructing sequencing library for pathogenic microorganism detection

[0192] In a fifth aspect, the present invention provides a method for constructing a sequencing library for pathogenic microorganism detection, comprising:

[0193] R1. Providing a sample to be tested, wherein the length of the host nucleic acid fragment in the sample to be tested is less than 200 bp, and the length of the nucleic acid fragment of the pathogenic microorganism that may be present in the sample to be tested is ≥ 1 kb, which can be 1-10 kb or 1 kb-5 kb;

[0194] R2. performing nucleic acid multiple displacement amplification on the sample to be tested to increase the proportion of nucleic acid fragments of pathogenic microorganisms that may be present in the sample to be tested, thereby obtaining multiple displacement amplification products;

[0195] R3. Construct a library for the multiple displacement amplification products to obtain a sequencing library.

[0196] In step R2, the sample to be tested can be subjected to electrophoretic gel recovery, magnetic bead fragment sorting, or molecular chromatography exchange column analysis. However, subsequent amplification is required, which is cumbersome and not conducive to the detection of trace pathogen nucleic acids. Research has found that the use of multiple displacement amplification in step R2 does not require additional amplification, effectively combining the long-fragment screening and amplification achieved by electrophoretic gel recovery, magnetic bead fragment sorting, or molecular chromatography exchange column analysis.

[0197] In some optional embodiments, the sample to be tested can be prepared by the method of the first aspect or come from the sample of the second aspect.

[0198] Application of the method of the first aspect, the sample of the second aspect, the method of the third aspect, the kit of the fourth aspect, or the method of the fifth aspect in pathogenic microorganism metagenomic sequencing, or in preparing a product for pathogenic microorganism metagenomic sequencing.

[0199] Kits for microbiology

[0200] In a fourth aspect, the present invention provides a kit for microorganisms, comprising:

[0201] Cell lysis solution, nuclease, and nuclease buffer are added to the sample to be tested at the same time, and after incubation at 37°C for 15 minutes, a nucleic acid inactivator is added for inactivation. The method does not require PBS washing and centrifugation, and nucleic acid extraction can be performed directly. The extracted nucleic acid further reduces the proportion of host nucleic acid by degrading the cell mitochondrial DNA. The cell lysis solution is Quillaja saponin, and the mass concentration of the saponin in the in vitro host cell mixed system is 1.5%-3%. The nuclease is M-SAN, and the final concentration in the sample to be tested is 700-900U / ml. The final concentration of the components of the nuclease buffer added to the sample to be tested is: NaCl, 125mM-250mM, MgCl 2， 4-15mM, PBS pH 7.2–8.7, Tris-HCl pH, 7.2-8.7 25-50mM. The nucleic acid inactivator is TCEP, and the final concentration in the sample to be tested is 10mM. The method for degrading mitochondrial DNA is CRISPR-Cas9.

[0202] Example

[0203] The following examples describe the present disclosure in more detail and are intended to be illustrative only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and used directly without further processing, and all instruments used in the examples are commercially available.

[0204] The bacteria and virus samples in the examples of this application are commercially available products.

[0205] Specific steps:

[0206] 1. De-hosting

[0207] Take 300 μL of mixed sample, add 100 μL of cell lysate (containing 10% saponin, the saponin was purchased from Sigma, product number S4521, from the bark of Quillaja sapodilla, containing the active ingredient Sapogenin 20-25%), 50 μL of 10× nuclease buffer (a mixed solution of 0.25 M Tris-HCl with 1.5 M NaCl and 0.1 M MgCl2, pH 7.2-8.7), and 10 μL of nuclease M-SAN (ArcticZymes, 70950-202, sufficient volume) in 1.5 mL of sterile EP, shake at 1000 rpm to mix, and react at 37°C for 15 min.

[0208] Add 50 μL of 10×TCEP (100 mM), mix by pipetting, and react at room temperature for 5 minutes to obtain a host-free sample.

[0209] 2. Nucleic Acid Extraction

[0210] The host nucleic acid-free sample obtained in step 1 was subjected to nucleic acid extraction using the VAMNE Magnetic Pathogen DNA / RNA Kit (Novozymes, catalog number RM601) to obtain total nucleic acid (gDNA). When viral RNA needs to be detected, the product needs to be reverse transcribed to obtain DNA.

[0211] 3. CRISPR-Cas9 for mitochondrial cleavage

[0212] CRISPR-Cas9 cleavage of mitochondria, 100:100:1 (Cas9:sg-RNA:DNA) cleavage site molar ratio:

[0213] A total of 30uL of Cas9 treatment system was added, and the cells were treated at 37°C for 30 minutes. 1uL of proteinase K was added and the cells were treated at 37°C for 15 minutes.

[0214] In addition, the sgRNA is a commercially available known sequence, and the length of the fragment formed after the final cutting of mitochondrial DNA is 100-200bp.

[0215] The amount of X and Y added depends on the mass of the nucleic acid. For example, X uL can be 1 uL or 2 uL, and Y uL can be 1 uL or 2 uL.

[0216] 4. PCR Amplification of DNA

[0217] 1. Prepare DNA fragmentation system in a sterile PCR tube.

[0218] The active components in the tagment buffer are: 2%-3% BSA, 0.05%-1.5% Tween-20, 0.1-1% SDS and 20mM-30mM DTT.

[0219] The reagent used to dilute the assembly product is 1×Tn5 dilution buffer.

[0220] 2. Use a pipette to pipette up and down to mix thoroughly (you can also use your index finger to flick the liquid to mix).

[0221] 3. Reaction procedure: 55℃ for 5 minutes, then place on ice for 2 minutes.

[0222] 4. Add 6uL of 6x termination buffer, mix well and let stand at room temperature for 5 minutes.

[0223] 5. Add 0.8× 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.

[0224] 6. Add 200uL of 80% ethanol to rinse the magnetic beads, incubate for 30s, remove the supernatant, and repeat once.

[0225] 7. Keep the PCR tube on the magnetic rack, use a 10uL pipette to remove the ethanol remaining at the bottom of the tube, and dry it at room temperature until no ethanol remains (beads appear frosted).

[0226] 8. Remove the PCR tube, add 25uL of nuclease-free water, mix the magnetic beads thoroughly, and incubate at room temperature for 2 minutes.

[0227] 9. Place the PCR tube back on the magnetic rack. After the solution is clarified, aspirate 24uL for subsequent PCR amplification to purify the amplified product.

[0228] Specific steps of PCR amplification:

[0229] Amplification system:

[0230] Amplification procedure:

[0231] The sequence of universal primer P' (10uM) is (5P'-ACTTGCCTGTCGCTCTATCTTC-3) and was obtained from Sangon Biotech (Shanghai) Co., Ltd.

[0232] Premix TaqTM (LA Taq TM Version 2.0) from Takara.

[0233] 5. Sequencing Library Construction

[0234] The product of step 4 was connected to the sequencing adapter according to the library construction kit QLK-V1.1.1 of Qitan Technology to construct the library.

[0235] 6. Nanopore Sequencing

[0236] The ligation product of step five was subjected to nanopore sequencing in the sequencer QNome3841 of Qitan Technology according to the sequencing kit QSK-V1.1.1 of Qitan Technology.

[0237] 7. Bioinformatics analysis of sequencing data.

[0238] Example 1

[0239] Human cells (10 6 / ml) and Escherichia coli (10 6 The cells were mixed with the host-removal method (1:1 / ml) according to step 1, and the host-removal method was used or not. Then, the nucleic acid extracted in step 2 was simultaneously tested by qPCR. After amplification in step 4, sequencing was performed in steps 5 to 7.

[0240] The results are shown in Figure 2, which shows the fluorescence quantitative qPCR results of this example. rpoA refers to the human gene, and cyaA refers to the E. coli gene. After host removal treatment (treated group), the CT value of the human gene changed by more than 10, indicating a 1000-fold host removal effect, while the CT value of E. coli did not change significantly.

[0241] Sequencing results showed that in the untreated group, the E. coli content was less than 1%, and the host DNA content was greater than 99%. After host removal, the E. coli content increased to 43%, while the host DNA content decreased to 38%. Using the method of this application, host removal significantly increased the proportion of bacterial metagenomics.

[0242] Example 2

[0243] Human cells (10 6 / ml) and Escherichia coli (10 5 / ml) and DNA phage T1

[0244] (10 7 / ml) and RNA phage MS2 (10 7 / ml) were mixed in a volume ratio of 1:1:1:1, and the host removal scheme (QT) of step 1 and the untreated scheme U1 were used respectively. Then, qPCR detection was performed simultaneously on the nucleic acid extracted in step 2. After amplification in step 4, steps 5 to 7 were sequenced. After host removal treatment using the method of the present application, the proportion of bacterial, DNA virus, and RNA virus metagenomics was significantly increased.

[0245] Comparative Example 1 (NBT)

[0246] The difference between this comparative example and Example 2 is that the host removal scheme in step 1 adopts the multi-step centrifugation washing host removal scheme involved in the literature (Charalampous, T., Kay, GL, Richardson, H. et al. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection. Nat Biotechnol 37, 783–792 (2019). https: / / doi.org / 10.1038 / s41587-019-0156-5).

[0247] Comparative Example 2 (VT)

[0248] The difference between this comparative example and Example 2 is that the host removal scheme in step 1 adopts the commercial host removal kit Novozymes FastPure Host Removal and Microbiome DNA Isolation Kit DC501-01.

[0249] Comparative Example 3 (ZT)

[0250] The difference between this comparative example and Example 2 is that the host removal scheme in step 1 is performed using a commercial host removal kit ZYMO brand ZT HostZERO Microbial DNA Kit D4310.

[0251] The comparative results of Example 2 and Comparative Examples 1-3 are shown in Figure 3, which shows the qPCR results of the human genome, DNA phage T1, and RNA phage MS2. In the figure, the three groups from left to right are the human genome, DNA phage T1, and RNA phage MS2. Compared with the untreated sample U1, only the host removal scheme QT in step 1 reduces the loss of DNA phage and RNA phage while removing the host from the human genome, and the CT value changes within 1. The other comparative examples 1-3 (NBT, VT, ZT) all lost a large amount of DNA phage and RNA phage.

[0252] From the metagenomic sequencing results in the right figure, it can be seen that after removing the host, the host removal scheme QT in step one not only improved the detection rate of Escherichia coli, but also significantly improved the detection rate of DNA phage and RNA phage.

[0253] Example 3: Detection of the de-hosting effect of different viruses

[0254] The artificially cultured viruses were gradiently diluted and then divided into two groups: a treated group (performed according to the above step 1) and an untreated group, and then nucleic acid extraction (performed according to the above step 2) and qPCR detection were performed.

[0255] The smaller the change in CT value, the less destructive effect the host removal method in step 1 of the present application has on different viruses.

[0256] Table 1. Changes in Ct values ​​of different viruses detected by qPCR in the treated group relative to the untreated group

[0257] The results are shown in Table 1. Compared with the untreated group, for RNA viruses, the Ct values ​​of influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, and coronavirus in the treated group did not change by more than 1; the Ct value of rhinovirus in the treated group changed by about 1-2; for DNA viruses, the Ct value of Epstein-Barr virus, mumps virus, and adenovirus in the treated group also changed by less than 1, that is, the host removal treatment method provided in step 1 had little destructive effect on different viruses.

[0258] Example 4: Simulated sample testing of host removal effect

[0259] 1. Bacteria simulation sample

[0260] First, human cells were counted under a microscope using a hemocytometer, and E. coli was counted using the LB plate counting method. 2 -10 4 CFU / mL) and human cells (10 5 -10 7 Cells were serially diluted and mixed, and then subjected to the aforementioned steps 1 and 2 for host removal and nucleic acid extraction, respectively. Untreated cells without host removal served as a control. The host-removed nucleic acids were subjected to nanopore library construction and sequencing in steps 4-7. The results are shown in Table 2.

[0261] The results show that in the untreated group, the human background 7 cells / mL, the concentration of E. coli was 10 2 -10 4 The number of sequences detected at CFU / mL was 0. The human background was 105 -10 6 When the number of cells / mL is 10, the number of sequences of E. coli is also 0. The reason is that the genome size of a human cell is 1000 times that of an E. coli genome. When the number of E. coli is much lower than that of human cells, the amount of sequencing data required is huge. After host removal, E. coli can be detected in all grouped samples, and the abundance is close to the change of dilution multiple. 5 -10 7 The lowest detection concentration (LOD) of Escherichia coli reached 10 2 CFU / mL.

[0262] Table 2. Nanopore sequencing results of bacterial LOD experiments

[0263] Note: The first digit of the two-digit grouping column in the table represents the order of magnitude of the host cell concentration after mixing, and the second digit represents the order of magnitude of the E. coli concentration after mixing.

[0264] Furthermore, after step 2, step 3 of CRISPR was added to remove host mitochondrial DNA, and then steps 4-7 of nanopore library construction and sequencing were performed. 5 -10 7 The lowest detection concentration (LOD) of Escherichia coli reached 10 CFU / mL under the human background of 10 cells / mL.

[0265] The above results show that the library constructed using the method of the present application has more readable sequences than the untreated group. When there are more readable sequences, the detection sensitivity and accuracy of microorganisms are greatly improved, and a sequencing library with more practical value is obtained.

[0266] 2. Virus simulation samples

[0267] In order to verify the LOD of the host-free system on the virus cell simulation sample, we 6 10 cells / mL were mixed with the human background. 2 -10 7 We used qPCR to verify the difference in ADV nucleic acid content before and after host removal.

[0268] Table 3. Virus cell LOD nanopore sequencing results

[0269] In 10 6Nanopore sequencing of adenovirus (ADV) virus serially diluted in a human background at a concentration of 10 cells / mL was performed to determine the number and percentage of characteristic adenovirus sequences detected. 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 unit, i.e., milliliters. "Copy / ml" indicates the number of copies per milliliter of solution.

[0270] As shown in Figure 4 and Table 3, the results show that host removal does not significantly reduce the ADV nucleic acid content and the detection effect shows a linear gradient distribution with the input amount. The nucleic acid after host removal was subjected to nanopore sequencing, and the results are shown in Table 3. Ideally, since the adenovirus ADV genome is only 30kb, while the human genome is 2×3Gb in size. Without host removal, 10 7 Copies / mL ADV virus and 10 6 The nucleic acid ratio of cells / mL of human cells is 1:20,000, and the virus accounts for 0.005%.

[0271] The actual sequencing results are also close to this ratio, which is 0.006%, indicating that viruses, which are microorganisms with small genomes, can be detected even if their copy numbers are high (10 6 ), and it is also difficult to identify by sequencing without removing the host.

[0272] After the host was removed, as shown in Figure 5, the detection LOD of adenovirus was found to be reduced to 10 4 copies / mL, greatly improving the sensitivity of pathogen detection. 6 cells / mL human background, (a) is untreated 10 7 The genome coverage of ADV is shown in Table 1. (b)-(e) show the genome coverage of ADV after host removal. 4 -10 7 The ADV genome coverage distribution of each adenovirus sample was compared in the sequencing results, and the results are shown in Figure 5. I found that even at adenovirus 10 4 / mL, only 10 characteristic sequences were detected, and the distribution of their sequences on the genome was also random, meeting the standard of detecting 3 characteristic sequences required for conventional virus identification.

[0273] Furthermore, after step 2, step 3 of CRISPR was added to remove host mitochondrial DNA, and then steps 4-7 of nanopore library construction and sequencing were performed. 6 The lowest detection concentration (LOD) of adenovirus reached 10 cells / mL under human background.3 copies / mL.

[0274] 3. Blood simulation sample

[0275] In order to verify the host-removal effect of the host-removal system in real clinical scenarios, negative blood samples (leukocytes 10 7 / ml) were added with different concentrations of four common clinical standard pathogens, and the host was removed and not removed, respectively. Then, nanopore sequencing of steps 2, 4-7 was performed. The results are shown in Table 4.

[0276] Table 4. Blood simulation sample LOD test

[0277] RPM value (a method for standardizing statistical data of second-generation sequencing, that is, the number of characteristic sequences aligned to the sample per million sequences).

[0278] As shown in Table 4, the RPM value of bacteria after host removal was significantly higher than that of the control group, and the RPM value of the control group was lower, and most bacteria were lower than the clinical RPM ≥ 10 judgment standard. This confirms that the effect of our host removal can significantly improve the sensitivity of pathogen detection to 10 in blood flow simulation samples. 2 CFU / mL pathogen.

[0279] Example 5: The proportion of pathogenic nucleic acids increased after removing mitochondrial nucleic acids

[0280] Human cells (10 6 / ml) and adenovirus ADV (10 7 / ml) were mixed at a volume ratio of 1:1, and treated according to the methods of steps 1 to 7 above. The results were compared with those without the host removal treatment in step 1 or the degraded mitochondrial DNA without step 3. The results are shown in Table 5.

[0281] Table 5

[0282] The results in Table 5 show that after host removal, the proportion of adenovirus increased from 0.0006% to 2.4%, while the proportion of human origin decreased from 99.6% to 97.3%. Mitochondrial origin accounted for 94.3% of the total after host removal. After further host removal using CRISPR, the proportion of ADV increased to 19.9%, the proportion of human origin decreased to 78.3%, and the proportion of mitochondrial origin decreased to 58.1%.

[0283] Example 6: Analysis of Short Fragments of Host Nucleic Acids after Host Removal

[0284] 1. The host cells, E. coli or a mixture of the two were treated according to the aforementioned steps 1 and 2, while the cells not subjected to step 1 were used as the untreated group.

[0285] Electrophoresis was performed to examine the length of nucleic acid fragments after extraction. The results are shown in Figure 6. In Figure 6, B1 and C1 represent individual cells without host and untreated; D1 and E1 represent individual bacteria without host and untreated; and F1 and G1 represent a mixture of bacterial cells without host and untreated. Compared to the untreated group, host-depletion treatment of the host cells alone resulted in shorter extracted nucleic acid fragments, approaching 100 bp. Host-depletion treatment of the bacteria alone did not significantly alter the extracted nucleic acid fragments. Compared to the untreated control, host-depletion treatment of the mixture resulted in fragment lengths primarily close to 100 bp. Therefore, after host depletion, host-derived nucleic acid fragments become short, while bacterial nucleic acid lengths remain largely unchanged.

[0286] 2. Analysis of interference of short fragments on sequencing results

[0287] The fragments of approximately 100 bp were evenly distributed on human chromosomes after next-generation sequencing analysis, proving that there is no similarity between short host fragments of approximately 100 bp after host removal. As shown in Figure 7, Figure a represents unclassified sequences; unclassified sequences refer to sequences that have not been uniquely identified as pathogenic species after sequence alignment. (b) Median unclassified length; (c) Median total read length. Compared with the untreated group, the proportion of unclassified sequences after host removal was higher. The median total sequence length after host removal was also lower than that of the untreated group. However, there was no significant difference in the median unclassified length between the host removal group and the untreated group, and the lengths were both shorter than the total sequence lengths of the two groups. This proves that short fragments can affect pathogen identification, resulting in a large number of sequences that fail to match the database. This proves that short fragments affect metagenome alignment.

[0288] 3. Treat the host cells according to steps 1 and 2 above, and use sufficient amounts of different nucleases, such as M-SAN, Benzonase, high-salt-tolerant universal nuclease, or HL-SAN. Meanwhile, the host cells that did not undergo step 1 were used as the untreated group.

[0289] When using Benzonase (Nanjing Novozymes, Catalog No. RM1022-00), the concentration of Benzonase in step 1 reaction is 500 U / mL, the concentration of NaCl is 0 mM, the concentration of MgCl2 is 6 mM, and the concentration of Tris-HCl is 20 mM, pH 8.5.

[0290] When using high-salt-tolerant universal nuclease (Yisheng Bio, Cat. No. 20159ES25), the concentration of high-salt-tolerant nuclease in step 1 is 500 U / mL, the concentration of NaCl is 500 mM, the concentration of MgCl2 is 5 mM, and the concentration of Tris-HCl is 25 mM, pH 8.5.

[0291] When using HL-SAN (ArcticZymes, Cat. No. 70910-202), in step 1, the concentration of HL-SAN is 500 U / mL, the concentration of NaCl is 500 mM, the concentration of MgCl2 is 10 mM, and the concentration of Tris-HCl is 25 mM, pH 8.5.

[0292] Electrophoresis was used to measure the length of nucleic acid fragments after extraction. The results are shown in Figure 8. In the figure, B1 and C1 were treated with M-SAN nuclease to remove the host; D1 and E1 were untreated; and F1 and G1 were treated with Benzonase nuclease to remove the host. Compared to the untreated group, all four nucleases resulted in shortening of the host nucleic acid, with the difference being that Benzonase nuclease degraded the host nucleic acid to form fragments of approximately 200 bp. In addition, high-salt-tolerant universal nuclease and HL-SAN degraded the host nucleic acid to form fragments of approximately 100-200 bp (Figure not provided).

[0293] Example 7: Effect of MDA on Enrichment of Long Fragments

[0294] 1. The human genome and E. coli genome, derived from human renal epithelial cells HEK293T, were broken down into long fragments L (approximately 10 kb) and short fragments S (approximately 100 bp), respectively, as shown in Figure 9. In the figure, B1 represents L cells; C1 represents L bacteria; D1 represents S cells; and E1 represents S bacteria.

[0295] As shown in Table 6, these four fragments were mixed in pairs at ratios of 1:10 and 10:1, then subjected to MDA amplification and nanopore sequencing. The results show that when the MDA substrates are all long fragments, the ratio of amplified products is close to the original ratio of the substrates. When the substrates contain short fragments, the amplified products are primarily derived from the long fragments and are not affected by the substrate ratio, demonstrating the selective amplification of long fragments by MDA.

[0296] Table 6. Nanopore sequencing data analysis results after MDA amplification

[0297] When the same sample was subjected to MDA amplification and Tn5 interruption amplification (step three above), the sequencing results, as shown in Table 6, showed that the MDA-amplified product had a higher data output on the nanopore sequencing platform. Due to the long fragments of the amplified products, the number of unclassified reads was significantly reduced. Due to the selective amplification of the host-free products, the products after MDA amplification had a lower proportion of human origin and a higher proportion of pathogens.

[0298] MDA amplification system preparation table:

[0299] The above reagents were obtained from Novezan Discover-sc Single Cell WGA Kit (N603).

[0300] 2. The nucleic acids of the mixed host and pathogen-removed simulated samples were subjected to MDA amplification, Tn5 amplification (step 4), and second-generation sequencing interruption amplification, and the products after MDA amplification were also interrupted by second-generation library construction. The MDA and Tn5 amplification products were then subjected to nanopore sequencing. The MDA amplification products were interrupted by second-generation sequencing and then subjected to second-generation sequencing together with the products of normal second-generation library construction. The results are shown in Figure 10. Among them, B1-D1 are the MDA amplification fragments of the host-removed product, E1-G1 are the Tn5 interruption amplification fragments, H1-B2 are the second-generation sequencing interruption amplification fragments, and C2-E2 are the second-generation sequencing interruption fragments after MDA amplification (b) Comparison of abundance after sequencing with different treatments. This shows that effective amplification products were obtained.

[0301] Example 8: Detection of fungi

[0302] In artificially cultured human cells (concentration of 10 6 Different concentrations (10 5 , 10 4 , 10 3 , 10 2 albicans (10, 10, or 1 copies / mL), and then divided into two treatment groups: a treated group (performed according to the above step 1) and an untreated group. Total nucleic acid was then extracted (performed according to the above step 2) and qPCR detection was performed, MDA amplification was performed in Example 7, and sequencing was performed in steps 5-7.

[0303] The qPCR test results showed that the Ct value of Candida albicans did not change by more than 1 before and after the host removal treatment in step 1, indicating that the host removal treatment method provided in step 1 had little destructive effect on the fungus.

[0304] The sequencing results showed that the LOD of Candida albicans was: 10 1 copies / mL.

[0305] Furthermore, after step 2, CRISPR was added to remove host mitochondrial DNA in step 3, and MDA amplification was performed in Example 7, and sequencing was performed in steps 5-7. 6 The LOD of Candida albicans can reach 10 cells / mL in a human background. 0 copies / mL.

[0306] Example 9: Detection of simulated blood samples

[0307] Negative blood samples from healthy people (leukocyte 10 7Four common clinical standard pathogens (Candida albicans - fungi, Klebsiella pneumoniae - Gram-negative bacteria, Enterococcus faecalis - Gram-positive bacteria, and bacteriophage T1 - DNA virus) were added to a 4% PBS (100 μg / ml) solution and subjected to host removal and non-host removal treatments, respectively. Then, steps 2 and 3, MDA amplification of Example 7, and nanopore sequencing of steps 5-7 were performed. The LOD results of the host removal treatment are as follows:

[0308] Virus is 10 3 Copies / ml, bacteria is 10 0 CFU / ml, fungi are 10 0 CFU / ml.

[0309] Example 10: Clinical application

[0310] In clinical practice, the detection rate of RNA viruses is low. Conventional virus detection methods such as immunological methods such as colloidal gold have low sensitivity. In a patient with mixed upper respiratory tract viral and bacterial infection in the hospital, the patient developed symptoms of upper respiratory tract infection. As shown in Table 7, we collected throat swabs SW and sputum samples SP from the patient, performed host removal and nanopore sequencing, and the results showed that the patient was infected with Hemophilus influenzae infection, and a small amount of influenza B virus sequences were found. The patient developed fever symptoms on the third day and underwent colloidal gold-based influenza B virus diagnosis, and was finally diagnosed with influenza B virus.

[0311] Table 7 Metagenomic sequencing results of a patient with mixed upper respiratory tract bacterial and viral infection

[0312] qPCR testing of the collected nucleic acids for influenza B virus revealed that the Ct value for influenza B decreased with the progression of the disease. The abundance of influenza B virus sequences in the nanopore sequencing results also increased accordingly. Over 80% viral coverage and 7x sequencing depth were achieved for influenza B virus in a sputum sample on day three. This result demonstrates the effectiveness of our host-removal method in diagnosing mixed bacterial and viral infections in clinical settings. It eliminates the risk of missing influenza B virus due to virus discarding by the host-removal method. Furthermore, the high speed of nanopore sequencing provides timely notification of infection status in patients, enabling physicians to provide targeted treatment.

[0313] Combining CRISPR / Cas9 cleavage of mitochondrial DNA with MDA selective amplification of long fragments can further enhance host elimination. This is because the nucleic acid fragments cleaved by CRISPR / Cas9 are also 100-200bp in length. As shown in Table 7, after CRISPR cleavage and subsequent MDA amplification, the human fraction of the sample significantly decreased from nearly 50% to 0.5%, while the bacterial fraction increased from 40% to over 85%, compared to the uncleaved sample.

[0314] To verify that MDA amplification is applicable to RNA-derived target sequence amplification, samples infected with mixed RNA viruses previously frozen in a refrigerator were host-free, and libraries were constructed and nanopore sequenced based on MDA.

[0315] Table 8. MDA-based database construction test

[0316] The results, shown in Table 8, effectively detected Haemophilus influenzae and influenza B virus, with abundances similar to those in Table 7, and their concentrations. The table shows the number of valid sequence reads for bacteria and viruses. This demonstrates that MDA amplification is applicable to RNA-derived target sequence amplification, effectively detecting bacteria and viruses, and that the resulting libraries have a high number of valid reads.

[0317] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present application, and such modifications or substitutions should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A sample preparation method for detecting microorganisms, wherein: include: Treatment step: taking an in vitro host cell sample, treating it with a host cell lysis reagent and a nuclease to degrade host nucleic acid, wherein the treatment is performed in a liquid having a salt concentration of 10-600 mM; Nuclease inactivation step: providing conditions to inactivate the nuclease; The step of obtaining microbial nucleic acid is as follows: extracting total nucleic acid to obtain the sample for detecting microorganisms.

2. The method according to claim 1, wherein: The method satisfies at least one of the following conditions: 1) In the treatment step, the nuclease is capable of degrading the host nucleic acid to a first nucleic acid length that is smaller than the second nucleic acid length of the microorganism obtained by extracting the total nucleic acid in the step of obtaining microbial nucleic acid, thereby forming a first length difference, and optionally, the first length difference is at least 100 bp, 2) In the treatment step, the length of the nucleic acid fragment after the host nucleic acid degradation is more than 35 bp, 3) In the treatment step, the length of the nucleic acid fragment after the host nucleic acid degradation is less than 900 bp; optionally, 35-300 bp; 4) In the step of obtaining microbial nucleic acid, the length of the nucleic acid fragment of the microorganism in the sample for detecting the microorganism is ≥ 1 kb, and can be 1-10 kb. 5) The nuclease is capable of degrading DNA and / or RNA, and the nuclease is M-SAN nuclease, Benzonase nuclease and / or high-salt-tolerant nuclease.

3. The method according to claim 1 or 2, wherein: The method satisfies at least one of the following conditions: 1) In the treatment step, the salt concentration is 15-590 mM or 19-565 mM; the salt includes MgCl2 and Tris-HCl, and the pH value of the liquid is 7-9; Optionally, the salt further comprises a neutral salt, and the concentration of the neutral salt in the liquid is 0-500 mM; 2) In the treatment step, the treatment with a host cell lysis reagent and a nuclease comprises incubating the ex vivo host cell sample with a host cell lysis reagent, a nuclease, and a nuclease buffer to degrade host nucleic acids; Optionally, the components and their concentrations in the nuclease buffer include: NaCl: 0-500 mM; MgCl2: 4-15 mM; Tris-HCl: 15-50 mM; pH of the nuclease buffer: 7-9; 3) In the treatment step, the host cell lysis reagent is a host cell membrane lysis reagent, which may be saponin; Optionally, the active ingredient in the saponin is used at a concentration of 0.3-0.7%, optionally 0.32-0.6%.

4. The method according to any one of claims 1 to 3, wherein: In the step of inactivating the nuclease, any one or more of heat inactivation, tris(2-carboxyethyl)phosphine, proteinase K, or EDTA is used to inactivate the nuclease, and / or, The step of obtaining microbial nucleic acid does not include centrifugation and / or washing steps before extracting nucleic acid, and / or, The extracted nucleic acid in the step of obtaining microbial nucleic acid includes DNA and / or RNA.

5. The method according to any one of claims 2 to 4, wherein: In the step of obtaining microbial nucleic acid, after extracting total nucleic acid, the method further includes: using the first length difference to further enrich or separate the nucleic acid of the microorganism obtained by extracting total nucleic acid in the step of obtaining microbial nucleic acid, so as to increase the proportion of nucleic acid of the microorganism in the sample for detecting microorganisms; and / or, In the step of obtaining microbial nucleic acid, the first length difference is maintained in the method of extracting total nucleic acid.

6. The method according to any one of claims 1 to 5, wherein: In the step of obtaining microbial nucleic acid, after extracting the total nucleic acid, the step of degrading the host's mitochondrial DNA and / or the host's ribosomal RNA is also included. Optionally, the length of the fragments after degradation of the host mitochondrial DNA and / or host ribosomal RNA is less than the length of the nucleic acid of the microorganism obtained by extracting the total nucleic acid in the step of obtaining microbial nucleic acid, forming a second length difference.

7. The method according to claim 6, wherein: The second length difference can be used to further enrich or separate the nucleic acid of the microorganism obtained by extracting nucleic acid in the step of obtaining microorganism nucleic acid, so as to increase the proportion of the nucleic acid of the microorganism in the sample for detecting the microorganism; Optionally, the method for degrading host mitochondrial DNA and / or host ribosomal RNA is specific degradation, which can be CRISPR / Cas9.

8. The method according to claim 6, wherein: The second length difference is less than or equal to the first length difference.

9. The method according to any one of claims 1 to 8, wherein: In the step of obtaining microbial nucleic acid, after the nucleic acid is extracted, a step of nucleic acid enrichment and / or amplification using method a or method b is included to increase the amount of nucleic acid in the sample for detecting microorganisms. Method a means randomly shearing the nucleic acid and then adding a universal amplification linker, and then performing PCR amplification. Optionally, the nucleic acid is randomly sheared and then adding a universal amplification linker using Tn5 enzyme; b Method represents enriching and / or amplifying the nucleic acid of the microorganism, and further optionally, using a multiple displacement amplification method.

10. The method according to any one of claims 1 to 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, Pseudomonas 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 bacteriophage, 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 sample for detecting microorganisms, wherein: The invention is prepared by the method described in any one of claims 1 to 10.

12. A method for detecting microorganisms, wherein: include: Obtaining a sample for detecting microorganisms obtained by the preparation method according to any one of claims 1 to 10, or a sample for detecting microorganisms according to claim 11; Performing metagenomic sequencing on the sample to determine the detection results of the microorganisms; The sequencing includes second-generation sequencing based on PCR amplification, or single-molecule sequencing based on nanopore, optionally, single-molecule sequencing based on nanopore.

13. The method according to claim 12, wherein: The upper limit of the ratio of the number of the host cells to the number of the microorganisms in the isolated host cell sample is: (10 4 -10 7 ):1; Optionally, the microorganism is a bacterium, and the upper limit of the number ratio is 10 7 :1; Optionally, the microorganism is a virus, and the upper limit of the number ratio is (10 4 -10 7 ):1; Optionally, the microorganism is a fungus, and the upper limit of the number ratio is 10 7 :1; Optionally, the concentration of the host cells in the isolated host cell sample is 1×10 4 -1×10 7 cells / mL; Optionally, the concentration of the microorganism in the isolated host cell sample is 1-1×10 7 CFU / mL.

14. A kit for detecting a sample of a microorganism, wherein: include: Host cell lysis reagents, nucleases, and nuclease buffers, Optionally, the salt in the nuclease buffer is used at a concentration of 10-600 mM; Optionally, the salt includes MgCl2, Tris-HCl and a neutral salt; Optionally, the components in the nuclease buffer and their concentrations are as follows: NaCl: 0-500 mM; MgCl2: 4-15 mM; pH of the nuclease buffer: 7-9; Optionally, the host cell lysis reagent comprises a host cell membrane lysis reagent, and optionally, the host cell membrane lysis reagent is saponin; Optionally, the active ingredient in the saponin is used at a concentration of: 0.3-0.7%; Optionally, the nuclease is capable of degrading DNA and / or RNA.

15. A method for constructing a sequencing library for pathogenic microorganism detection, wherein: include: R1. Providing a sample to be tested, wherein the length of the nucleic acid fragment of the host in the sample to be tested is <200 bp, and the length of the nucleic acid fragment of the pathogenic microorganism that may be present in the sample to be tested is ≥1 kb, which can be 1-10 kb; R2. performing nucleic acid multiple displacement amplification on the sample to be tested to increase the proportion of nucleic acid fragments of pathogenic microorganisms that may be present in the sample to be tested, and obtaining multiple displacement amplification products; R3. Construct a library for the multiple displacement amplification products to obtain a sequencing library.

16. The method according to claim 15, wherein: The sample to be tested is prepared by the method described in any one of claims 1 to 10 or the sample described in claim 11.

17. Use of the method according to any one of claims 1 to 10, the sample according to claim 11, the method according to claim 12 or 13, the kit according to claim 14, or the method according to claim 15 or 16 in pathogenic microorganism metagenomic sequencing, or in the preparation of a product for pathogenic microorganism metagenomic sequencing.

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