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

Abstract

The invention provides a sample preparation method for microbiological detection, a sample, a detection method, a kit and application. The method comprises the following steps: a treatment step: taking an in-vitro host cell sample, and treating the in-vitro host cell sample with a host cell lysis reagent and nuclease to degrade host nucleic acid, the treatment being carried out in a liquid with a salt concentration of 10-600mM; a nuclease inactivation step: providing conditions to inactivate the nuclease; and obtaining microbial nucleic acid: extracting total nucleic acid to obtain the sample for detecting the microorganisms. According to the method disclosed by the invention, the interference of the host cell genome on the sample detection sensitivity can be effectively reduced in metagenome sequencing, and effective sequencing data of microorganisms in a detection result is improved, so that the positive detection rate of the microorganisms and the sensitivity of detecting the microorganisms are improved.

Description

Technical Field

[0001] The present application belongs to the technical field of biological sample preparation, and specifically relates to a sample preparation method, a sample, a detection method, a kit and an application for microbial detection. Background Art

[0002] Microorganisms are widely present in nature, usually including viruses, bacteria, fungi, protozoa and some algae, etc. Rapid detection of microorganisms is of great significance for related tests.

[0003] 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, and it is difficult to detect low-abundance pathogenic microorganism sequences, which often leads 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. Summary of the invention

[0004] 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 are convenient for metagenomic sequencing and have high detection accuracy; the samples, detection methods, kits and applications obtained based on the sample preparation method also have corresponding advantages.

[0005] In a first aspect, an embodiment of the present application provides a method for preparing a sample for detecting microorganisms, comprising:

[0006] a 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;

[0007] a nuclease inactivation step, providing conditions to inactivate the nuclease;

[0008] The step of obtaining microbial nucleic acid is to extract total nucleic acid to obtain the sample for detecting microorganisms.

[0009] 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.

[0010] In some optional embodiments, the method satisfies at least one of the following conditions:

[0011] 1) In the treatment step, the nuclease 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 total nucleic acid in the step of obtaining microbial nucleic acid, thereby forming a first length difference;

[0012] 2) In the treatment step, the length of the nucleic acid fragment after the host nucleic acid degradation is more than 35 bp,

[0013] 3) In the treatment step, the length of the nucleic acid fragment 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;

[0014] 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, which can be 1-10 kb or 1 kb-5 kb,

[0015] 5) The nuclease is capable of degrading DNA and / or RNA, and the nuclease is M-SAN nuclease and / or Benzonase nuclease, and can further be M-SAN nuclease.

[0016] In some optional embodiments, the method satisfies at least one of the following conditions:

[0017] 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;

[0018] 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;

[0019] Optionally, the components in the nuclease buffer and their 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;

[0020] 3) The host cell lysis reagent is a host cell membrane lysis reagent, which may further be saponin, or Quillaja sylvestris bark saponin. More optionally, the active ingredient in the saponin is used at a concentration of 1.5-3.5%, or 1.6-3.0%, or 1.7-2.7%.

[0021] 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.

[0022] The step of obtaining microbial nucleic acid does not include centrifugation and / or washing steps before extracting nucleic acid.

[0023] The extracted nucleic acid in the step of obtaining microbial nucleic acid includes DNA and / or RNA.

[0024] In some optional embodiments, in the step of obtaining microbial nucleic acid, after extracting the 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,

[0025] The first length difference is maintained during the method of extracting nucleic acid.

[0026] In some optional embodiments, in the step of obtaining microbial nucleic acid, after extracting the total nucleic acid, a step of degrading the host mitochondrial DNA and / or the host ribosomal RNA is also included.

[0027] Optionally, the fragment length of the host mitochondrial DNA and / or host ribosomal RNA after degradation is less than the nucleic acid length of the microorganism obtained by extracting the total nucleic acid in the step of obtaining microbial nucleic acid, forming a second length difference.

[0028] 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 nucleic acid of the microorganism in the sample for detecting microorganisms;

[0029] Optionally, the method for degrading host mitochondrial DNA and / or host ribosomal RNA is specific degradation, which may further be CRISPR, and may further be CRISPR / Cas9.

[0030] In some optional embodiments, the second length difference is less than or equal to the first length difference.

[0031] 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.

[0032] 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;

[0033] b Method represents enrichment and / or amplification of nucleic acid of the microorganism, further optionally, using a multiple displacement amplification (MDA) method.

[0034] In some optional embodiments, the microorganisms include bacteria, fungi and / or viruses, and the viruses include DNA viruses and / or RNA viruses.

[0035] 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 haemolyticus, Staphylococcus capitis, and Streptococcus agalactiae (Group B Streptococcus).

[0036] 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.

[0037] In some optional embodiments, the DNA virus includes at least one of a DNA bacteriophage, an Epstein-Barr virus, a mumps virus, and an adenovirus.

[0038] In some optional embodiments, the RNA virus includes at least one of an RNA bacteriophage, influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, and coronavirus.

[0039] 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.

[0040] 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;

[0041] Performing metagenomic sequencing on the sample to determine the detection results of the microorganisms;

[0042] The sequencing includes second-generation sequencing based on PCR amplification, or single-molecule sequencing based on nanopore, optionally, single-molecule sequencing based on nanopore.

[0043] 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 2 -10 4 ):1;

[0044] Optionally, the microorganism is a bacterium, and the upper limit of the number ratio is 10 4 :1;

[0045] Optionally, the microorganism is a virus, and the upper limit of the number ratio is 10 3 :1;

[0046] Optionally, the microorganism is a fungus, and the upper limit of the number ratio is 10 3 :1;

[0047] Optionally, the concentration of the host cells in the isolated host cell sample is 1×10 4 -1×10 7 cells / mL;

[0048] The concentration of the microorganism in the isolated host cell sample is 1×10 2 -1×10 7 CFU / mL.

[0049] In a fourth aspect, the present application provides a kit for detecting a sample of a microorganism, comprising: a host cell lysis reagent, a nuclease, and a nuclease buffer,

[0050] Optionally, the salt in the nuclease buffer is used at a concentration of 10-600 mM, 15-590 mM or 19-565 mM,

[0051] Optionally, the salt includes MgCl2, Tris-HCl and a neutral salt,

[0052] Optionally, the components in the nuclease buffer and their 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;

[0053] Optionally, the host cell lysis reagent includes a host cell membrane lysis reagent, and optionally, the host cell membrane lysis reagent is saponin, and further optionally, it is Quillaja sylvestris bark saponin. More optionally, the active ingredient in the saponin is used at a concentration of 1.5-3.5%, optionally 1.6-3.0%, and further optionally 1.7-2.7%.

[0054] Optionally, the nuclease is capable of degrading DNA and / or RNA, optionally M-SAN.

[0055] In a fifth aspect, the present application embodiment provides a method for constructing a sequencing library for pathogenic microorganism detection, comprising:

[0056] 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 or 1 kb-5 kb;

[0057] 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;

[0058] R3. Construct a library for the multiple displacement amplification products to obtain a sequencing library.

[0059] 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.

[0060] 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 the preparation of a product for pathogenic microorganism metagenomic sequencing.

[0061] Compared with the prior art, this application has at least the following beneficial effects:

[0062] 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 proportion of pathogenic nucleic acids is increased by reducing the damage to pathogens to improve the sensitivity of sequencing. 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

[0063] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application will be briefly introduced below. 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 paying creative work.

[0064] Figure 1 A flow chart showing a method for preparing a sample for detecting microorganisms according to Example 1 of the present application is shown;

[0065] Figure 2 The human cells (10 6 / ml) and Escherichia coli (10 6 / ml) Fluorescence quantitative qPCR results;

[0066] Figure 3 The qPCR results of human genes, DNA phage T1 and RNA phage M of the present application example are shown;

[0067] Figure 4 The quantitative results of LOD of virus cells in the embodiment of the present application are shown;

[0068] Figure 5 The adenovirus genome coverage of the present application example is shown;

[0069] Figure 6 The length of the host nucleic acid after the host is removed in the embodiment of the present application is shown;

[0070] Figure 7 It shows that the short fragments in the embodiment of the present application affect the metagenome comparison analysis;

[0071] Figure 8 It shows that the host nucleic acid becomes shorter after being treated with different nucleases in the examples of the present application;

[0072] Fig. 9 The distribution diagram of long and short fragments of bacterial cells in the embodiment of the present application is shown;

[0073] Fig.10 The effect of using MDA to amplify long fragments on simulated samples in an embodiment of the present application is shown. DETAILED DESCRIPTION

[0074] In order to make the application purpose, technical solution and beneficial technical effect of the present application clearer, the present application is further described in detail in conjunction with the embodiments below. It should be understood that the embodiments described in this specification are only for explaining the present application, not for limiting the present application.

[0075] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unclearly recorded range; and any lower limit can be combined with other lower limits to form an unclearly recorded range, and any upper limit can be combined with any other upper limit to form an unclearly recorded range. In addition, although not clearly recorded, each point or single value between the endpoints of the range is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unclearly recorded range.

[0076] In the description of the present application, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number, and the “multiple” in “one or more” means two or more than two.

[0077] The above application content of the present application is not intended to describe each disclosed embodiment or each implementation in the present application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided by a series of examples, which can be used in various combinations. In each example, enumeration is only used as a representative group and should not be interpreted as exhaustive.

[0078] The traditional method of culturing and identifying microorganisms has the advantages of short cycle, wide coverage, and easy operation when conducting metagenomic sequencing. At the same time, metagenomic sequencing also faces a huge challenge, that is, the sample contains a large amount of host DNA. For example, when the host is human, it is very difficult to detect low-abundance pathogen sequences in the background of high human DNA, not to mention more in-depth analysis such as microbial typing and drug resistance genes through metagenomic sequencing.

[0079] How to efficiently remove the human DNA background and maximize the retention of the 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.

[0080] 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 sample nucleic acids 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. The removal efficiency is usually low, the effect is not ideal, and some non-host eukaryotic organisms such as fungi or parasites will also 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 will be lost. (4) If the host cells are human cells, 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, and then cell membrane-impermeable propidium iodide azide (PMA) 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, which can no longer be amplified and complete the subsequent sequencing library construction process. This method has a high removal efficiency, but it 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.

[0081] 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 utilize recombinant proteins that degrade related nucleic acids to reduce or remove host genome mitochondrial DNA, which can effectively reduce host genome DNA in metagenomic sequencing and improve the effective sequencing data of microorganisms in the sample.

[0082] Method for preparing samples for detecting microorganisms

[0083] In a first aspect, the present application provides a method for preparing a sample for detecting microorganisms, such as Figure 1 As shown, including:

[0084] a 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;

[0085] a nuclease inactivation step, providing conditions to inactivate the nuclease;

[0086] The step of obtaining microbial nucleic acid is to extract total nucleic acid to obtain the sample for detecting microorganisms.

[0087] The inventors found that in order to minimize the damage to microorganisms during the host removal process, a 10-600mM salt system was used, but this also resulted in the host nucleic acid fragments after nuclease degradation being not a few bp in length, but around 100bp or 200bp. The fragments were evenly distributed on human chromosomes through second-generation sequencing analysis, proving that there was no similarity between short host fragments of around 100bp after host removal.

[0088] 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. The present application pays attention to and classifies its proportion before sequencing to reduce the proportion of host nucleic acid, thereby improving the accuracy of sequencing.

[0089] In order to retain the nucleic acid of pathogenic microorganisms as much as possible, a salt concentration system of 10-600mM and conventional means of extracting total nucleic acid were adopted to make obvious differences in the length of microbial and host nucleic acid degradation fragments, 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.

[0090] The method of the present application, on the one hand, increases the proportion of pathogen nucleic acid 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.

[0091] According to the embodiments of the present application, the nuclease of the present application is a class of nucleic acid degrading enzymes, which are enzymes 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 (RNaseT1): 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 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 and degradation rate of the chains.

[0092] According to the present application embodiment, the host cell lysis reagent is a reagent for lysing (dissolving) host cells to release target proteins or other cell components. These reagents usually contain a series of components, which are intended to destroy cell membranes, nuclear membranes and organelle membranes, thereby releasing organelles, proteins and nucleic acids in cells.

[0093] The components of common host cell lysis reagents include at least one of the following reagents: osmotic agents, cationic / anionic detergents, protease inhibitors, nuclease inhibitors and buffers. As an example, osmotic agents: such as mannitol, glucose, etc., 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, Tween20, SDS (sodium dodecyl sulfate), etc., are used to destroy the cell membrane, nuclear membrane and organelle membrane to release the cell contents. As an example, protease inhibitors: such as phenylmethylsulfonylfluoride (PMSF), etc., are used to protect proteins in cells from degradation. As an example, nuclease inhibitors: such as ribonuclease inhibitors (RNase inhibitors), etc., are used to protect nucleic acids in cells from degradation. As an example, buffers include PBS buffers. Provide appropriate pH and ion concentration to maintain suitable reaction conditions.

[0094] 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, thereby achieving degradation of host nucleic acids.

[0095] In some embodiments, during the processing step, the host cells can be mammalian cells, such as dog cells, monkey cells, mouse cells, cat cells, and can be human cells, and can further be human cerebrospinal fluid cells or human thoracic and abdominal cells.

[0096] In some embodiments, in 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.

[0097] In some optional embodiments, the nuclease in the processing step can degrade the host nucleic acid to a first nucleic acid length that is smaller than a second nucleic acid length of the microorganism obtained by the extracted nucleic acid in the step of obtaining microbial nucleic acid, thereby forming a first length difference.

[0098] According to an embodiment of the present application, the first length difference may range from 100 bp to 9.6 kb.

[0099] 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.

[0100] 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.

[0101] 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, bp, 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, 33 5bp, 340bp, 345bp, 350bp, 355bp, 360bp, 365bp, 370bp, 375bp, 380bp, 385bp, 390bp, 395bp, 400bp, 405bp, 410b p, 415bp, 420bp, 425bp, 430bp, 435bp, 440bp, 445bp, 450bp, 455bp, 460bp, 465bp, 470bp, 475bp, 480bp, 485bp, 4 Any numerical value among 90bp, 495bp, 500bp, 505bp, 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 composed of these values.

[0102] According to the embodiments of the present application, the length of the fragment after the host nucleic acid degradation may 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.

[0103] 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, and can be 1-10 kb or 1 kb-5 kb.

[0104] Optionally, 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, 2.9 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 4.1 kb, 4. 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 composed of these values.

[0105] In the related art, the length of the second generation sequencing is about 35-600bp, and nanopore sequencing has no length limit, and can be long or short. In the step of obtaining microbial nucleic acid, there are more relatively long fragments in the nucleic acid fragments of the microorganism, which is conducive to improving the accuracy of sequencing comparison. And the length of the nucleic acid fragment of the microorganism in the step of obtaining microbial nucleic acid is related to the extraction method and / or enrichment method. The enrichment method is preferably MDA.

[0106] 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 may further be M-SAN nuclease.

[0107] It has been found that using different nucleases to fully degrade the host nucleic acid, the degradation fragments obtained are of different sizes. The degradation fragments obtained by Benzonase nuclease are about 200bp, and those obtained by M-SAN nuclease are about 100bp. 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.

[0108] In some optional embodiments, the method satisfies at least one of the following conditions:

[0109] 1) In the treatment step, the treatment is carried out in a liquid having a salt concentration of 10-600 mM, optionally 19-565 mM;

[0110] 2) using a host cell lysis reagent and nuclease treatment, including incubating an ex vivo host cell sample with a host cell lysis reagent, a nuclease, and a nuclease buffer to degrade host nucleic acids;

[0111] Optionally, the components in the nuclease buffer and their 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;

[0112] The host cell lysis reagent is a host cell membrane lysis reagent, which may further be saponin, further may be Quillaja sylvestris saponin, and more optionally, the active ingredient in the saponin is used at a concentration of 1.5-3.5%, optionally 1.6-3.0%, further optionally 1.7-2.7%.

[0113] It can be understood that the mass concentration of saponin in the in vitro host cell mixed system is within the above range.

[0114] 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.

[0115] 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.

[0116] 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 using tris(2-carboxyethyl)phosphine.

[0117] The step of obtaining microbial nucleic acid does not include centrifugation and / or washing steps before extracting nucleic acid.

[0118] The extracted nucleic acid in the step of obtaining microbial nucleic acid includes DNA and / or RNA.

[0119] Tris(2-carboxyethyl)phosphine (TCEP) is a reducing agent that can denature polymeric protein structures, such as protein complexes or polymers with large molecular weights. By destroying the non-covalent interactions between proteins, TCEP can denature polymers into individual subunits. It can inactivate nucleases.

[0120] 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 avoiding the loss of pathogen samples, especially pathogens such as viruses, to the greatest extent.

[0121] Proteinase K has the function of decomposing proteases, which can effectively increase the utilization rate of protease activity and improve the body's immunity and immune effect. Proteinase K is a protein-dissolving enzyme isolated from Candida albicans. It has high activity and is used for the separation of plasmids or genomic DNA. It can directly enhance the body's absorption and utilization of proteins.

[0122] Ethylenediaminetetraacetic acid (EDTA) is an organic compound with the chemical formula C 10 H 16 N2O8 is a white powder at room temperature and pressure, which can inactivate nucleases.

[0123] In some optional embodiments, in the step of obtaining microbial nucleic acid, after extracting the 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 the extracted 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,

[0124] The first length difference is maintained during the method of extracting nucleic acid.

[0125] It can be understood that: the host nucleic acid (which can be understood as a nucleic acid fragment of the host genome) can be made to be at a shorter length without affecting the length of the nucleic acid of the microorganism to be detected. The first length difference between the length of the nucleic acid of the microorganism and the nucleic acid of the host is maintained for subsequent detection.

[0126] According to the embodiments of the present application, a long-fragment nucleic acid extraction kit can be used, the main principle or difference from an ordinary kit is that column extraction and severe mechanical grinding are avoided, and the fragments are kept long.

[0127] In some optional embodiments, in the step of obtaining microbial nucleic acid, after extracting the nucleic acid, a step of degrading the host mitochondrial DNA and / or the host ribosomal RNA is also included.

[0128] 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.

[0129] 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 nucleic acid of the microorganism in the sample for detecting microorganisms;

[0130] It can be understood that: the nucleic acid in the host's mitochondria can be made to be at a shorter length without affecting the length of the nucleic acid of the microorganism to be detected. The second length difference between the length of the nucleic acid of the microorganism and the mitochondrial nucleic acid of the host is maintained for subsequent detection. Optionally, the method for degrading the host mitochondrial DNA and / or the host ribosomal RNA is specific degradation, which can further be CRISPR, and further can be CRISPR / Cas9.

[0131] 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.

[0132] 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.

[0133] 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; or

[0134] b Method represents enrichment and / or amplification of nucleic acid of the microorganism, further optionally, using a multiple displacement amplification (MDA) method.

[0135] It has been found that compared with method a, the amount of data obtained by nanopore sequencing after amplification using method b is larger. Since the amount of data obtained is larger, it is beneficial to improve the accuracy of detection. Due to the difference in the length of the amplified product fragments, the number of unclassified reads is significantly reduced. Since the product after the host is removed is selectively amplified in one step, the product after MDA amplification has a lower human origin ratio and a higher pathogen ratio after sequencing. In the embodiment of the present application, the nucleic acid of the microorganism is still a long fragment relative to the host nucleic acid fragment after the host is removed.

[0136] MDA amplification refers to an isothermal amplification method in which multiple sites are located on DNA. Its full name is Multiple Displacement Amplification. It is a special DNA amplification technology that uses phi29 DNA polymerase to amplify DNA under isothermal conditions. By taking advantage of the fact that MDA amplification preferentially amplifies long fragments and has a less obvious amplification effect on short fragments, it is possible to achieve selective amplification of the nucleic acid of the microorganism to be detected based on the second length difference or the first length difference.

[0137] In some optional embodiments, the microorganisms include bacteria, fungi and / or viruses, and the viruses include DNA viruses and / or RNA viruses.

[0138] 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 haemolyticus, Staphylococcus capitis, and Streptococcus agalactiae (Group B Streptococcus).

[0139] 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.

[0140] In some optional embodiments, the DNA virus includes at least one of a DNA bacteriophage, an Epstein-Barr virus, a mumps virus, and an adenovirus.

[0141] In some optional embodiments, the RNA virus includes at least one of an RNA bacteriophage, influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, and coronavirus.

[0142] In some embodiments, the method includes adding cell lysate, nuclease, 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 host nucleic acid ratio by degrading cell mitochondrial DNA. The cell lysate 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 mixed system is 700-900U / ml.

[0143] The components in the nuclease buffer and their 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.

[0144] The final concentration of each component of the nuclease buffer added to the mixed system is: NaCl concentration is 125mM-250mM, MgCl2 concentration is 4-15mM, Tris-HCl pH 7.2-8.7, its concentration is 25-50mM. The nucleic acid inactivator is TCEP, and the final concentration in the mixed system is 10mM. The method for degrading mitochondrial DNA is CRISPR-Cas9.

[0145] According to the embodiments of the present application, the product name of the nuclease M-SAN may be M-SAN HQ (Bioprocessing grade), Triton FREE, 25-25k, 70950-202, may be from the brand Arctic Zymes, and may be stored at -20°C.

[0146] According to the embodiments of the present application, the preparation method of the present application allows the host DNA to be released and degraded quickly without long time consumption, high cost, low efficiency, extraction bias, and loss of some microorganisms. During the downstream DNA library construction process, the host DNA sequencing reads are further specifically reduced to more effectively reduce the host DNA background and improve the effective sequencing data of other microorganisms.

[0147] sample

[0148] 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.

[0149] According to the embodiments of the present application, the amount of microbial nucleic acid that may be detected in the above sample, or the absence of the above microorganisms that may be present, is higher in detection accuracy when the above sample is used for metagenomic sequencing.

[0150] Detection methods for microorganisms

[0151] In a third aspect, the present invention provides a method for detecting microorganisms, comprising:

[0152] 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;

[0153] Performing metagenomic sequencing on the sample to determine the detection results of the microorganisms;

[0154] The sequencing includes second-generation sequencing based on PCR amplification, or single-molecule sequencing based on nanopore, optionally, single-molecule sequencing based on nanopore.

[0155] According to the embodiments of the present application, after the host genomic DNA is effectively removed 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.

[0156] 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 2 ):1;

[0157] Optionally, the microorganism is a bacterium, and the upper limit of the number ratio is 10 4 :1;

[0158] Optionally, the microorganism is a virus, and the upper limit of the number ratio is 10 3 :1;

[0159] Optionally, the microorganism is a fungus, and the upper limit of the number ratio is 103 :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;

[0160] The concentration of the microorganism in the isolated host cell sample is 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 or 1×10 7 CFU / mL.

[0161] In some embodiments, the method further comprises:

[0162] Performing nucleic acid adapter ligation on the sample, so that the nucleic acid fragments ligated by the nucleic acid adapter are physically and / or chemically enriched to obtain a sample to be sequenced;

[0163] Metagenomic sequencing is performed on the samples to be sequenced to determine the detection results of the microorganisms.

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

[0165] Identify the type of microorganism,

[0166] Perform metagenomic sequencing on the sample to obtain a sequencing sequence;

[0167] According to the type of the microorganism and the sequencing sequence, it is determined whether the type of the microorganism is contained.

[0168] According to the embodiments of the present application, the relevant testing personnel have clear suspected types of microorganisms in the sample and have roughly determined the types of microorganisms. Software or other tools can be used to compare and analyze the sequencing sequence with the sequence of the type of microorganism, so as to determine whether the sample contains microorganisms related to the previously suspected type based on the comparison results.

[0169] In some embodiments, the method further comprises:

[0170] Perform metagenomic sequencing on the sample to obtain a sequencing sequence;

[0171] The sequencing sequence is analyzed to obtain an analysis result, so as to determine whether the sample contains microorganisms based on the analysis result.

[0172] According to the embodiment of the present application, when the test personnel are uncertain about the type of microorganism in the sample, they can use software to analyze the sequencing sequence to obtain the analysis result, and use manual comparison or other software to compare and combine other information to determine whether the sample contains microorganisms.

[0173] In some embodiments, the method further comprises:

[0174] Perform metagenomic sequencing on the sample to obtain a sequencing sequence;

[0175] Allowing the sequencing sequence to be analyzed to obtain an analysis result;

[0176] In response to the analysis result, the detection result of the microorganism in the sample is output.

[0177] According to the embodiment of the present application, when the test personnel are uncertain about the type of microorganism in the sample, they can use software to analyze the sequencing sequence to obtain the analysis result. The software is used for comparison, and the software determines and outputs the test result of whether the sample contains microorganisms.

[0178] In some embodiments, the method further comprises:

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

[0180] According to the embodiment of the present application, when the test personnel are uncertain about the type of microorganism in the sample, they can use software to analyze the sequencing sequence to obtain the analysis result. The software is used for comparison, and the software determines and outputs the test result of whether the sample contains microorganisms. If it contains microorganisms, the type of the relevant microorganism is also displayed.

[0181] Kit for testing samples for microorganisms

[0182] In a fourth aspect, the present application provides a kit for detecting a sample of a microorganism, comprising: a host cell lysis reagent, a nuclease, and a nuclease buffer,

[0183] Optionally, the salt in the nuclease buffer is used at a concentration of 10-600 mM, 15-590 mM or 19-565 mM,

[0184] Optionally, the salt includes MgCl2, Tris-HCl and a neutral salt,

[0185] Optionally, 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.

[0186] Optionally, the host cell lysis reagent includes a host cell membrane lysis reagent, and optionally, the host cell membrane lysis reagent is saponin, and further optionally, it is Quillaja sylvestris bark saponin. More optionally, the active ingredient in the saponin is used at a concentration of 1.5-3.5%, optionally 1.6-3.0%, and further optionally 1.7-2.7%.

[0187] Optionally, the nuclease is capable of degrading DNA and / or RNA, optionally M-SAN.

[0188] Method for constructing sequencing library for pathogenic microorganism detection

[0189] In a fifth aspect, the present application embodiment provides a method for constructing a sequencing library for pathogenic microorganism detection, comprising:

[0190] 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 or 1 kb-5 kb;

[0191] 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;

[0192] R3. Construct a library for the multiple displacement amplification products to obtain a sequencing library.

[0193] In step R2, the sample to be tested can be subjected to electrophoresis gel recovery, magnetic bead fragment sorting, or molecular chromatography exchange column, but further amplification is required later, and the steps are cumbersome, which is not conducive to the detection of trace pathogenic nucleic acids. Studies have found that in step R2, the use of multiple displacement amplification does not require additional amplification, which is equivalent to combining the long fragment screening and amplification achieved by electrophoresis gel recovery, magnetic bead fragment sorting, or molecular chromatography exchange column operations.

[0194] 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.

[0195] 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 the preparation of a product for pathogenic microorganism metagenomic sequencing.

[0196] Kits for Microbiology

[0197] In a fourth aspect, the present application provides a kit for microorganisms, comprising:

[0198] Cell lysate, nuclease, and nuclease buffer are added to the sample to be tested at the same time, and a nucleic acid inactivator is added to inactivate the sample after incubation at 37°C for 15 minutes. 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 cell mitochondrial DNA. The cell lysate is a saponin from Quillaja sylvestris bark, 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 each component of the nuclease buffer added to the sample to be tested is: NaCl 125mM-250mM, MgCl2 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.

[0199] Example

[0200] The following examples more specifically describe the disclosure of the present application, which are intended for illustrative purposes only, as it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the disclosure of the present application. Unless otherwise stated, 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 can be used directly without further processing, and the instruments used in the examples are commercially available.

[0201] The bacteria and virus samples in the examples of this application are commercially available products.

[0202] Specific steps:

[0203] 1. De-hosting

[0204] Take 300 μL of mixed sample, add 100 μL of cell lysis buffer (containing 10% saponin, the saponin was purchased from Sigma, product number S4521, from the bark of Quillaja saponaria, 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), 10 μL of nuclease M-SAN (Arctic Zymes, 70950-202, sufficient amount) in 1.5 mL of sterile EP, shake at 1000 rpm to mix, and react at 37°C for 15 min.

[0205] Add 50 μL of 10×TCEP (100 mM), mix by pipetting, and react at room temperature for 5 min to obtain a host-free sample.

[0206] 2. Nucleic Acid Extraction

[0207] The host nucleic acid-free sample obtained in step 1 is subjected to nucleic acid extraction using the VAMNE Magnetic Pathogen DNA / RNA Kit (Novozymes, catalog number RM601), and total nucleic acid (gDNA) is finally obtained. When viral RNA needs to be detected, the product needs to be reverse transcribed to obtain DNA.

[0208] 3. CRISPR-Cas9 for mitochondrial cleavage

[0209] CRISPR-Cas9 cleavage of mitochondria, 100:100:1 (Cas9: sg-RNA: DNA) cleavage site molar ratio:

[0210]

[0211]

[0212] Cas9 treatment system 30uL, 37℃ treatment for 30min, add 1uL proteinase K,

[0213] Treat at 37°C for 15 min.

[0214] In addition, 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] 4. PCR Amplification of DNA

[0216] 1. Prepare the DNA fragmentation system in a sterile PCR tube.

[0217]

[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 mix thoroughly by pumping up and down (you can also use your index finger to flick the tube to mix). 3. React at 55°C for 5 minutes, then place on ice for 2 minutes.

[0221] 4. Add 6uL of 6x termination buffer, mix well and let stand at room temperature for 5 minutes.

[0222] 5. Add 0.8× balanced Mix DNA Clean Beads and incubate at room temperature for 5 minutes; place the PCR tube on the magnetic rack and remove the supernatant after the solution is clarified.

[0223] 6. Add 200uL of 80% ethanol to rinse the beads, incubate for 30s and remove the supernatant, repeat once.

[0224] 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 there is no ethanol residue (beads appear frosted).

[0225] 8. Remove the PCR tube, add 25uL of nuclease free water, mix the magnetic beads thoroughly, and incubate at room temperature for 2 minutes.

[0226] 9. Place the PCR tube back on the magnetic rack and wait for the solution to become clear before taking out 24uL for later use.

[0227] PCR amplification was continued and the amplified product was purified.

[0228] Specific steps of PCR amplification:

[0229] Amplification system:

[0230]

[0231] Amplification Procedure:

[0232] program time 72℃ 8min 98℃ 1min 98℃ 10s 60℃ 15s 72℃ 1min(30cycles) 72℃ 7min 12℃ Forever

[0233] The sequence of Universal primer P' (10uM) is (5P'-ACTTGCCTGTCGCTCTATCTTC-3), which is from Shanghai Biotech Co., Ltd.

[0234] Premix Taq TM (LA Taq TM Version 2.0) from Takara Corporation.

[0235] 5. Sequencing library construction

[0236] The product of step 4 was connected to the sequencing adapters according to the library construction kit QLK-V1.1.1 of Qitan Technology to construct the library.

[0237] 6. Nanopore sequencing

[0238] According to the sequencing kit QSK-V1.1.1 of Qi Tan Technology, the ligation product of step five was subjected to nanopore sequencing in the sequencer QNome3841 of Qi Tan Technology.

[0239] 7. Bioinformatics analysis of sequencing data.

[0240] Example 1

[0241] Human cells (10 6 / ml) and Escherichia coli (10 6 / ml) at a volume ratio of 1:1, and the host removal method and the host removal method in step 1 are used, and then the nucleic acid extracted in step 2 is simultaneously tested by qPCR. Other steps are carried out according to the steps in Example 1, such as amplification in step 4 followed by sequencing in steps 5 to 7.

[0242] The results are as follows Figure 2 The figure shows the fluorescence quantitative qPCR results of this example, where rpoA refers to the human gene and cyaA refers to the E. coli gene. After the host removal treatment (treated group), the human gene CT value changed by more than 10, which is considered to have a 1000-fold host removal effect, while the CT value of E. coli did not change significantly.

[0243] The sequencing results showed that in the untreated group, the E. coli detection amount was <1%, and the host DNA accounted for >99%. After the host was removed, the E. coli detection amount increased to 43%, while the host DNA detection amount decreased to 38%. The proportion of bacterial metagenomes was significantly increased after the host was removed using the method of the present application.

[0244] Example 2

[0245] Human cells (10 6 / ml) and Escherichia coli (105 / ml) and DNA phage T1 (10 7 / ml) and RNA bacteriophage MS2 (10 7 / ml) were mixed in a volume ratio of 1:1:1:1, and the host removal scheme (QT) of step one and the untreated scheme U1 were used respectively. Then, qPCR detection was performed synchronously on the nucleic acid extracted in step 2. After amplification in step 4, sequencing in steps 5 to 7 was performed. After host removal treatment using the method of the present application, the proportion of the metagenomics of bacteria, DNA viruses, and RNA viruses was significantly increased.

[0246] Comparative Example 1 (NBT)

[0247] 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. Nanoporemetagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection. Nat Biotechnol 37, 783–792 (2019).

[0248] https: / / doi.org / 10.1038 / s41587-019-0156-5).

[0249] Comparative Example 2 (VT)

[0250] The difference between this comparative example and Example 2 is that the host removal scheme in step 1 adopts a commercial host removal kit, Novagen FastPure Host Removal and Microbiome DNA Isolation Kit DC501-01.

[0251] Comparative Example 3 (ZT)

[0252] The difference between this comparative example and Example 2 is that the host removal scheme in step 1 is carried out using a commercial host removal kit ZYMO brand ZT HostZERO Microbial DNA Kit D4310.

[0253] The comparison results of Example 2 and Comparative Examples 1-3 are as follows Figure 3As shown, the qPCR results of human genome, DNA phage T1 and RNA phage MS2 are shown. In the figure, the three groups from left to right are 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. Other comparative examples 1-3 (NBT, VT, ZT) all lose a large amount of DNA phage and RNA phage.

[0254] 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 greatly improved the detection rate of DNA phage and RNA phage.

[0255] Example 3: Detection of the de-hosting effect of different viruses

[0256] 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, followed by nucleic acid extraction (performed according to the above step 2) and qPCR detection.

[0257] The smaller the change in CT value, the smaller the destructive effect of the host removal method in step 1 of the present application on different viruses.

[0258] Table 1. Changes in Ct values ​​of different viruses detected by qPCR in the treated group relative to the untreated group

[0259] Virus Name Virus Type Ct value changes Epstein-Barr virus DNA viruses 0.26 Mumps virus DNA viruses -0.99 Adenovirus type 1 DNA viruses 0.08 Influenza A_Generic RNA viruses 0.87 Influenza A / H1 RNA viruses 0.44 Influenza A / H3 RNA viruses 0.98 Influenza B RNA viruses 0.17 Parainfluenza virus type 1 RNA viruses 0.97 Parainfluenza virus type 2 RNA viruses 0.38 Parainfluenza virus type 3 RNA viruses -0.34 Parainfluenza virus type 4 RNA viruses 0.58 Respiratory syncytial virus RNA viruses 0.73 Coronavirus 229E RNA viruses 0.72 Coronavirus NL63 RNA viruses 0.88 Coronavirus OC43 RNA viruses -0.13 Rhinovirus type 1 RNA viruses 1.36 Rhinovirus type 2 RNA viruses 2.25

[0260] As shown in the results 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.

[0261] Example 4: Simulated sample test for host removal effect

[0262] 1. Bacteria simulation samples

[0263] First, human cells were counted under a microscope using a hemocytometer, and E. coli were counted using the LB plate counting method. 2 -10 4 CFU / mL) and human cells (10 5 -10 7Cells / mL) were gradiently diluted and mixed, and the host removal and nucleic acid extraction of the above steps 1 and 2 were performed respectively, with the untreated cells without host removal as a control. The nucleic acid after host removal was subjected to nanopore library construction and sequencing of steps 4-7, and the results are shown in Table 2.

[0264] The results show that in the untreated group, the human background was 10 7 Under the condition of 10 cells / mL, the concentration of E. coli was 2 -10 4 The number of detected sequences was 0 when the CFU / mL was 10. 5 -10 6 When the number of cells / mL was 10, the number of sequences of E. coli was 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 in all grouped samples can be detected, and the abundance is close to the change of dilution multiple. 5 -The lowest detection concentration (LOD) of E. coli reached 10 in a human background of 10 cells / mL 2 CFU / mL.

[0265] Table 2. Nanopore sequencing results of bacterial LOD experiments

[0266]

[0267] Note: The first digit of the two-digit number in the group 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 after mixing.

[0268] Furthermore, after step 2, step 3 of CRISPR was added to remove the host mitochondrial DNA, and then the nanopore library construction and sequencing of steps 4-7 were performed. 5 -10 cells / mL human background, the lowest detection concentration (LOD) of Escherichia coli reached 10 CFU / mL.

[0269] The above results show that the library constructed by 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.

[0270] 2. Virus simulation samples

[0271] In order to verify the LOD of the host-free system on the virus cell simulation sample, we 6 10 cells / mL of human background were mixed with 2 -10 7We used qPCR to verify the difference in ADV nucleic acid content before and after host removal.

[0272] Table 3. Virus cell LOD nanopore sequencing results

[0273]

[0274] In 10 6 Nanopore sequencing was performed on ADV viruses diluted in gradients in a human background of 10 cells / mL, and the number and proportion of characteristic sequences of adenovirus detected. Among them, the term "copy / ml" is usually used to describe the concentration unit in a substance or solution. "Copy" refers to the number of a molecule or substance, usually the number of gene copies or the number of virus copies. "ml" refers to the volume unit, that is, milliliters. "Copy / ml" means the number of copies contained in each milliliter of solution.

[0275] like Figure 4 As shown in Table 3, the results show that host removal does not significantly reduce the ADV nucleic acid content and the detection effect and input amount show a linear gradient distribution. The nucleic acid after host removal was sequenced by nanopore, and the results are shown in Table 3. Ideally, since the adenovirus ADV genome is only 30kb, and the human genome is 3Gb in size. Without host removal, 10 7 Copies / mL ADV virus and 10 6 The nucleic acid ratio of cells / mL human cells is 1:20000, and the virus accounts for 0.005%.

[0276] The actual sequencing result is 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.

[0277] After de-hosting, Figure 5 As shown in Figure 2, the detection LOD of adenovirus can be reduced to 10 4 copies / mL, greatly improving the sensitivity of pathogen detection. Figure 5 In 10 6 Cells / mL human background, (a) is untreated 10 7 Genome coverage of ADV copies / mL.

[0278] (b)-(e) are respectively 10 4 -10 7The ADV genome coverage distribution of each adenovirus sample was compared. Figure 5 I found that even in 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.

[0279] Furthermore, after step 2, step 3 of CRISPR was added to remove the host mitochondrial DNA, and then the nanopore library construction and sequencing of steps 4-7 were performed. 6 The lowest detection concentration (LOD) of adenovirus reached 10 cells / mL under human background. 3 copies / mL.

[0280] 3. Blood simulation sample

[0281] In order to verify the host-removal effect of the host-removal system in real clinical scenarios, four common clinical standard pathogens were added to negative blood samples of healthy people at different concentrations, and the samples were treated with host removal and non-host removal, respectively. Then, nanopore sequencing of steps two and four to seven was performed. The results are shown in Table 4.

[0282] Table 4. Blood simulation sample LOD test

[0283]

[0284] RPM value (a method for standardizing statistical data of next-generation sequencing, i.e. the number of characteristic sequences aligned to samples per million sequences).

[0285] 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, most of the bacteria were lower than the clinical RPM ≥ 10 judgment standard, and false positives could not be ruled out. This confirms that the effect of our host removal on blood flow simulation samples can significantly improve the sensitivity of pathogen detection to 10 2 CFU / mL pathogen.

[0286] Example 5: The proportion of pathogenic nucleic acid increases after removing mitochondrial nucleic acid

[0287] Human cells (10 6 / ml) and adenovirus ADV (10 7 / ml) were mixed at a volume ratio of 1:1, and processed according to the methods of steps 1 to 7 above, and compared with the host removal treatment without step 1 or the degraded mitochondrial DNA without step 3. The results are shown in Table 5.

[0288] Table 5

[0289]

[0290] The results in Table 5 show that after host removal, the proportion of adenovirus increased from 0.0006% to 2.4%, and the proportion of human origin decreased from 99.6% to 97.3%, of which mitochondria accounted for 94.3% 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 mitochondria decreased to 58.1%.

[0291] Example 6: Analysis of short fragments of host nucleic acid after host removal

[0292] 1. The host cells, E. coli or a mixture of the two are treated according to the aforementioned steps 1 and 2, and the cells not subjected to step 1 are used as an untreated group.

[0293] Electrophoresis was used to detect the length of nucleic acid fragments after nucleic acid extraction. The results were as follows Figure 6 As shown, Figure 6 Among them, B1 and C1 are single cells without host and untreated; D1 and E1 are single bacteria without host and untreated; F1 and G1 are mixed bacterial cells without host and untreated. Compared with the untreated group, the extracted nucleic acid fragments will become shorter and close to 100bp when the host cells are treated with host alone; the extracted nucleic acid fragments will not change significantly when the bacteria are treated with host alone; the fragment length of the mixture is still mainly distributed close to 100bp compared with the untreated control after the host is treated. Therefore, after removing the host, the host-derived nucleic acid becomes a short fragment, and the length of the bacterial nucleic acid remains basically unchanged.

[0294] 2. Analysis of the interference of short fragments on sequencing results

[0295] The fragment of about 100 bp was evenly distributed on the human chromosome through the second generation sequencing analysis, proving that there is no similarity between the host short fragments of about 100 bp after the host is removed. Figure 7 As shown, Figure a represents unclassified; where unclassified refers to sequences that have not been uniquely identified as pathogenic species after sequence alignment. (b) Median length of unclassified sequences; (c) Median length of total reads. The proportion of unclassified sequences after host removal treatment is higher than that of the untreated group. The median length of total sequences after host removal treatment is also lower than that of the untreated group. However, there is no significant difference in the median length of unclassified sequences when comparing the host removal group with the untreated group, and the lengths are all less than the total sequence lengths of the two groups. This proves that short fragments will affect pathogen identification, resulting in a large number of sequences that fail to match the database. This proves that short fragments affect the alignment of metagenomes.

[0296] 3. The host cells were treated according to the aforementioned steps 1 and 2, and sufficient amounts of different nucleases M-SAN or Benzonase were used respectively. Meanwhile, the cells not subjected to step 1 were used as the untreated group.

[0297] Electrophoresis was used to detect the length of nucleic acid fragments after nucleic acid extraction. The results were as follows Figure 8 As shown in the figure, B1 and C1 are M-SAN nuclease-depleted hosts; D1 and E1 are untreated; F1 and G1 are Benzonase-depleted hosts. Compared with the untreated group, both nucleases have the phenomenon of shortening host nucleic acids, and the difference is that Benzonase nuclease degrades host nucleic acids to form fragments of about 200 bp.

[0298] Example 7: Effect of MDA enrichment of long fragments

[0299] 1. The human genome and E. coli genome derived from human renal epithelial cells HEK293T were respectively converted into long fragments L (nearly 10 kb) and short fragments S of nearly 100 bp, such as Fig. 9 In the figure, B1 is L cell; C1 is L bacteria; D1 is S cell; E1 is S bacteria.

[0300] As shown in Table 6, these four fragments were mixed in pairs at a ratio of 1:10 and 10:1, and then MDA amplification and nanopore sequencing were performed. From the results, it can be seen that when the MDA amplification substrates are all long fragments, the ratio of the amplified products is close to the original ratio of the substrates. When the amplification substrates contain short fragments, the amplified products are mainly derived from the long fragments and are not affected by the substrate ratio, which proves that MDA has the effect of selectively amplifying long fragments.

[0301] Table 6. Analysis results of nanopore sequencing data after MDA amplification

[0302]

[0303]

[0304] When the same sample was subjected to MDA amplification and Tn5 interruption amplification (the above step three), according to the sequencing results, as shown in Table 6, we found that the product amplified by MDA had 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. Since the product after host removal was selectively amplified in one step, the product after MDA amplification had a lower proportion of human origin and a higher proportion of pathogens.

[0305] MDA amplification system preparation table:

[0306]

[0307] The above reagents were from Novezan Discover-sc Single Cell WGA Kit (N603).

[0308] 2. The nucleic acids of the mixed host and the simulated samples after pathogen removal were amplified by MDA, Tn5 (step 4), and interrupted by second-generation sequencing. The products of MDA amplification were also interrupted by second-generation library construction. The MDA and Tn5 amplification products were sequenced by nanopore, and the MDA amplification products were interrupted by second-generation sequencing and then sequenced by the products of normal second-generation library construction. The results are as follows: Fig.10 As shown. Among them, B1-D1 is the MDA amplification fragment after host removal, E1-G1 is the Tn5 interruption amplification fragment, H1-B2 is the second generation sequencing interruption amplification fragment, and C2-E2 is the second generation sequencing interruption fragment after MDA amplification (b) Comparison of abundance after sequencing with different treatments. It shows that effective amplification products are obtained.

[0309] Example 8: Detection of fungi

[0310] In artificially cultured human cells (concentration 10 6 Different concentrations (10 / mL) were mixed 5 , 10 4 , 10 3 , 10 2 albicans (10, 10, or 1 copies / mL), and then divided into 2 groups for treatment: a treated group (performed according to the aforementioned step 1) and an untreated group, and then total nucleic acid extraction (performed according to the aforementioned step 2) and qPCR detection were performed, MDA amplification was performed in Example 7, and sequencing was performed in steps 5-7.

[0311] 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.

[0312] The sequencing results showed that the LOD of Candida albicans was: 10 3 copies / mL.

[0313] 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 Cells

[0314] / mL human background, the LOD of Candida albicans can reach 10 2 copies / mL.

[0315] Example 9: Clinical application

[0316] 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 influenza hemophilus infection, and a small amount of influenza B virus sequences were found. The patient developed fever symptoms on the third day and underwent a colloidal gold-based influenza B virus diagnosis, and was finally diagnosed with influenza B virus.

[0317] Table 7 Metagenomic sequencing results of a patient with mixed upper respiratory tract bacterial and viral infection

[0318]

[0319]

[0320] The collected nucleic acid was tested for influenza B virus qPCR, and it was found that as the course of the disease worsened, the Ct value of the patient's influenza virus decreased. The sequence abundance of influenza B virus in the nanopore sequencing results also increased accordingly. In the sputum sample on the third day, more than 80% virus coverage and 7 times sequencing depth of influenza B virus were obtained. This result proves the effectiveness of our host removal in the diagnosis of clinical mixed bacterial and viral infections. The virus will not be discarded due to the host removal method, resulting in missed detection of influenza B virus. The nanopore sequencing speed is fast, which can promptly indicate the patient's infection status, allowing doctors to provide precise treatment for patients.

[0321] Combining the two methods of CRISPR / Cas9 cutting mitochondrial DNA and MDA selective amplification of long fragments can further improve the host removal effect. Because the nucleic acid fragments cut by CRISPR / Cas9 are also 100-200bp in length. As shown in Table 7, after CRISPR cutting of the sample and then MDA amplification, it can be seen that compared with the uncut sample, the proportion of human origin has been significantly reduced from nearly 50% to 0.5%, and the proportion of bacteria has increased from 40% to more than 85%.

[0322] In order to verify that MDA amplification is applicable to RNA-derived target sequence amplification, the mixed RNA virus-infected samples previously frozen in the refrigerator were host-free, and library construction and nanopore sequencing were performed based on MDA.

[0323] Table 8. MDA-based database construction test

[0324]

[0325]

[0326] The results are shown in Table 8, which can effectively detect Haemophilus influenzae and influenza B virus and their contents, which are close to those in Table 7 in abundance, and the table shows the number of effective sequences (reads) of bacteria and viruses. This shows that MDA amplification can be applied to RNA-derived target sequence amplification, can effectively detect bacteria and viruses, and the resulting library has more effective reads.

[0327] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope 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 and / or Benzonase 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: 1.5-3.5%, optional 1.6-3.0%.

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 2 -10 4 ):1; Optionally, the microorganism is a bacterium, and the upper limit of the number ratio is 10 4 :1; Optionally, the microorganism is a virus, and the upper limit of the number ratio is 10 3 :1; Optionally, the microorganism is a fungus, and the upper limit of the number ratio is 10 4 :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 1×10 2 -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: 1.5-3.5%; 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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