Method and kit for efficient unbiased removal of host nucleic acid in sample

Abstract

The present application provides a method for efficient unbiased removal of host nucleic acids (e.g., animal-derived nucleic acids and / or plant-derived nucleic acids) from a microbial sample (e.g., a clinical sample comprising microorganisms), as well as a kit for implementing the method.

Description

Technical Field

[0001] The present application relates to the field of detection in the field of molecular biology. Specifically, the present application relates to a method for efficiently and unbiasedly removing host nucleic acids (e.g., animal-derived nucleic acids and / or plant-derived nucleic acids) from microbial samples (e.g., clinical samples containing microorganisms), and a kit for implementing the method. Background Art

[0002] Metagenomics is a technology that uses high-throughput sequencing and bioinformatics analysis of all microbial DNA (or cDNA transcribed from RNA) in a sample to obtain the composition, function and genetic diversity of microbial communities. This technology is widely used in microecological research and is of great significance to agricultural production, food safety and human health. In addition, in terms of pathogen detection, metagenomics technology can achieve rapid identification and diagnosis of pathogenic microorganisms, especially emerging or rare pathogenic microorganisms, improve the sensitivity and accuracy of detection, help trace the origin of the epidemic, monitor the development of the epidemic, and guide clinical precision drug use.

[0003] However, many sample types contain a large amount of host nucleic acid. For example, in human nasopharyngeal swabs, oropharyngeal swabs, saliva, alveolar lavage fluid, cerebrospinal fluid, vaginal swabs and other sample types, the proportion of microbial nucleic acid in the total nucleic acid is less than 10%, or even as low as 0.01%-1%. This results in the host sequence in the sequencing data far exceeding the microbial sequence, greatly increasing the sequencing cost and time cost, increasing the difficulty of analysis, reducing the sensitivity of microbial detection, and limiting the application of metagenomics.

[0004] Among the current methods for removing host nucleic acids, differential centrifugation, methylated antibodies, and PMA cannot effectively remove host nucleic acids, and the proportion of host sequences in sequencing data is not significantly different from that before removal; methods such as flow cytometry and microfluidics are costly, (or) take a long time to operate, and are easily contaminated by exogenous microorganisms; although the differential lysis method has a high host removal efficiency, water, chemical reagents, and additional centrifugation operations may also lead to the loss of microorganisms, which has a significant impact on the composition of the microbial community.

[0005] The current methods for removing host nucleic acids still have many defects, and there is an urgent need to develop a new method for removing host nucleic acid contamination to reduce the proportion of host nucleic acids, improve the sensitivity of microbial detection, provide more accurate microbial composition information and richer functional information, while simplifying the steps and shortening the operation time. Summary of the invention

[0006] Based on a large number of experimental studies, the inventors of the present application have developed a new method for removing host nucleic acids (e.g., animal-derived nucleic acids and / or plant-derived nucleic acids) from microbial samples (e.g., clinical samples containing microorganisms). The method can efficiently remove host nucleic acids (e.g., animal-derived nucleic acids and / or plant-derived nucleic acids) from microbial samples (e.g., clinical samples containing microorganisms), and the operation process is simple and gentle, and can minimize the loss of microbial nucleic acids in the sample and reduce the preference for different microorganisms. That is, the method of the present application can retain the nucleic acids of microbial origin to the greatest extent while efficiently removing host nucleic acids, and the method of the present application does not affect the microbial composition in the sample, thereby facilitating metagenomic analysis.

[0007] Therefore, in a first aspect, the present application provides a method for removing host nucleic acids in a microbial sample, comprising the following steps:

[0008] (1) providing a sample to be processed containing a microorganism, wherein the sample to be processed is suspected of containing host nucleic acid;

[0009] (2) passing the sample to be processed through a filter membrane, wherein the pore size of the filter membrane is in the range of 5-10 μm;

[0010] (3) Using nuclease to remove free nucleic acid in the sample obtained in step (2).

[0011] It is easy for a person skilled in the art to understand that in order to avoid interference with the analysis results of the microorganisms, it is significantly advantageous to specifically remove the host nucleic acid in the microbial sample.

[0012] In certain embodiments, the host nucleic acid is an animal-derived nucleic acid and / or a plant-derived nucleic acid. The animal-derived nucleic acid refers to a nucleic acid derived from an animal, including nucleic acids present in animal cells in a sample and nucleic acids from animals free in a sample. The animal may be a human or other mammal (e.g., livestock (e.g., pigs, cows), pets (e.g., cats, dogs), etc.). The plant-derived nucleic acid refers to a nucleic acid derived from a plant, including nucleic acids present in plant cells in a sample and nucleic acids from plants free in a sample. In certain embodiments, the plant is selected from crops or ornamental plants. In certain embodiments, the plant-derived nucleic acid refers to a nucleic acid derived from a plant root system.

[0013] As used herein, the term "microorganism" has the meaning generally understood by those skilled in the art. In certain embodiments, the microorganism is selected from bacteria, archaea, fungi, actinomycetes, algae, viruses, mycoplasmas, chlamydia, and any combination thereof. In certain embodiments, the microorganism is selected from bacteria, archaea, fungi, viruses, and any combination thereof. In certain embodiments, the microorganism is a bacterium.

[0014] In certain embodiments, the pore size of the filter membrane in step (2) is in the range of 5-6 μm, 5-7 μm, 5-8 μm, 5-9 μm, 6-7 μm, 6-8 μm, 6-9 μm, 6-10 μm, 7-8 μm, 7-9 μm, 7-10 μm, 8-9 μm, 8-10 μm or 9-10 μm.

[0015] In some embodiments, the pore size of the filter membrane is 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm. In some embodiments, the pore size of the filter membrane is 10 μm.

[0016] In certain embodiments, in step (2), passing the sample to be processed through the filter membrane can effectively remove animal cells and / or plant cells that may be present in the sample to be processed.

[0017] In certain embodiments, the host nucleic acid is DNA and / or RNA.

[0018] In certain embodiments, the host nucleic acid is DNA.

[0019] In certain embodiments, the host nucleic acid is RNA.

[0020] In certain embodiments, in step (3) of the method, the nuclease is selected from DNase, RNase, universal nuclease or any combination thereof.

[0021] As used herein, "universal nuclease" refers to a class of nucleases that can degrade various forms of (double-stranded, single-stranded, linear, circular, native or denatured) DNA and RNA under very broad conditions. In certain exemplary embodiments, the universal nuclease is selected from Benzonase and Denarase.

[0022] In certain embodiments, the nuclease comprises TURBO TM DNase, Benzonase, Denarase, DNaseI, RNase I, HL-SAN DNase or any combination thereof.

[0023] In certain embodiments, the nuclease comprises TURBO TM DNase and Benzonase.

[0024] In certain embodiments, in step (3) of the method, the sample obtained in step (2) is treated with the nuclease at a temperature of 25-42° C. (e.g., 37° C.) to remove free nucleic acids in the sample. In certain embodiments, in step (3) of the method, the sample obtained in step (2) is treated with the nuclease at a temperature of 25-42° C. (e.g., 37° C.) for 0.5-4 h (e.g., 0.5-3 h, 2-3 h, 2-4 h, 3 h) to remove free nucleic acids in the sample.

[0025] In certain embodiments, the sample to be processed is derived from bronchoalveolar lavage fluid, oropharyngeal swab sample, sputum, saliva, blood, plasma, cerebrospinal fluid, pleural effusion, bile, pus, nasopharyngeal swab sample, skin swab sample, vaginal swab sample, amniotic fluid, aqueous humor, synovial fluid, urine, animal tissue homogenate, or plant tissue homogenate.

[0026] It should be noted that the inventors of the present application found during the research process that dissolving the sample in physiological saline containing glycerol can improve the retention of microbial nucleic acids, reduce the impact on the microbial composition in the sample, and / or improve the removal of host nucleic acids.

[0027] In certain embodiments, in step (1), the sample to be processed contains glycerol and sodium chloride.

[0028] In certain embodiments, the volume fraction of glycerol in the sample to be processed is 15%-40% (e.g., 15%-25%, 15%-30%, 20%-25%, 20%-30%, 20%-40%, 25%-30%, 25%-40%, 25%).

[0029] In certain embodiments, in step (1), the volume fraction of glycerol in the sample to be processed is 25%.

[0030] In certain embodiments, in step (1), the concentration of sodium chloride in the sample to be processed is 0.8-1.0 g / 100 ml (eg, 0.9 g / 100 ml).

[0031] In certain embodiments, in step (1), the osmotic pressure value of the sample to be processed is substantially the same as the osmotic pressure value of plasma or tissue fluid of a mammal (eg, human).

[0032] In certain embodiments, in step (1), the concentration of sodium chloride in the sample to be processed is 0.9 g / 100 ml.

[0033] In certain embodiments, in step (1), the sample to be processed is a fresh sample or a frozen sample.

[0034] In certain embodiments, the sample is derived from a viscous and inhomogeneous sample (e.g., bronchoalveolar lavage fluid, sputum). In certain embodiments, the method provides the sample to be processed in step (1) by pre-treating the viscous and inhomogeneous sample (e.g., liquefaction treatment).

[0035] In certain embodiments, the pretreatment comprises the following steps:

[0036] (i) mixing the viscous inhomogeneous sample with sputum digestion fluid to form a mixture;

[0037] (ii) the mixture is mixed thoroughly and incubated at a temperature of 25-42° C. (eg, 37° C.) for 10-60 min (eg, 15-30 min).

[0038] In certain embodiments, the sputasol (also commonly referred to as sputasol) contains dithiothreitol (DTT). In certain embodiments, the sputasol contains dithiothreitol (DTT) and phosphate. In certain embodiments, the sputasol can be purchased from manufacturers such as Thermo and Oxoid.

[0039] In certain embodiments, in the pretreatment step (ii), the mixture is incubated at 25-42° C. (eg, 37° C.) for 10-60 min (eg, 15-30 min) under shaking conditions.

[0040] In certain embodiments, in the mixture, the volume fraction of the sputum digestion fluid is 1%-10% (e.g., 1%-4%, 1%-5%, 1%-8%, 3%-4%, 3%-5%, 3%-8%, 3%-10%, 3.75%).

[0041] In certain embodiments, the volume fraction of the sputum digestive fluid in the mixture is 3.75%.

[0042] In certain embodiments, after step (3), the method further comprises a step of inactivating nucleases in the sample obtained in step (3).

[0043] In certain embodiments, the method uses proteinase K to inactivate nucleases in the sample.

[0044] In certain embodiments, the method inactivates nucleases in the sample by treating the sample with proteinase K at a temperature of 20-60°C (e.g., 56°C) for 5-30 min (e.g., 5-15 min, 5-20 min, 10-15 min, 10-20 min, 10-30 min, 15-20 min, 15-30 min, 15 min).

[0045] In certain embodiments, the method does not comprise the step of lysing the microorganism to release microorganism-derived nucleic acid.

[0046] In certain embodiments, the method does not comprise the step of lysing the animal cells and / or plant cells to release the animal cell and / or plant cell-derived nucleic acids.

[0047] In a second aspect, the present application provides a method for extracting nucleic acid of a microorganism in a sample, comprising the following steps:

[0048] (a) providing a sample containing microorganisms, wherein the sample is a sample processed according to the method of the first aspect;

[0049] (b) lysing the microorganisms in the sample to release nucleic acids derived from the microorganisms;

[0050] (c) extracting nucleic acid derived from the microorganism, which includes separation and purification of nucleic acid.

[0051] In certain embodiments, the nucleic acid is a nucleic acid derived from a microorganism; for example, the nucleic acid is DNA (e.g., genomic DNA and / or plasmid DNA) and / or RNA (e.g., genomic RNA, rRNA, mRNA, microRNA and / or lncRNA) derived from a microorganism.

[0052] In certain embodiments, the nucleic acid is DNA (eg, genomic DNA and / or plasmid DNA) derived from a microorganism.

[0053] In certain embodiments, the nucleic acid is RNA (eg, genomic RNA, rRNA, mRNA, microRNA and / or lncRNA) derived from a microorganism.

[0054] In certain embodiments, prior to step (b), the method further comprises a step of inactivating nucleases in the sample.

[0055] In certain embodiments, the method uses proteinase K to inactivate nucleases in the sample.

[0056] In certain embodiments, the method inactivates nucleases in the sample by treating the sample with proteinase K at a temperature of 20-60°C (e.g., 56°C) for 5-30 min (e.g., 5-15 min, 5-20 min, 10-15 min, 10-20 min, 10-30 min, 15-20 min, 15-30 min, 15 min).

[0057] In a third aspect, the present application provides a kit comprising a filter membrane and a nuclease;

[0058] Wherein, the filter membrane and nuclease are as defined in the first aspect.

[0059] In certain embodiments, the kit further comprises: one or more of glycerol, glycerol-sodium chloride mixed solution, sodium chloride, proteinase K, a buffer of the nuclease, a reagent for lysing microorganisms, and a reagent for nucleic acid separation and purification.

[0060] In certain embodiments, the kit further comprises: one or more of glycerol, glycerol-sodium chloride mixed solution, sodium chloride, proteinase K, a buffer of the nuclease, a reagent for lysing microorganisms, a reagent for nucleic acid separation and purification, and sputum digestion fluid.

[0061] In certain embodiments, the sputasol (also commonly referred to as sputasol) contains dithiothreitol (DTT). In certain embodiments, the sputasol contains dithiothreitol (DTT) and phosphate.

[0062] In a fourth aspect, the present application provides use of the method of the first aspect or the second aspect, or the kit of the third aspect for performing metagenomic analysis.

[0063] Advantageous Effects of the Invention

[0064] Compared with the prior art, the method for removing host nucleic acids (e.g., animal-derived nucleic acids and / or plant-derived nucleic acids) in microbial samples (e.g., clinical samples containing microorganisms) provided in the present application can efficiently remove host nucleic acids (e.g., animal-derived nucleic acids and / or plant-derived nucleic acids) in microbial samples (e.g., clinical samples containing microorganisms), and the operation process is simple and gentle, and can minimize the loss of microbial nucleic acids in the sample and reduce the preference for different microorganisms. That is, the method of the present application can retain the nucleic acids of microbial origin to the greatest extent while efficiently removing host nucleic acids, and the method of the present application does not affect the microbial composition in the sample, thereby facilitating metagenomic analysis.

[0065] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1: The top 20 species composition of 4 bronchoalveolar lavage fluid samples after treatment with the two methods in the experimental group or the control group.

[0067] Figure 2 : Top 20 species composition of 4 oropharyngeal swab samples after treatment with 2 methods in the experimental group or the control group.

[0068] Figure 3 : To evaluate the relationship between microbial Ct values ​​and the accuracy of microbial composition in bronchoalveolar lavage fluid samples.

[0069] Figure 4 : To evaluate the relationship between microbial Ct values ​​and the accuracy of microbial composition in oropharyngeal swab samples. DETAILED DESCRIPTION

[0070] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0071] Unless otherwise specified, the molecular biology experimental methods used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compiled Guide to Molecular Biology Experiments, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturers. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0072] Materials and Equipment

[0073] Glycerol (Sigma-Aldrich, 50046), sodium chloride, nuclease-free water (Invitrogen, AM9930), sputum digestion fluid (Sputasol, Oxoid, SR0233), 5μm-10μm filter membrane, TURBO DNase (Thermo Fisher, AM2238), Benzonase (MERCK, E8263), Proteinase K (QIAgen, 19133), DNeasy PowerSoil Pro Kit (QIAgen, 47014), TaqMan Fast Advanced Master Mix (ABI, 4444557), QIAseq FX DNA Library Kit (QIAgen, 180479), vortex mixer, oscillating metal bath, handheld centrifuge (Kopjia, D1008), Jingxin tissue grinder (Jingxin, JXFSTPRP-CLN-24), high-speed centrifuge (Thermo Scientific, 75002425).

[0074] Reagent preparation

[0075] 50% glycerol saline: Add 0.9g sodium chloride to 50ml water and stir to mix, add 50ml glycerol, mix thoroughly, and filter with a 0.22μm filter membrane.

[0076] 25% glycerol saline: Add 0.9g sodium chloride to 75ml water and stir to mix, add 25ml glycerol, mix thoroughly, and filter with a 0.22μm filter membrane.

[0077] Sputum digestion solution working solution: add 0.75ml sputum digestion solution stock solution to 9.25ml nuclease-free water and mix well.

[0078] Example 1: Experimental steps and effects of the present method for selectively removing host nucleic acids from bronchoalveolar lavage fluid samples

[0079] 1. Sample preparation: Collect 4 bronchoalveolar lavage fluid samples, 1 ml for each sample; add an equal volume of 50% glycerol saline, mix well, let stand at room temperature for 30 minutes, and store at -80℃ for long-term storage.

[0080] 2. Sample liquefaction: Add an equal volume of sputum digestion working solution to the viscous and inhomogeneous bronchoalveolar lavage fluid sample, vortex briefly to mix, and incubate in a 37°C shaking metal bath at 600 rpm for 15-30 minutes until there is no visible precipitation.

[0081] 3. Sample packaging: After each sample is thoroughly mixed, divide it into 3 equal volumes of 200 μL.

[0082] 4. Experimental Group

[0083] (1) Removal of host nucleic acid from bronchoalveolar lavage fluid samples:

[0084] A. Removal of host cells: Pass 200 μL of sample through a 10 μm filter;

[0085] B. Removal of host nucleic acid: Add 22 μL TURBO buffer to the filtered sample, followed by 2 μL Benzonase and 8 μL TURBO DNase, mix by pipetting, and incubate at 37°C in a shaking metal bath at 600 rpm for 3 hours;

[0086] C. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0087] (2) DNA extraction from bronchoalveolar lavage fluid samples:

[0088] A. Bead beating: Add 800 μL of lysis buffer CD1 (from DNeasy Power Soil Pro Kit) to the sample from which host nucleic acid has been removed. Grind the sample using a Jingxin grinder at 30 Hz for 5 minutes, rest for 1 minute, and continue grinding for another 5 minutes.

[0089] B. DNA extraction: Follow the DNeasy PowerSoil Pro Kit instructions. Centrifuge the homogenate tube at 15000g for 1 minute; aspirate 600μL supernatant, add 200μL CD2 solution, vortex mix for 5 seconds, and centrifuge at 15000g for 1 minute; transfer 700μL supernatant to a new collection tube, add 600μL CD3 solution, vortex mix for 5 seconds; add 650μL lysate to the MB Spin Column, centrifuge at 15000g for 1 minute, discard the supernatant, add the remaining lysate to the MB Spin Column again, centrifuge at 15000g for 1 minute, and discard the supernatant; carefully place the MB Spin Column in a clean 2ml collection tube; add 500μL EA solution to the MB Spin Column, centrifuge at 15000g for 1 minute, and discard the supernatant; add 500μL C5 solution to the MB Spin Column, centrifuge at 15000g for 1 minute, and discard the supernatant; carefully transfer the MB Spin Place the column in a clean 2 ml collection tube and centrifuge at 16000 g for 2 minutes; add 50 μL of C6 solution to the silica gel membrane and incubate at room temperature for 5 minutes to redissolve the DNA; centrifuge at 15000 g for 1 minute and collect the DNA solution into a centrifuge tube.

[0090] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FX DNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0091] 5. Control group 1

[0092] (1) DNA extraction from bronchoalveolar lavage fluid samples (original samples, without host nucleic acid removal): A. Bead beating: refer to the steps of "bead beating" above.

[0093] B. DNA extraction: refer to the steps of "DNA extraction" above.

[0094] (2) QPCR was used to detect the concentration of bacterial and human DNA in the samples, and the QIAseq FX DNA Library Kit was used to prepare the metagenomic library and sequence the samples.

[0095] 6. Control group 2

[0096] (1) Removal of free nucleic acids from bronchoalveolar lavage fluid samples (removal of free host and microbial nucleic acids in samples):

[0097] A. Remove free nucleic acids: Add 22 μL TURBO buffer to the aliquoted sample, then add 2

[0098] μL Benzonase and 8μL TURBO DNase, mix by pipetting, and incubate at 37℃ in a shaking metal bath at 600rpm for 3 hours;

[0099] B. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0100] (2) DNA extraction from bronchoalveolar lavage fluid samples:

[0101] A. Bead-beating wall breaking: refer to the steps of "bead-beating wall breaking" above.

[0102] B. DNA extraction: refer to the steps of "DNA extraction" above.

[0103] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FX DNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0104] Example 1 test results:

[0105] Table 1: Host and bacterial quantitative Ct values:

[0106]

[0107] Table 2: Proportion of host and microbial sequences (%)

[0108]

[0109] As can be seen from the qPCR results in Table 1, the host nucleic acid removal method of the present application can remove 99.97% of the host genomic DNA, while the microbial content is not affected or accompanied by less loss; Table 2 Metagenomic sequencing results show that using the method of the present application, the proportion of human nucleic acid decreased by 9.11%-67.10%, the proportion of microorganisms increased by 3.18%-67.36%, and the microbial enrichment was 22.76-390.23 times; We calculated the Jensen-Shannon Distance between the control group 2 and the experimental group in each sample

[0110] (JSD), which ranges from 0.02-0.19, Figure 1 It can be observed that the abundance of the main components between the control group 2 and the experimental group is relatively similar, indicating that the method of the present application will not affect the main composition structure of the sample.

[0111] In summary, the method of the present application can significantly reduce human DNA in bronchoalveolar lavage fluid samples, increase the proportion of microorganisms, and maintain the accuracy of the microbial composition of the samples.

[0112] Example 2: Experimental steps and effects of the present method for selectively removing host nucleic acid from oropharyngeal swab samples

[0113] 1. Sample preparation: Collect 4 oropharyngeal swab samples, use 1 ml of 25% glycerol saline for each sample, vortex mix for 1 minute, let stand at room temperature for 30 minutes, and store at -80℃ for long-term storage

[0114] 2. Sample packaging: After each sample is thoroughly mixed, divide it into 3 equal volumes of 200 μL.

[0115] 3. Experimental Group

[0116] (1) Oropharyngeal swab samples to remove host nucleic acid:

[0117] A. Removal of host cells: Pass 200 μL of sample through a 10 μm filter;

[0118] B. Removal of host nucleic acid: Add 22 μL TURBO buffer to the filtered sample, then add 2 μL Benzonase and 8 μL TURBO DNase, mix by pipetting, and incubate at 37°C in a shaking metal bath at 600 rpm for 3 hours;

[0119] C. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0120] (2) DNA extraction from oropharyngeal swab samples:

[0121] A. Bead breaking: refer to Example 1.

[0122] B. DNA extraction: refer to Example 1.

[0123] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FXDNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0124] 4. Control group 1

[0125] (1) DNA extraction from oropharyngeal swab samples (original samples, without host nucleic acid removal):

[0126] A. Bead beating: Perform a bead beating step to extract DNA from the oropharyngeal swab samples packaged above.

[0127] Refer to Example 1 for steps.

[0128] B. DNA extraction: refer to Example 1.

[0129] (2) QPCR was used to detect the concentration of bacterial and human DNA in the samples. Subsequently, the metagenomic library was prepared using the QIAseq FX DNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0130] 7. Control Group 2

[0131] (1) Removal of free nucleic acid from oropharyngeal swab samples (removal of free host and microbial nucleic acid in the sample):

[0132] A. Removal of free nucleic acids: Add 22 μL TURBO buffer to the aliquoted sample, then add 2 μL Benzonase and 8 μL TURBO DNase, mix by pipetting, and incubate at 37°C in a shaking metal bath at 600 rpm for 3 hours;

[0133] B. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0134] (2) DNA extraction from oropharyngeal swab samples:

[0135] A. Bead beating: The sample from which free nucleic acid has been removed is subjected to a bead beating step for DNA extraction. The steps are as in Example 1.

[0136] B. DNA extraction: refer to Example 1.

[0137] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FX DNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0138] Example 2 test results:

[0139] Table 3: Host and bacterial quantitative Ct values:

[0140]

[0141] Table 4: Proportion of host and microbial sequences (%)

[0142]

[0143]

[0144] As can be seen from the qPCR results in Table 3, the host nucleic acid removal method of the present application can remove more than 99.46% of the host genomic DNA, while the microbial content is not affected or accompanied by less loss; Table 4 Metagenomic sequencing results show that using the method of the present application, the proportion of human nucleic acid decreased by 59.54%-77.48%, the proportion of microorganisms increased by 42.95%-57.54%, and the microbial enrichment was 3.29-13.42 times; We calculated the Jensen-Shannon Distance between the control group 2 and the experimental group in each sample

[0145] (JSD), which ranges from 0.01-0.07, Figure 2 It can be observed that the abundance of the main components between the control group 2 and the experimental group is similar, indicating that the method of the present application will not affect the main microbial composition structure of the oropharyngeal swab sample and has no preference. In summary, the method of the present application can significantly reduce the contamination of human DNA in oropharyngeal swab samples, increase the proportion of microorganisms, and maintain the accuracy of the sample microbial composition.

[0146] Example 3: The detection limit of the present method for selectively removing host nucleic acids from BALF samples

[0147] 1. Sample preparation: Collect 35 bronchoalveolar lavage fluid samples, 1 ml for each sample; add an equal volume of 50% glycerol saline, mix well, let stand at room temperature for 30 minutes, and store at -80℃ for long-term storage.

[0148] 2. Sample liquefaction: Add an equal volume of sputum digestion working solution to the viscous and inhomogeneous bronchoalveolar lavage fluid sample, vortex briefly to mix, and incubate in a 37°C shaking metal bath at 600 rpm for 15-30 minutes until there is no visible precipitation.

[0149] 3. Sample packaging: After each sample is thoroughly mixed, divide it into 3 equal volumes of 200 μL.

[0150] 4. Experimental Group

[0151] (1) Removal of host nucleic acid from bronchoalveolar lavage fluid samples:

[0152] A. Removal of host cells: Pass 200 μL of sample through a 10 μm filter;

[0153] B. Removal of host nucleic acid: Add 22 μL TURBO buffer to the filtered sample, then add 2 μL Benzonase and 8 μL TURBO DNase, mix by pipetting, and incubate at 37°C in a shaking metal bath at 600 rpm for 3 hours;

[0154] C. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0155] (2) DNA extraction from bronchoalveolar lavage fluid samples:

[0156] A. Bead beating: refer to Example 1.

[0157] B. DNA extraction: refer to Example 1.

[0158] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FXDNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0159] 5. Control Group

[0160] (1) Removal of free nucleic acids from bronchoalveolar lavage fluid samples (removal of free host and microbial nucleic acids in samples):

[0161] A. Remove free nucleic acids: Add 22 μL TURBO buffer to the aliquoted sample, then add 2

[0162] μL Benzonase and 8μL TURBO DNase, mix by pipetting, and incubate at 37℃ in a shaking metal bath at 600rpm for 3 hours;

[0163] B. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0164] (2) DNA extraction from bronchoalveolar lavage fluid samples:

[0165] A. Bead beating: The sample from which free nucleic acid has been removed is subjected to a bead beating step for DNA extraction. The steps are as in Example 1.

[0166] B. DNA extraction: refer to Example 1.

[0167] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FXDNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0168] The results are as follows Figure 3 As shown, Figure 3 The results show that when the Ct value of the experimental group is lower than 30 (microbial load is higher than 124.22 pg / ml BALF), the JSD distance between the experimental group and the control group is lower than 0.25, and the method of this application does not affect the accuracy of the microbial composition; when the Ct value is higher than 30 (microbial load is lower than 124.22

[0169] When the microbial concentration was 124.22 pg / ml BALF, the accuracy of the sample microbial composition decreased significantly as the microbial concentration decreased, indicating that the detection limit of the method of the present application in bronchoalveolar lavage fluid was 124.22 pg / ml BALF.

[0170] Example 4: Detection limit of the present method in oropharyngeal swab samples

[0171] 1. Sample preparation: 34 oropharyngeal swab samples with different microbial loads were collected. Each sample was mixed with 1 ml of 25% glycerol saline, vortexed for 1 minute, left at room temperature for 30 minutes, and stored at -80°C for a long time.

[0172] 2. Sample packaging: After each sample is thoroughly mixed, divide it into 3 equal volumes of 200 μL.

[0173] 3. Experimental Group

[0174] (1) Oropharyngeal swab samples to remove host nucleic acid:

[0175] A. Removal of host cells: Pass 200 μL of sample through a 10 μm filter;

[0176] B. Removal of host nucleic acid: Add 22 μL TURBO buffer to the filtered sample, then add 2 μL Benzonase and 8 μL TURBO DNase, mix by pipetting, and incubate at 37°C in a shaking metal bath at 600 rpm for 3 hours;

[0177] C. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0178] (2) DNA extraction from oropharyngeal swab samples:

[0179] A. Bead breaking: refer to Example 1.

[0180] B. DNA extraction: refer to Example 1.

[0181] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FX DNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0182] 4. Control Group

[0183] (1) Removal of free nucleic acid from oropharyngeal swab samples (removal of free host and microbial nucleic acid in the sample):

[0184] A. Removal of free nucleic acids: Add 22 μL TURBO buffer to the aliquoted sample, then add 2 μL Benzonase and 8 μL TURBO DNase, mix by pipetting, and incubate at 37°C in a shaking metal bath at 600 rpm for 3 hours;

[0185] B. Nuclease inactivation: Add 20 μL proteinase K to the above sample, shake in a metal bath at 56°C, 600 rpm, and incubate for 15 minutes;

[0186] (2) DNA extraction from oropharyngeal swab samples:

[0187] A. Bead beating: The sample from which free nucleic acid has been removed is subjected to a bead beating step for DNA extraction. The steps are as in Example 1.

[0188] B. DNA extraction: refer to Example 1.

[0189] (3) QPCR was used to detect the concentration of bacterial and human DNA in the samples. The metagenomic library was then prepared using the QIAseq FX DNA Library Kit and high-throughput sequencing was performed using the Illumina Novaseq 6000PE150 platform.

[0190] The results are as follows Figure 4 As shown, Figure 4 It shows that when the Ct value of the experimental group is lower than 31 (microbial load is higher than 64.54pg / swab), the JSD distance between the experimental group and the control group is lower than 0.25, and the method of the present application does not affect the accuracy of the microbial composition; when the Ct value is higher than 31 (microbial load is lower than 64.54pg / swab), as the microbial concentration decreases, the accuracy of the sample microbial composition decreases significantly, indicating that the detection limit of the method of the present application in oropharyngeal swabs is 64.54pg / swab.

[0191] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for removing host nucleic acid from a microbial sample, comprising the following steps: (1) providing a sample to be processed containing a microorganism, wherein the sample to be processed is suspected of containing host nucleic acid; (2) passing the sample to be processed through a filter membrane, wherein the pore size of the filter membrane is in the range of 5-10 μm; (3) Using nuclease to remove free nucleic acid in the sample obtained in step (2).

2. The method of claim 1, wherein The pore size of the filter membrane in step (2) is in the range of 5-6 μm, 5-7 μm, 5-8 μm, 5-9 μm, 6-7 μm, 6-8 μm, 6-9 μm, 6-10 μm, 7-8 μm, 7-9 μm, 7-10 μm, 8-9 μm, 8-10 μm or 9-10 μm; Preferably, the pore size of the filter membrane is 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm; preferably, the pore size of the filter membrane is 10 μm.

3. The method of claim 1 or 2, wherein The host nucleic acid is DNA and / or RNA.

4. The method of any one of claims 1 to 3, wherein In step (3), the nuclease is selected from DNase, RNase, universal nuclease or any combination thereof; Preferably, the nuclease comprises TURBO TM DNase, Benzonase, Denarase, DNase I, RNase I, HL-SAN DNase or any combination thereof; Preferably, the nuclease comprises TURBO TM DNase and Benzonase.

5. The method of any one of claims 1 to 4, wherein The sample to be processed is derived from bronchoalveolar lavage fluid, oropharyngeal swab sample, sputum, saliva, blood, plasma, cerebrospinal fluid, pleural effusion, bile, pus, nasopharyngeal swab sample, skin swab sample, vaginal swab sample, amniotic fluid, aqueous humor, synovial fluid, urine, animal tissue homogenate or plant tissue homogenate.

6. The method of any one of claims 1 to 5, wherein In step (1), the sample to be processed contains glycerol and sodium chloride; Preferably, in the sample to be processed, the volume fraction of the glycerol is 15%-40% (e.g., 15%-25%, 15%-30%, 20%-25%, 20%-30%, 20%-40%, 25%-30%, 25%-40%, 25%); and / or, in the sample to be processed, the concentration of the sodium chloride is 0.8-1.0 g / 100 ml (e.g., 0.9 g / 100 ml); Preferably, in the sample to be processed, the volume fraction of the glycerol is 25%, and / or, in the sample to be processed, the concentration of the sodium chloride is 0.9 g / 100 ml.

7. A method for extracting nucleic acid from microorganisms in a sample, comprising the following steps: (a) providing a sample containing microorganisms, wherein the sample is a sample processed according to the method according to any one of claims 1 to 6; (b) lysing the microorganisms in the sample to release nucleic acids derived from the microorganisms; (c) extracting nucleic acid derived from the microorganism, which includes separation and purification of nucleic acid.

8. The method of claim 7, wherein Prior to step (b), the method further comprises the step of inactivating nucleases in the sample; Preferably, the method uses proteinase K to inactivate nucleases in the sample.

9. A kit comprising a filter membrane and a nuclease; in, The filter membrane is as defined in claim 1 or 2; the nuclease is as defined in claim 4; Preferably, the kit further comprises: one or more of glycerol, glycerol-sodium chloride mixed solution, sodium chloride, proteinase K, a buffer of the nuclease, a reagent for lysing microorganisms, and a reagent for separating and purifying nucleic acids.

10. Use of the method of any one of claims 1 to 6, the method of claim 7 or 8, or the kit of claim 9 for performing metagenomic analysis.

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