Physical host removal method for respiratory tract sample

Abstract

The invention relates to a physical host removal method for a respiratory tract sample, and belongs to the technical field of pathogen detection. The invention aims to combine a physical method to remove hosts and a chemical method to remove hosts, establish an efficient and broad-spectrum respiratory tract sample host removing method by optimizing physical homogenization parameters and combining the chemical method, and break through the bottleneck in the prior art. The detection method disclosed by the invention has the advantages that 1) the detection method can be suitable for sputum samples without adding treatment steps; 2) the human source removal efficiency is high, and compared with a chemical method for removing hosts widely applied at present, the host removal efficiency is high, and the number of microbial sequences is high; and 3) the operation is simple and convenient, and hosts can be removed within a short time.

Description

Technical Field

[0001] This invention relates to a physical host removal method for respiratory samples, belonging to the field of pathogen detection technology. Background Technology

[0002] In respiratory samples used for infection detection, the high content of host DNA can easily interfere with the detection of pathogens. Therefore, host removal treatment is usually required before pathogen detection. Current mainstream host removal methods often use chemical reagents for lysis due to the significant differences in cellular structural stability and mechanical strength between human cells and pathogens. However, such methods often use highly potent lysis reagents that can easily damage structurally fragile pathogens, leading to some being missed in subsequent detections and affecting detection sensitivity. CN112322701A discloses a denaturing agent combination (saponin / TritonX-100 / digitalis saponin / CHAPS) that releases nucleic acids by disrupting the human cell membrane structure, followed by DNase I digestion of host DNA. However, the strong lysis properties of saponins in this scheme can easily damage structurally fragile pathogens (such as mycoplasma and viruses), leading to missed detections. CN117965526A optimizes the lysis system by adding alkyl glycosides to the saponin / Tween buffer to improve host removal rate and integrates the lysis-binding steps to simplify the process. However, it still relies on the principle of chemical lysis, which poses an uncontrollable risk of damage to pathogenic microorganisms (such as viruses and parasites) that do not have cell wall protection.

[0003] All of the above chemical methods require a balance in lysis intensity. Insufficient intensity will result in incomplete removal of the host, while excessive intensity will damage the pathogen. In particular, for complex samples such as sputum, the mucus components may interfere with the lysis efficiency and exacerbate pathogen loss. Summary of the Invention

[0004] This patent aims to combine physical and chemical methods for host removal, and by optimizing physical homogenization parameters and combining them with chemical methods, to establish an efficient and broad-spectrum method for host removal of respiratory samples, thus breaking through the existing technical bottlenecks.

[0005] A host removal method for respiratory samples includes the following steps:

[0006] (a) The respiratory sample is mixed with a lysis system containing grinding beads and nuclease;

[0007] (b) The mixture obtained in step (a) is subjected to mechanical shear force to homogenize it in order to selectively lyse the host cells in the sample and release the host nucleic acid;

[0008] (c) Incubate under preset conditions to allow the nuclease to degrade the host nucleic acid;

[0009] (d) Add a stop solution to terminate the activity of the nuclease and obtain a host-free sample.

[0010] The homogenization rotation speed in step (b) is 4-5 m / s.

[0011] The homogenization process consists of 2-4 cycles, each lasting 30-60 seconds.

[0012] The pyrolysis system also contains the surfactant Triton X-100.

[0013] The final concentration of Triton X-100 in the pyrolysis system is 0.01%-0.1% (v / v).

[0014] The lysis system also contains saponins.

[0015] The final concentration of the saponin in the pyrolysis system is 0.05-0.2% (w / v).

[0016] The incubation temperature in step (c) is 35-40℃ and the incubation time is 5-15 min.

[0017] When the respiratory sample is a viscous sample such as sputum, the lysis system also contains DTT.

[0018] It also includes a step to predict the proportion of pathogen sequences in the host-free sample, which includes:

[0019] Based on the total nucleic acid T extracted after host removal treatment, the samples were divided into a high nucleic acid group and a low nucleic acid group. When the total nucleic acid T was greater than 50 ng, the sample was classified as the high nucleic acid group; when the total nucleic acid T was less than or equal to 50 ng, the sample was classified as the low nucleic acid group. The predicted pathogen sequence proportion P in the corresponding group was calculated using the following formula:

[0020] Wherein, P is the predicted proportion of pathogen sequences, P0 is the average value of the initial pathogen nucleic acid proportion determined by fitting data from the high or low nucleic acid group to which the sample belongs, which is obtained by averaging the known sample measurement values; E is the amount of nuclease added in step (a); and k is an undetermined coefficient characterizing the efficiency of a unit nuclease in degrading host nucleic acids.

[0021] The beneficial effects of this invention are as follows: The advantages of the detection method of this invention include: 1) It can be applied to sputum samples without adding processing steps. 2) It has high human removal efficiency; compared with the currently widely used chemical methods for host removal, it has high host removal efficiency and high microbial sequence count. 3) It is simple to operate and can complete host removal within 18 minutes. Attached Figure Description

[0022] Figure 1 It is a homogenization and host removal process;

[0023] Figure 2 It is a process that combines chemical and physical methods. Detailed Implementation

[0024] This patent establishes a physical host removal process suitable for respiratory samples (including but not limited to bronchoalveolar lavage fluid and sputum). Based on the structural differences between animal and microbial cells, a relatively weak mechanical shear force combined with a lysis buffer is used to selectively lyse host cells. The released host nucleic acids are degraded by an added totipotent nuclease, while having minimal impact on the microbial cells. The sample after host nucleic acid removal can be processed with nucleic acid extraction reagents to obtain high-purity genomic DNA.

[0025] This invention primarily employs a physical bead milling method for removing host nucleic acids. Using a homogenizing speed of 4-5 m / s for 30 seconds to 1 minute, host cells are lysed without significant impact on microbial cells. Furthermore, combining this method with low concentrations of saponins and Triton X-100 detergents effectively enhances host cell lysis efficiency, thereby increasing the number of pathogen sequences detected. Simultaneously, the addition of DTT to the system makes this invention applicable to viscous samples such as sputum. Therefore, the method for removing host nucleic acids using this invention can efficiently remove host nucleic acids while avoiding damage to fragile microbial cells. This method is compatible with viscous samples such as sputum, requires no additional steps, is simple to operate, highly efficient, and reproducible.

[0026] This invention proposes a method for removing host nucleic acid. According to an embodiment of the invention, the method includes: (1) adding a sample containing host nucleic acid into a Lysing matrix D tube, mixing the nuclease Mix and transferring it to a cell mechanical lysis apparatus; (2) incubating after the reaction to obtain a reaction solution; and (3) mixing the reaction solution with a stop solution.

[0027] According to an embodiment of the present invention, based on 0.4 mL of the sample, the amount of nuclease Mix added is 50 μL, the incubation time of the nuclease is 10 min, and the amount of stop solution added is 50 μL. This allows for more effective lysis of host cells, release of host nucleic acids, lysis of host nucleic acids under the action of nucleases, and termination of the reaction using a stop solution to remove host nucleic acids while avoiding damage to microbial cells.

[0028] According to an embodiment of the present invention, the homogenization rotation speed in step (1) is 4-5 m / s, the time for one cycle is 30 s-1 min, and the number of cycles is 2-4. This is to sufficiently destroy the host cells and release the host nucleic acids therein.

[0029] According to an embodiment of the present invention, in step (2), the incubation temperature is 37°C and the time is 10 minutes. This is to fully lyse the host cell, release the host nucleic acid therein, and then lyse the host nucleic acid under the action of nucleases to achieve the purpose of removing the host nucleic acid.

[0030] According to an embodiment of the present invention, in step (3), the terminating solution is EDTA. This is to ensure sufficient termination of the nuclease and sufficient collection of microbial cells.

[0031] In another aspect, the present invention proposes a method for further removing host nucleic acids. According to an embodiment of the present invention, the method includes adding low concentrations of saponins and Triton X-100 to the host removal method described above, in order to further remove host nucleic acids from the sample to be tested. Thus, the method of the present invention can achieve the goal of accurately detecting microbial nucleic acids.

[0032] In embodiments of the present invention, the concentration of the saponin described above is 0.1%, and the concentration of Triton X-100 is 0.01% to 0.1%.

[0033] Example 1

[0034] Sample types: bronchoalveolar lavage fluid and sputum (except for Example 2, all test samples were bronchoalveolar lavage fluid samples); Procedure: Take 0.4 mL of bronchoalveolar lavage fluid / sputum sample and place it in a homogenization tube. Then add 1.5 μL of totipotent nuclease (simultaneously, 0.8 μL and 3.0 μL were used for parallel experiments) and 50 μL of nuclease reaction buffer (both the totipotent nuclease and nuclease reaction buffer were purchased from Nanjing Difeng Medical Equipment Co., Ltd., and the nuclease reaction buffer contained Mg2+). Homogenize at 5 m / s-30 s-3 cycles (5 m / s is the linear velocity of the homogenizer; the instrument is FastPrep-24, purchased from MPBiomedicals; the homogenization cycle number is 3, with each cycle lasting 30 s and an interval of 30 s between cycles). Then transfer to a metal bath and incubate at 37°C for 10 min. Add 50 μL of 0.5 M EDTA and incubate for another 10 min. Then centrifuge at high speed to remove the supernatant to obtain the host-free sample.

[0035] Downstream reactions: nucleic acid extraction, metagenomic sequencing (suitable for MGI-200 platform sequencing, SE50 sequencing strategy).

[0036] In the following tests, six samples were used. The host-free samples were homogenized and disrupted, followed by extraction and library construction. The resulting library samples were subjected to next-generation metagenomic sequencing. After data processing and quality control, the extracted nucleic acid concentration, the proportion of human sequences, and the proportion of pathogen sequences are shown in the table below:

[0037] Table 1

[0038]

[0039] Table 2

[0040]

[0041] Table 3

[0042]

[0043] Example 2

[0044] The pretreatment method for the experimental group was physical host removal followed by homogenization; the pretreatment method for the control group was chemical debonding followed by homogenization; all other experimental conditions were kept consistent.

[0045] Experimental procedure:

[0046] Sample preparation:

[0047] Two mL of sputum samples were taken from five patients with clinical infectious diseases and placed in centrifuge tubes. The patients with clinical infectious diseases were selected from Dify Medical Laboratory.

[0048] Experimental procedure:

[0049] 1. Extraction process (mainly host removal process, including host removal pretreatment, homogenization lysis, and magnetic bead extraction); The method in this invention supports various types of respiratory samples, including bronchoalveolar lavage fluid and sputum. The samples in the following examples are derived from bronchoalveolar lavage fluid and sputum.

[0050] 1.1 Pre-host processing

[0051] 1.1.1 Sampling: Samples from the same clinically infected patient were mixed and then 0.2 mL and 0.4 mL were taken respectively and used as experimental samples for the control group and the experimental group.

[0052] 1.1.2 Lysis of human cells:

[0053] Experimental group: Take 0.4 mL of sample, add DTT to a final concentration of 0.02 M, 1.5 μL of pluripotent nuclease and nuclease buffer to Lysing Matrix D tube, place it in a cell mechanical lysis apparatus for mechanical lysis, and immediately place it in a metal bath at 37 °C for 10 min after completion.

[0054] The experiment for the control group: 0.2 mL of sample was added to 0.2 mL of sample dilution buffer (containing DTT and Triton X-100) and proteinase K, and then incubated in a metal bath at 60 °C for 10 min. Then, 1 × PBS and 0.5 mL of host removal reagent mix (containing saponins, Tween-20, pluripotent nuclease and nuclease buffer) were added, vortexed and mixed, and then incubated in a metal bath at 37 °C for 10 min.

[0055] 1.1.3 Collecting microbial cells:

[0056] Experimental group: Immediately after lysing human cells, add 50 μL of 0.5 M EDTA and incubate for 10 min. Transfer all supernatant to centrifuge tubes, centrifuge at 16000 g for 3 min at room temperature, discard the supernatant and retain the precipitate.

[0057] The experiment in the control group: After lysing human cells, 200 μL of 0.5 M EDTA was added, and the mixture was incubated in a metal bath at 37°C for 10 min. The mixture was then centrifuged at 16000 g at room temperature for 3 min, and the supernatant was discarded while the precipitate was retained.

[0058] 1.2 Homogeneous lysis (control group & experimental group)

[0059] 1.2.1 Homogeneous lysis: Resuspend the microbial cell pellet with 1 mL PBS and transfer all liquid to a LysingMatrix E tube. Place the tube in a cell mechanical lysis apparatus for mechanical lysis. After completion, centrifuge at 16000g for 5 min and transfer the supernatant to a 5 mL centrifuge tube.

[0060] 1.3 Extraction using magnetic beads

[0061] 1.3.1 Add proteinase K and lysis buffer, mix well and incubate at room temperature for 20 min.

[0062] 1.3.2 Nucleic acid adsorption: Add the pre-washed magnetic beads to a 5 mL centrifuge tube, place it on a multi-tube vortex mixer for vortex resuspension, and incubate at room temperature for 10 min.

[0063] 1.3.3 Purification of nucleic acids: Add washing buffer 1, washing buffer 2, and anhydrous ethanol to 5 mL centrifuge tubes respectively for nucleic acid purification.

[0064] 1.3.4 Drying and transferring nucleic acid: The purified nucleic acid was placed in a metal bath at 56°C and dried with the lid off. Then, enzyme-free water was added to wash and transfer the nucleic acid.

[0065] 1.3.5 Quantitative transfer to library construction process: Nucleic acid is quantified using Qubit.

[0066] 2. DNA library construction process using the interruption method

[0067] 2.1 Sample intake confirmation: The sample intake is 10 ng. Take the corresponding volume of DNA according to the DNA concentration and add water to 25 μL. If it is less than 10 ng, take 25 μL directly.

[0068] 2.2 Enzyme digestion, end repair and A addition: Add 5 μL of Smearase mix to 25 μL of sample on an ice plate, mix well by pipetting, and perform enzyme digestion, end repair and A addition reaction.

[0069] 2.3 Adapter ligation: Prepare the ligation mix (including Ligation Enhancer + Fast T4 DNA Ligase + adapter), add the ligation mix to the sample after the reaction in step 2.2 on an ice plate, mix by blowing and aspirating, and then carry out the ligation reaction.

[0070] 2.4 0.8× Magnetic bead purification: Add 40 μL of purification magnetic beads to the ligation product, mix by blowing and aspiration, let stand at room temperature, place on a magnetic rack, remove the supernatant by magnetic aspiration, and add 50 μL of PCR mix to resuspend the magnetic beads.

[0071] 2.5 PCR amplification: After mixing the sample from step 4 by pipetting and aspirating, run the PCR reaction program for 12 cycles.

[0072] 2.6 1.0× Magnetic bead purification: Add 50 μL of purified magnetic beads to the amplified product, mix by pipetting and aspirating, let stand at room temperature for 3 min, place on a magnetic rack and aspirate for 1 min, and discard the supernatant; wash with 200 μL of 80% ethanol for 30 s, discard the supernatant, and wash twice in total; remove residual ethanol, air dry on a magnetic rack for 2 min; add 50 μL of enzyme-free water to elute, mix by pipetting and aspirating, let stand at room temperature for 3 min, place on a magnetic rack and aspirate for 1 min, and then transfer 45 μL of library.

[0073] 2.7 Concentration determination: The library was quantified using Qubit and diluted according to the required concentration (below 20 ng / μL).

[0074] 3. Library sequencing (DIFSEQ-200 sequencer, SE50, 20M sequencing throughput)

[0075] 4. Bioinformatics Analysis Process

[0076] Bioinformatics analysis requires feedback of basic quality control data such as Raw_Reads, HQ_Reads, GC percentage, Q30%, and Dup%; it also involves comparing and filtering pathogen reads and host reads (normally human reads) from the HQ_Reads for the detection target, and calculating their respective percentages in the HQ_Reads.

[0077] Test results:

[0078] Table 4

[0079]

[0080] The table above shows the differences in extraction yield, the proportion of human sequences, and the proportion of pathogen sequences between the experimental and control groups. It is evident that the control group showed significantly better results in removing human-derived sequences. The table below shows the pathogen sequence data for each sample in the two groups (throughput normalized to 20M). It is clear that the number of pathogen sequences detected in the experimental group was significantly greater than that in the control group.

[0081] Table 5

[0082]

[0083] It is evident that this method is applicable to sputum samples.

[0084] Example 3

[0085] The chemical host removal method was further combined with this method, and the remaining steps were consistent with those in the experimental group of Example 2.

[0086] The Lysing Matrix D tubes in Experiment Group 1 need to be filled with omnipotent nuclease and nuclease buffer.

[0087] In experimental group 2, an additional 0.01% of Triton X-100 needs to be added;

[0088] In experimental group 3, an additional 0.1% of Triton X-100 needs to be added;

[0089] All other experimental conditions were kept consistent.

[0090] Sample preparation:

[0091] Two mL of bronchoalveolar lavage fluid was taken from five patients with clinical infectious diseases and placed in centrifuge tubes. The patients with clinical infectious diseases were selected from Dify Medical Laboratory.

[0092] Experimental procedure:

[0093] Consistent with the experimental group in Example 2

[0094] The composition of homogenization tube D in the corresponding experimental group is as follows:

[0095] Table 6

[0096]

[0097] Test results:

[0098] Table 7

[0099]

[0100] The table above shows the sample extraction yield of different experimental groups. It can be seen that adding Triton X-100 significantly reduces the nucleic acid yield.

[0101] Table 8

[0102]

[0103] Table 9

[0104]

[0105]

[0106] Tables 8 and 9 show the proportions of human and pathogen sequences. It can be seen that adding Triton X-100 significantly reduces the proportion of human sequences and increases the proportion of pathogen sequences in the samples.

[0107] Table 10

[0108]

[0109] It is evident that adding Triton X-100 to the physical host removal medium (Lysing Matrix D tube) is beneficial for pathogen detection, and the optimal Triton X-100 concentration is 0.1%.

[0110] Example 4

[0111] Further, saponins were added to the Lysing MatrixD tubes to enhance the dehumanization effect, and the remaining steps were consistent with the experimental group in Example 2.

[0112] The Lysing Matrix D tubes in Experiment Group 1 need to be filled with omnipotent nuclease and nuclease buffer.

[0113] In experimental group 2, an additional 0.1% of saponin needs to be added;

[0114] In experimental group 3, Triton X-100 0.1% and saponin 0.1% need to be added additionally;

[0115] All other experimental conditions were kept consistent.

[0116] The composition of homogenization tube D in the corresponding experimental group is as follows:

[0117] Table 11

[0118]

[0119] Test results:

[0120] Table 12

[0121]

[0122] It can be seen that the extraction yield decreased slightly after adding saponins and Triton X-100.

[0123] Table 13

[0124]

[0125] Table 14

[0126]

[0127] It is evident that the simultaneous addition of Triton X-100 and saponins significantly reduced the proportion of human sequences and increased the proportion of pathogen sequences in the samples.

[0128] Table 15

[0129]

[0130]

[0131] It is evident that adding saponins and Triton X-100 under physical host removal conditions yields better results in actual testing.

[0132] Example 5:

[0133] The Lysing Matrix D tubes in Experiment Group 1 need to be filled with omnipotent nuclease and nuclease buffer.

[0134] Experimental group 2 requires the addition of 0.1% Triton X-100 and 0.1% saponin;

[0135] The three samples included in this embodiment were negative bronchoalveolar lavage fluid, and the pathogen count added to each 1.2 mL sample is shown in the table below:

[0136] Table 16

[0137] Pathogen name Dosage (CFU) Candida albicans 3×10^5 Aspergillus fumigatus 3×10^5 Streptococcus pneumoniae 3×10^4 Staphylococcus epidermidis 3×10^4 Acinetobacter baumannii 3×10^6 Klebsiella pneumoniae 3×10^6 Pseudomonas aeruginosa 3×10^6

[0138] After the pathogen was added to the sample, it was divided into three groups, with each sample in each group having a volume of 0.4 mL.

[0139] All other experimental conditions were kept consistent.

[0140] The composition of homogenization tube D in the corresponding experimental group is as follows:

[0141] Table 17

[0142]

[0143] Test results:

[0144] Table 18

[0145]

[0146]

[0147] It is evident that the addition of saponins and Triton X-100 significantly reduced the proportion of human sequences.

[0148] Table 19 Pathogen Sequence Count (RPM 20M)

[0149]

[0150] It is evident that the number of each pathogen sequence added in experimental group 2 was significantly higher than that in experimental group 1.

[0151] Example 6

[0152] To better assess the proportion of pathogen sequences in samples after host removal treatment, this patent also constructs a prediction method for predicting the proportion of pathogen sequences. This method estimates the proportion of pathogen sequences in the sample based on the total amount of nucleic acid and the amount of nuclease added after each homogenization and host removal process. Let the total amount of nucleic acid after each homogenization and host removal be T, and the amount of nuclease added be E. Furthermore, let the initial total nucleic acid amount in the sample be T0, and the initial proportion of pathogen nucleic acid be P0. Then the following relationship holds:

[0153] Initial pathogen nucleic acid content Pathogen0 = P0·T0

[0154] Initial human nucleic acid quantity Human0 = (1-P0)·T0

[0155] Nucleases primarily degrade human nucleic acids, with less degradation of pathogen nucleic acids. Assuming the degradation rate of pathogen nucleic acids is very small and negligible, the degradation rate of human nucleic acids is directly proportional to the amount of nuclease added, E. Therefore, the amount of human nucleic acid degraded is:

[0156] Human degraded =k·E·Human0

[0157] Where k is the parameter to be fitted, representing the degradation efficiency of human nucleic acid per unit amount of nuclease added. After degradation, the remaining amount of human nucleic acid is: Human final =Human0-Human degraded = (1-kE)·Human0; The amount of pathogen nucleic acid remains approximately unchanged: Pathogen final ≈Pathogen0=P0T0.

[0158] The total nucleic acid content after host removal is the sum of the pathogen nucleic acid content and the remaining human nucleic acid content:

[0159] T = Pathogen final+Human final =P0T0+(1-kE)(1-P0)T0

[0160] Rearranging the above equation, we get: T=T0[P0+(1-kE)(1-P0)]

[0161] The relationship between the initial total nucleic acid quantity T0 and the total nucleic acid quantity T after the experiment was obtained:

[0162]

[0163] The proportion of pathogen nucleic acid is:

[0164]

[0165] Substitute the T0 obtained above into:

[0166]

[0167] When performing parametric regression, the samples were divided into a high nucleic acid content group (greater than 50) and a low nucleic acid content group (less than 50) based on the total amount of nucleic acid in the samples. The initial proportion of pathogen nucleic acid in each group was determined by averaging the data fitting results of each group. The comparison between predicted and calculated values ​​in the high and low nucleic acid content groups is shown in the table below:

[0168] Table 20

[0169]

[0170] The model parameters are summarized below:

[0171] Table 21

[0172] Fitting groups <![CDATA[P0(%)]]> k(μL⁻¹) Overall RMSE (%) Low nucleic acid group 33.81 -846.40 8.40 High nucleic acid group 34.07 846.40 1.70

[0173] It can be seen that this prediction model can predict the proportion of pathogen sequences in low nucleic acid content and high nucleic acid content groups respectively, and the calculation error is less than 10% under different experimental group conditions.

Claims

1. A method for dehosting a respiratory tract sample, comprising, The method comprises the following steps: (a) mixing the respiratory sample with a lysis system comprising grinding beads and a nuclease; (b) homogenizing the mixture obtained in step (a) by applying mechanical shear force to selectively lyse host cells in the sample and release host nucleic acids; (c) incubating under preset conditions to allow the nuclease to degrade the host nucleic acids; (d) adding a termination solution to terminate the activity of the nuclease to obtain a dehosted sample.

2. The method of claim 1, wherein, The homogenization speed in step (b) is 4-5 m / s.

3. The method of claim 2, wherein, The homogenization process comprises 2-4 cycles, and each cycle lasts for 30-60 s.

4. The method of claim 1, wherein, The lysis system further comprises a surfactant Triton X-100.

5. The method of claim 4, wherein, The final concentration of Triton X-100 in the lysis system is 0.01%-0.1% (v / v).

6. The method of any one of claims 1, 4, or 5, wherein, The lysis system further comprises a saponin.

7. The method of claim 6, wherein, The final concentration of saponin in the lysis system is 0.05-0.2% (w / v).

8. The method of claim 1, wherein, The temperature of the incubation in step (c) is 35-40℃, and the time is 5-15 min.

9. The method of claim 1, wherein, When the respiratory sample is a viscous sample such as sputum, the lysis system further comprises DTT.

10. The method of claim 1, wherein, A step of predicting the proportion of pathogen sequences in the dehosted sample is further included, which comprises: According to the total amount T of nucleic acid extracted after dehosting treatment, the sample is divided into a high nucleic acid group and a low nucleic acid group, wherein when the total amount T of nucleic acid is greater than 50 ng, the sample is classified into a high nucleic acid group, and when the total amount T of nucleic acid is less than or equal to 50 ng, the sample is classified into a low nucleic acid group; and the predicted pathogen sequence proportion P in the corresponding group is calculated by the following formula: Wherein, P is the predicted pathogen sequence proportion, P0 is the average value of the initial pathogen nucleic acid proportion determined according to the data fitting of the high nucleic acid group or the low nucleic acid group to which the sample belongs, is obtained by averaging the measured values of the known samples; E is the amount of the nuclease added in step (a); k is a pending coefficient representing the degradation efficiency of unit nuclease on host nucleic acid.

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