A blood cell pathogenic microorganism de-host nucleic acid extraction kit and method

Abstract

The present application relates to the field of biotechnology, in particular to a blood cell pathogenic microorganism host nucleic acid extraction kit and method, comprising cell lysis solution, DNA enzyme, DNA enzyme buffer, proteinase K, lysozyme, cell wall lyase, glass beads and extraction lysis solution;The cell lysis solution is saponin and beta octyl thioglucoside solution;The DNA enzyme is Ultra Nuclease;The DNA enzyme buffer comprises MgCl2, Tris-HCl and BSA;Low concentration of saponin and low concentration of beta octyl thioglucoside are used, the detection of various pathogenic microorganisms is considered under the condition of ensuring good host removal effect. The chemical method and physical method are combined to break the wall of microbial cells, the positive detection rate of pathogenic microorganisms in clinic is improved, and the blank of pathogenic microorganism missed detection caused by the detection of plasma sample cfDNA at present is made up.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, particularly to the field of molecular detection in microbial molecular biology, and specifically to a kit and method for extracting host-deactivated nucleic acid from blood cell pathogens. Background Technology

[0002] Bloodstream infection (BSI) is an infection caused by various pathogenic microorganisms (including bacteria and fungi) invading the bloodstream, including bacteremia and sepsis. Bacteremia refers to the transient entry of bacteria into the bloodstream without obvious clinical symptoms of toxemia; sepsis, on the other hand, is a systemic inflammatory response syndrome caused by the co-entry of pathogenic microorganisms and toxins into the bloodstream. Despite significant advancements in medical science, bloodstream infection remains an increasingly serious problem in global public health. 40% of patients with sepsis or septic shock are caused by bloodstream infection. 20% of ICU infections are due to bloodstream infection. When infection control is delayed or antibiotic administration is not timely in these patients, treatment outcomes are usually poor.

[0003] Currently, blood culture is the gold standard for diagnosing bloodstream infections. However, blood culture relies on clinical prediction, has a long culture cycle, and a low positive rate. Metagenomic next-generation sequencing (mNGS) technology has attracted significant attention in the field of infectious diseases due to its advantages such as short cycle time, high sensitivity, and comprehensive detection range, and has been widely used in the detection of bloodstream infections. Currently, the industry primarily collects cell-free nucleic acid (cfDNA) from patient plasma for mNGS detection of bloodstream infections, improving the positive detection rate. However, pathogens exist both in plasma as cfDNA and as particles mixed within blood cells; detecting only plasma can lead to false negatives. Therefore, it is necessary to detect pathogen targets within blood cells.

[0004] Most clinical samples, such as bronchoalveolar lavage fluid, sputum, and blood cells, contain a large number of human cells. Since the human cell genome is 10-10,000 times larger than that of pathogenic microorganisms, over 90% of the sequencing data obtained from metagenomic sequencing are human sequences. This significantly impacts the sensitivity of metagenomic pathogen detection. Detecting low-abundance pathogen sequences in a high-human DNA background is extremely difficult, let alone detecting some difficult-to-break pathogens, such as Gram-positive bacteria, Cryptococcus, and Mycobacterium tuberculosis.

[0005] Currently, there are some patent reports on host removal methods for bronchoalveolar lavage fluid, sputum, and respiratory samples, but no research reports on host removal for blood cells. The publicly disclosed methods for eliminating host DNA mainly include the following, but all have some shortcomings: 1) A gentle digestion method using a combination of surfactants and DNase releases the host genome into solution, obtaining microorganisms with intact cell wall structures, thus achieving host-degraded microbial nucleic acid extraction. This method involves a long PBS washing time for DNase, is complex, and inevitably leaves residual DNase and results in the loss of pathogenic microorganisms; 2) Some reports indicate that several surfactants are used at very high concentrations to increase the efficiency of removing human-derived DNA. As a result, some cellularly vulnerable pathogens such as Gram-negative bacteria, viruses, mycoplasma, and chlamydia are lost; 3) Regarding the centrifugation required by the previous method, some reports have suggested changing the method for removing DNase residues by using EDTA to terminate the process instead of PBS washing, with all DNase used for subsequent extraction. However, residual EDTA can significantly affect the enzymes used in subsequent extractions, and this method has low removal efficiency; 4) Chemical methods based on osmotic lysis and propidium iodide treatment are used to remove host contamination. This method also requires a long time and a specific light source, and is not currently widely used; 5) For repetitive sequences widely present in the human genome, capture probes are designed in advance and then used for removal from the human genome. This method is very inefficient, and the capture probes need to be prepared in advance, making the process complex.

[0006] Therefore, there is an urgent need to provide a host-de-intervention extraction kit and method for blood cells that is compatible with various microbial extraction methods, and is simple and efficient for extracting difficult-to-break microorganisms. Summary of the Invention

[0007] The purpose of this invention is to address the current urgent need by providing a kit and method for extracting host-free nucleic acid from blood cell pathogens.

[0008] To achieve the above objectives, one aspect of the present invention is to provide a host-free nucleic acid extraction kit for blood cell pathogens, the kit comprising cell lysis buffer, DNase, DNase buffer, proteinase K, lysozyme, lysozyme, glass beads, and extraction lysis buffer;

[0009] The cell lysis buffer was a mixed solution of saponins and β-octyl thioglucosinolate.

[0010] The DNA enzyme is Ultra Nuclease;

[0011] The DNA enzyme buffer includes MgCl2, Tris-HCl, and BSA;

[0012] The extraction lysis buffer includes Tris-HCl, sodium chloride, guanidine hydrochloride, and SDS.

[0013] Furthermore, the mass percentage concentration of saponins in the cell lysate was 1.6%, and the mass percentage concentration of β-octyl glucosinolate was 0.4%.

[0014] Furthermore, the pH of the DNase buffer is 6-9, and the pH of the extraction lysis buffer is 6-7.

[0015] Current methods for removing host cells using saponins typically employ high working concentrations of 2.5% or higher. Such high concentrations significantly damage microbial cells, particularly Gram-negative bacteria, chlamydia, mycoplasma, and viruses. These pathogens have fragile cellular structures that are easily destroyed during host cell lysis, thus affecting detection. This invention innovatively utilizes low concentrations of saponins and β-octyl glucosinolates during host cell lysis. Extensive optimization testing has been conducted to adjust the ratio of these two components, ensuring effective removal of host cells without excessively high concentrations that could damage the fragile pathogens.

[0016] Another aspect of the present invention is to provide a method for extracting host nucleic acid from blood cell pathogenic microorganisms, the method comprising the following steps:

[0017] S1. Host lysis: Add cell lysis buffer to the sample tube, invert to mix at room temperature, centrifuge and remove the supernatant;

[0018] S2. Host nucleic acid removal: Add DNase buffer and DNase to the sample tube, mix and incubate; add proteinase K, mix thoroughly and incubate, centrifuge and discard the supernatant after the reaction is complete, no PBS washing is required;

[0019] S3. Microbial cell disruption: After adding lysozyme and cell wall lysis enzyme, the precipitate is resuspended and then transferred to a centrifuge tube containing glass beads. The mixture is then incubated and vortexed to mix.

[0020] S4. Microbial Nucleic Acid Extraction: After centrifugation, add extraction lysis buffer and proteinase K to the centrifuge tube, vortex to mix, and incubate. After high-speed centrifugation, transfer all supernatant to a new centrifuge tube, add anhydrous ethanol, mix by inverting, and transfer the entire solution to the adsorption column already loaded into the collection tube. Centrifuge to remove the liquid in the collection tube, and place the adsorption column back into the collection tube. Add washing buffer WS1 to the adsorption column, centrifuge to remove the liquid in the collection tube, and place the adsorption column back into the collection tube. Add washing buffer WS2 to the adsorption column, centrifuge to remove the liquid in the collection tube, and place the adsorption column back into the collection tube. Centrifuge to remove the liquid in the collection tube, and place the adsorption column completely dry at room temperature before placing it in a new centrifuge tube. Add nuclease-free water to the adsorption column, place at room temperature, centrifuge, and collect the DNA solution for storage at -20°C.

[0021] Furthermore, the sample type is blood cells.

[0022] Furthermore, the cell lysis buffer is a solution of saponin and β-octyl glucosinolate; the DNase is UltraNuclease; the DNase buffer includes MgCl2, Tris-HCl, and BSA; and the extraction lysis buffer includes Tris-HCl, sodium chloride, guanidine hydrochloride, and SDS.

[0023] Furthermore, the final concentration of the saponin is 0.8%, and the final concentration of β-octyl thioglucosinolate is 0.2%.

[0024] Furthermore, the final concentration of the Ultra Nuclease is 2-5 U / μL, and the final concentrations of each component in the DNA enzyme buffer are MgCl2 1-10 mM, Tris-HCl 10-50 mM, and BSA 0.1-0.5 mg / ml, respectively.

[0025] Furthermore, the final concentration of the lysozyme is 5-20 mg / ml, and the final concentration of the carapace lyase is 0.1-0.5 U / μL.

[0026] Furthermore, the final concentrations of each component in the extraction lysis buffer are Tris-HCl 40-100mM, sodium chloride 100-500mM, guanidine hydrochloride 2-4M, and SDS 0.5%-2% by mass.

[0027] In the host removal process of this invention, Ultra Nuclease is used. Ultra Nuclease is a pluripotent nuclease that can degrade not only DNA but also RNA. When this method is used to detect RNA pathogens, it can also improve the detection rate of RNA pathogens.

[0028] The cell walls of Gram-positive bacteria contain a high proportion of peptidoglycan, while the cell walls of fungi contain a high proportion of chitin. Lysozyme and lysosomal enzymes can specifically act on peptidoglycan and chitin. In the cell wall disruption of microorganisms in this invention, the combined application of lysozyme and lysosomal enzymes results in more complete lysis of pathogen cells.

[0029] Furthermore, this invention combines chemical methods (lysozyme and cell wall lysin) and physical methods (mechanical grinding with glass beads) to disrupt microbial cells, resulting in a more thorough dual disruption effect and a higher pathogen detection rate.

[0030] The technical effects and advantages of this invention are as follows:

[0031] 1. This invention is specifically developed for blood cell sample types, filling the gap caused by the current practice of only detecting cfDNA in plasma samples, which leads to missed detection of pathogenic microorganisms;

[0032] 2. This invention creatively uses low concentrations of saponins and low concentrations of β-octyl glucosinolates during the host lysis process, and after extensive optimization testing, the ratio of the two has been adjusted to ensure the removal of host cells without the concentration being too high and damaging the structurally fragile pathogen.

[0033] 3. In the host removal process of this invention, Ultra Nuclease is used, which is a pluripotent nuclease that can degrade not only DNA but also RNA. When this method is used to detect RNA pathogens, it can also improve the detection rate of RNA pathogens.

[0034] 4. This invention combines chemical methods (lysozyme and cell wall lysin) and physical methods (mechanical grinding with glass beads) to break down microbial cells, resulting in a more thorough double cell wall breaking effect and a higher pathogen detection rate. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the process for extracting host nucleic acid from pathogenic microorganisms according to the present invention. Detailed Implementation

[0036] To better understand the usage of this invention, specific implementation methods and embodiments will be shown below, thereby providing a clear and complete description of the technical solution. Obviously, the described implementation method is merely one embodiment of this invention, and not all of them.

[0037] Unless otherwise specified, the experimental methods used in the following implementation methods and examples are conventional methods. Unless otherwise specified, the experimental materials used in the following implementation methods and examples were all purchased from conventional biochemical reagent stores. Quantitative experiments were all performed in triplicate, and the results were averaged.

[0038] The specific implementation steps are as follows; the process can be referred to. Figure 1 :

[0039] 1. Nucleic acid extraction

[0040] S1. Host lysis: Take 600 μL of blood cell sample into a 1.5 ml sample tube, add 600 μL of cell lysis buffer, mix by inverting at room temperature for 10 min, centrifuge at 10000 rpm for 3 min and discard the supernatant.

[0041] S2. Host Nucleic Acid Removal: Add 197 μL of DNase buffer and 3 μL of DNase to the sample tube, mix thoroughly, and incubate at 37°C for 15 min. The final concentrations of each component are: Ultra Nuclease 2 U / μL, MgCl2 2 mM, Tris-HCl 20 mM, and BSA 0.1 mg / ml. Then add 20 μL of proteinase K, mix thoroughly, and incubate at 56°C for 10 min to achieve a final concentration of 2 mg / ml. After the reaction, centrifuge and discard the supernatant; no PBS washing is required.

[0042] S3. Microbial cell disruption: Add 10 μL of lysozyme and 10 μL of cell wall lysis enzyme to resuspend the precipitate. Transfer the resuspended solution to a centrifuge tube containing 0.2 g of glass beads. Incubate the centrifuge tube at 37 °C for 30 min. Vortex to mix for 15 min to achieve a final concentration of 5 mg / ml for lysozyme and a final concentration of 0.2 U / μL for cell wall lysis enzyme.

[0043] S4. Microbial nucleic acid extraction: After a short centrifugation of the centrifuge tube, add 200 μL of extraction lysis buffer and 20 μL of proteinase K, vortex to mix, and incubate at 56 °C for 10 min to achieve the following final concentrations: Tris-HCl 50 mM, sodium chloride 200 mM, guanidine hydrochloride 4 M, and SDS 0.5%. After high-speed centrifugation, transfer all supernatant to a new centrifuge tube, add 200 μL of anhydrous ethanol, mix by inverting, and then transfer the entire solution to the adsorption column already loaded into the collection tube. Centrifuge at 12000 rpm for 30 seconds to remove the liquid from the collection tube, and place the adsorption column back into the collection tube. Add 500 μL of washing buffer WS1 to the adsorption column, centrifuge at 12000 rpm for 30 seconds to remove the liquid from the collection tube, and place the adsorption column back into the collection tube. Add 500 μL of washing buffer WS2 to the adsorption column, centrifuge at 12000 rpm for 30 seconds to remove the liquid from the collection tube, and place the adsorption column back into the collection tube. Centrifuge at 12000 rpm for 2 minutes to remove the liquid from the collection tube, and allow the adsorption column to dry completely at room temperature before placing it into a new centrifuge tube. Add 25 μL of nuclease-free water to the adsorption column, incubate at room temperature for 5 minutes, centrifuge at 12000 rpm for 1 minute, and collect the DNA solution for storage at -20°C.

[0044] 2. Library Construction

[0045] The DNA library preparation kit used in this method is the One ShotMax DNALib Prep Kit for MGI (catalog number PDM601) from Nanjing Shijian Medical Laboratory Co., Ltd.

[0046] The specific process is as follows:

[0047] (1) Take out Frag Enzyme Mix and ERA Enzyme Mix, thaw them and gently tap them with your fingers to mix them. Do not vortex. Thaw and mix FEA Reaction Buffer. Briefly centrifuge all reagents to collect them to the bottom of the tube and place them on ice for later use.

[0048] (2) Set the PCR instrument according to Table 1, and set the hot cover temperature to 80 degrees; start the program, and pause when the temperature reaches 4 degrees.

[0049] Table 1 Fragmentation Procedure

[0050] Temperature (°C) time 4 1min 37 22min 72 30min 4 Hold

[0051] (3) Prepare the reaction system as shown in Table 2 in the centrifuge tube containing the sample DNA placed on ice:

[0052] Table 2 Fragmented reaction system

[0053] Components Volume (μl) DNA 28.8 FEA Reaction Buffer 10 ERA Enzyme Mix 1.2 Frag Enzyme Mix 10 Total 50

[0054] (4) Use a pipette to gently blow and mix 10-15 times. Note that this should be done on ice and do not vortex.

[0055] (5) After a brief centrifugation to the bottom of the tube, immediately transfer it to a pre-cooled PCR instrument and click Resume to run the program again;

[0056] (6) When the program reaches the final 4℃ Hold step, remove the tubes from the PCR instrument. Take out Ligation Buffer, DNA Adapter X, and DNA Ligase, and prepare the adapter ligation reaction system as shown in Table 3:

[0057] Table 3 Connection Reaction System

[0058]

[0059]

[0060] Gently pipette and mix 10-15 times, then briefly centrifuge to collect the mixture at the bottom of the tube.

[0061] Note: Please refer to Table 4 for the number of connectors used.

[0062] Table 4 Recommended Quantity of Connectors

[0063] Input DNA Pre-dilution factor 100ng-200ng Undiluted 25ng-100ng 1:2 5ng-25ng 1:10 100pg-5ng 1:30

[0064] (7) Place the instrument on the PCR machine and execute the procedure in Table 5:

[0065] Table 5 Connection Reaction Procedure

[0066] Temperature (°C) time 20 15min 4 Hold

[0067] (8) After the process is complete, add 64 μl of magnetic beads (make sure to equilibrate at room temperature for more than 30 min) and mix thoroughly.

[0068] (9) Let it sit at room temperature for 5 minutes. Note that you should not place it on the magnetic rack during this time.

[0069] (10) Place it on a magnetic rack and let it stand to clarify, then discard the supernatant;

[0070] (11) Add 200 μl of 80% ethanol, let stand for 30 seconds and then discard the supernatant;

[0071] (12) Add 200 μl of 80% ethanol, let stand for 30 seconds and then discard the supernatant;

[0072] (13) After rapid centrifugation, discard the residual ethanol with a 10 μl pipette, place at room temperature and air dry;

[0073] (14) Remove the tube from the magnetic rack, add 21.5 μl of nuclease-free water to resuspend the magnetic beads, mix thoroughly, place at room temperature for 2 min, centrifuge quickly for 5 sec, place on the magnetic rack for 2 min, take 20 μl of supernatant and transfer to a new PCR tube, do not touch the magnetic beads;

[0074] (15) Thaw the PCR Primer Mix for MGI and HiFi Amplification Mix, invert and mix well, briefly centrifuge to collect the mixture at the bottom of the tube, and prepare the reaction in Table 6 in a clean PCR tube:

[0075] Table 6 PCR amplification reaction system

[0076] Components Volume (μl) Previous product 20 PCR Primer Mix for MGI 5 HiFi Amplification Mix 25 Total 50

[0077] (16) Use a pipette to gently mix the liquid (do not shake to mix), and briefly centrifuge to collect the reaction solution to the bottom of the tube;

[0078] (17) Perform the PCR reaction shown in Table 7:

[0079] Table 7 PCR Amplification Reaction Procedure

[0080]

[0081] (18) After the process is complete, add 32.5 μl of magnetic beads to the 50 μl of PCR product and mix well.

[0082] (19) Let it sit at room temperature for 5 minutes. Note that you should not place it on the magnetic rack during this time.

[0083] (20) After placing the supernatant on a magnetic rack and allowing it to stand and clarify, transfer the supernatant to a clean PCR tube and discard the magnetic beads.

[0084] (21) Add 25 μl of magnetic beads to the supernatant above and mix well;

[0085] (22) Let it sit at room temperature for 5 minutes. Note that you should not place it on the magnetic rack during this time.

[0086] (23) Place it on a magnetic rack and let it stand to clarify, then discard the supernatant;

[0087] (24) Add 200 μl of 80% ethanol, let stand for 30 seconds and then discard the supernatant;

[0088] (25) Add 200 μl of 80% ethanol, let stand for 30 seconds, and then discard the supernatant;

[0089] (26) After rapid centrifugation, discard the residual ethanol with a 10 μl pipette, place at room temperature and air dry;

[0090] (27) Remove the tube from the magnetic rack, add 20 μl of nuclease-free water to resuspend the magnetic beads, mix thoroughly, let stand at room temperature for 2 min, centrifuge quickly for 5 sec, place on the magnetic rack for 2 min, take the supernatant and transfer it to a new PCR tube, do not touch the magnetic beads.

[0091] 3. DNB Preparation

[0092] 3.1 Reagent Preparation

[0093] Take out the library, TE buffer, DNB preparation buffer, DNB polymerase I mixture and DNB termination buffer, place them on an ice box for about 0.5 hours to thaw, then vortex for 5 seconds, briefly centrifuge and place on an ice box for later use.

[0094] 3.2 Preparation process

[0095] (1) Pool sample: Prepare on ice using 0.2 mL eight-tube or PCR tubes; the total library intake is 120 fmol, and the system is prepared according to Table 8:

[0096] Table 8 Library pooling reaction system

[0097] Components Added volume (μl) dsDNA library X TE buffer 20-X DNB preparation buffer 20 Total volume 40

[0098] (2) Mix the reaction mixture using a vortex mixer, centrifuge for 5 seconds using a mini centrifuge, and then place it in a PCR instrument for reaction. The reaction conditions are shown in Table 9.

[0099] Table 9. Reaction Procedures After Library Pooling

[0100] Temperature ℃ Time (min) Heat-sealed lid (105℃) On 95 3 40 3 4 Hold

[0101] (3) Take out the DNB polymerase II mixture and place it on an ice box. Centrifuge briefly for 5 seconds and place it on an ice box for later use.

[0102] (4) Once the PCR instrument reaches 4°C, remove the PCR tubes, centrifuge for 5 seconds using a mini centrifuge, and then add the components listed in Table 10 on ice:

[0103] Table 10 DNA Polymerase Reaction System

[0104] Components Added volume (μl) DNB polymerase I mixture 40 DNB polymerase II mixture 4

[0105] (5) Gently mix the reaction mixture with a pipette, centrifuge for 5 seconds in a mini centrifuge, and immediately place it in a PCR instrument. The reaction conditions are shown in Table 11.

[0106] Table 11 DNB Reaction Procedure

[0107] Temperature ℃ Time (min) Heat-sealed lid (40℃) On 30 25 4 Hold

[0108] (6) Once the temperature of the PCR instrument reaches 4℃, immediately add 20μL of DNB termination buffer and slowly mix by pipetting with a wide-mouth pipette tip 5-8 times. Do not shake or vigorously pipet.

[0109] 4. DNB Concentration Measurement

[0110] After DNB preparation is completed, use The concentration was measured using the ssDNA Assay Kit. A concentration of 8 ng / μL or higher was considered acceptable; samples with unacceptable concentrations needed to be prepared again.

[0111] 5. Sequencing

[0112] 6. Bioinformatics analysis of sequencing data

[0113] Example 1

[0114] Tests for different mass percentage concentrations of saponins or β-octyl glucosinolates in cell lysates

[0115] Eight parallel tubes of 600 μl blood cell samples were collected and numbered according to Table 12. The samples were compared using different concentrations of saponins and β-octyl glucosinolates extracted from the samples. The sample analysis process—nucleic acid extraction, library construction, sequencing, and data analysis—was performed according to the prescribed method. The different concentrations are shown in Table 12.

[0116] Table 12 Cell lysate numbers and formulations for individual saponins / β-octyl thioglucosinolates

[0117]

[0118]

[0119] The experimental results are shown in Table 13. As the concentration of both lysis buffers increased, the host cell lysis effect strengthened; however, above a certain concentration, the host cell removal effect weakened. The results indicate that 1.5% saponin showed the best host cell removal effect. β-octyl glucosinolate also had some removal effect, but it was not as significant as that of saponin.

[0120] Table 13 Comparison of host lysis effects of individual saponin / β-octyl thioglucosinolate lysis buffers

[0121] serial number Total number of reads Host read count (percentage) 1 12809321 12320005(96.18％) 2 22208874 19514938(87.87％) 3 21985476 8077464(36.74％) 4 25339591 11078469(43.72％) 5 27099319 25587177(94.42％) 6 24440377 22294512(91.22％) 7 13931522 8948217(64.23％) 8 40943086 33647028(82.18％)

[0122] Example 2

[0123] Tests of saponins and β-octyl glucosinolates at different mass percentage concentrations in cell lysates

[0124] Ten parallel tubes of 600 μl blood cell samples were taken and numbered according to Table 14. The results of extracting different concentrations of saponins and β-octyl glucosinolates were compared with those of 1.5% saponins. The sample underwent nucleic acid extraction, library construction, sequencing, and data analysis according to the prescribed method. The different concentration ratios are shown in Table 14.

[0125] Table 14 Cell lysis buffer numbers and formulations with different saponin / β-octyl thioglucosinolate ratios

[0126] serial number Cell lysis buffer final concentration formulation 1 1.5% saponins 2 0.8% saponins + 0.2% β-octyl glucosinolates 3 0.8% saponins + 0.3% β-octyl glucosinolates 4 0.8% saponins + 0.4% β-octyl glucosinolates 5 1.0% saponin + 0.2% β-octyl glucosinolate 6 1.0% saponin + 0.3% β-octyl thioglucosinolate 7 1.0% saponin + 0.4% β-octyl thioglucosinolate 8 1.5% saponins + 0.2% β-octyl thioglucosinolates 9 1.5% saponins + 0.3% β-octyl thioglucosinolates 10 1.6% saponins + 0.4% β-octyl thioglucosinolates

[0127] The experimental results are shown in Table 15. High concentrations of lysis buffer have a stronger lysis effect but cause greater damage to structurally fragile microorganisms. The best combination is 0.8% saponin + 0.2% β-octyl glucosinolate, which can detect a variety of microorganisms.

[0128] Table 15 Comparison of host lysis effects of lysis buffers with different saponin / β-octyl thioglucosinolate ratios

[0129]

[0130] Example 3

[0131] Five clinical blood cell samples were randomly selected, and metagenomic extraction, library construction, and next-generation sequencing were performed according to the method of this invention. The next-generation sequencing results of samples without host removal were used as controls. The results are shown in Table 16 below.

[0132] Table 16 Comparison of second-generation metagenomic sequencing results using host removal and non-host removal methods.

[0133]

[0134] Results show that the host removal process of this invention can effectively enrich various microbial cells in the sample, such as circovirus, human herpesvirus type 1, human herpesvirus type 5, Acinetobacter juncus, Escherichia coli, Mycoplasma pneumoniae, and Aspergillus niger, compared with the non-host removal process. It has almost no damage to structurally fragile pathogens and the overall microbial abundance is significantly improved.

[0135] Conclusion: This invention relates to a host removal extraction kit and method for blood cell samples. It utilizes low concentrations of saponins and β-octyl glucosinolates, ensuring effective host removal while also detecting various pathogens. The combined use of chemical methods (lysozyme and cell wall lysin) and physical methods (mechanical grinding with glass beads) to disrupt microbial cells improves the positive detection rate of pathogens in clinical practice and fills the gap left by current methods that only test plasma sample cfDNA, which leads to missed detections of pathogens.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall fall within the scope of the present invention.

Claims

1. A reagent kit for extracting host-free nucleic acid from blood cell pathogens, characterized in that, The kit includes cell lysis buffer, DNase, DNase buffer, proteinase K, lysozyme, lysozyme, glass beads, and extraction lysis buffer; The cell lysis buffer was a mixed solution of saponins and β-octyl thioglucosinolate. The DNA enzyme is Ultra Nuclease; The DNA enzyme buffer includes MgCl2, Tris-HCl, and BSA; The extraction lysis buffer includes Tris-HCl, sodium chloride, guanidine hydrochloride, and SDS; The final concentration of the saponin is 0.8%, and the final concentration of β-octyl glucosinolate is 0.2%. The final concentration of the Ultra Nuclease is 2-5 U / μL, and the final concentrations of each component in the DNA enzyme buffer are MgCl2 1-10 mM, Tris-HCl 10-50 mM, and BSA 0.1-0.5 mg / ml, respectively. The final concentration of the lysozyme is 5-20 mg / ml, and the final concentration of the carapace lyase is 0.1-0.5 U / μL; The final concentrations of each component in the extraction lysis buffer are Tris-HCl 40-100 mM, sodium chloride 100-500 mM, guanidine hydrochloride 2-4 M, and SDS 0.5%-2% by mass.

2. The host-free nucleic acid extraction kit for blood cell pathogens according to claim 1, characterized in that, The pH of the DNase buffer is 6-9, and the pH of the extraction lysis buffer is 6-7.

3. A method for extracting host-free nucleic acid from hematologic pathogens, characterized in that, The method includes the following steps: S1. Host lysis: Add cell lysis buffer to the sample tube, invert to mix at room temperature, centrifuge and remove the supernatant; S2. Host nucleic acid removal: Add DNase buffer and DNase to the sample tube, mix and incubate; add proteinase K, mix thoroughly and incubate, centrifuge and discard the supernatant after the reaction is complete, no PBS washing is required; S3. Microbial cell disruption: After adding lysozyme and cell wall lysis enzyme, the precipitate is resuspended and then transferred to a centrifuge tube containing glass beads. The mixture is then incubated and vortexed to mix. S4. Microbial Nucleic Acid Extraction: After centrifugation, add extraction lysis buffer and proteinase K to the centrifuge tube, vortex to mix, and incubate. After high-speed centrifugation, transfer all supernatant to a new centrifuge tube, add anhydrous ethanol, mix by inverting, and transfer the entire solution to the adsorption column already loaded into the collection tube. Centrifuge to remove the liquid in the collection tube, and place the adsorption column back into the collection tube. Add washing buffer WS1 to the adsorption column, centrifuge to remove the liquid in the collection tube, and place the adsorption column back into the collection tube. Add washing buffer WS2 to the adsorption column, centrifuge to remove the liquid in the collection tube, and place the adsorption column back into the collection tube. Centrifuge to remove the liquid in the collection tube, and place the adsorption column completely dry at room temperature before placing it in a new centrifuge tube. Add nuclease-free water to the adsorption column, place at room temperature, centrifuge, and collect the DNA solution for storage at -20°C. The cell lysis buffer is a solution of saponins and β-octyl glucosinolates; the DNase is Ultra Nuclease; the DNase buffer includes MgCl2, Tris-HCl, and BSA; the extraction lysis buffer includes Tris-HCl, sodium chloride, guanidine hydrochloride, and SDS. The final concentration of the saponin is 0.8%, and the final concentration of β-octyl glucosinolate is 0.2%. The final concentration of the Ultra Nuclease is 2-5 U / μL, and the final concentrations of each component in the DNA enzyme buffer are MgCl2 1-10 mM, Tris-HCl 10-50 mM, and BSA 0.1-0.5 mg / ml, respectively. The final concentration of the lysozyme is 5-20 mg / ml, and the final concentration of the carapace lyase is 0.1-0.5 U / μL; The final concentrations of each component in the extraction lysis buffer are Tris-HCl 40-100 mM, sodium chloride 100-500 mM, guanidine hydrochloride 2-4 M, and SDS 0.5%-2% by mass.

4. The method for extracting host-free nucleic acid from hematologic pathogens according to claim 3, characterized in that, The sample type is blood cells.

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