Reagent combination and kit for removing host nucleic acid and method for removing host nucleic acid

Abstract

The invention provides a reagent combination and kit for removing host nucleic acid and a method for removing the host nucleic acid. The reagent combination for removing the host nucleic acid comprises a lysis solution, a nuclease solution and a stop solution, the lysate contains saponin, NaCl and MgCl2, the concentration of the saponin is 0.001%-5% by volume, the concentration of the NaCl is 0.005 M to 5 M, and the concentration of the MgCl2 is 0.1 mM to 100 mM. The reagent composition for removing the host nucleic acid can effectively remove the host nucleic acid and avoid damage to microbial cells, and the method is simple and convenient to operate, short in time consumption, high in efficiency, high in repeatability and suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biology. Specifically, this invention provides reagent combinations and kits for removing host nucleic acids, as well as methods for removing host nucleic acids. Background Technology

[0002] Metagenomics involves extracting DNA from all microorganisms in environmental samples to construct metagenomic libraries, and then using genomic research strategies to study the genetic composition and community function of all microorganisms contained in the environmental samples. With the rapid development of gene sequencing technology, metagenomic next-generation sequencing (mNGS) is widely used in clinical practice, enabling rapid and accurate detection of pathogenic microorganisms in clinical samples. However, clinical mNGS technology has certain limitations. Because most clinical samples contain a large amount of host cells and free host nucleic acids, with some samples containing over 90% host nucleic acid, the mNGS sequencing data is mostly composed of host nucleic acid sequences, wasting sequencing costs and interfering with the detection of pathogenic microorganisms.

[0003] To address this issue, specific methods for removing host nucleic acids from samples can effectively reduce the proportion of host nucleic acids and increase the proportion of microorganisms, thereby reducing sequencing costs, increasing microbial detection rates, and facilitating the rapid and accurate detection of pathogenic microorganisms. Therefore, host nucleic acid removal is essential and has significant economic and social value.

[0004] However, existing reagents for removing host nucleic acids have drawbacks such as complex operation, long time consumption, inconsistent performance, high price, and limited selectivity. There is an urgent need to develop products and methods with stable performance and high host removal rate. Summary of the Invention

[0005] This invention aims to at least partially address the technical problems existing in the prior art. To this end, this invention proposes reagent combinations, kits, and their applications for removing host nucleic acids, methods for removing host nucleic acids, and methods for detecting microbial nucleic acids. The reagent combinations for removing host nucleic acids using this invention can effectively remove host nucleic acids while avoiding damage to microbial cells. The methods are simple to operate, quick, efficient, and highly reproducible, making them suitable for widespread application.

[0006] In one aspect of the invention, a reagent combination for removing host nucleic acids is provided. According to an embodiment of the invention, the reagent combination for removing host nucleic acids comprises: a lysis buffer, a nuclease solution, and a stop solution; the lysis buffer contains saponin, NaCl, and MgCl2, wherein the concentration of the saponin is 0.001–5% by volume, the concentration of the NaCl is 0.005–5 M, and the concentration of the MgCl2 is 0.1–100 mM.

[0007] Based on the structural differences between animal and microbial cells, the inventors of this invention employ a mild lysis buffer to selectively lyse host cells. The released host nucleic acids are degraded by nucleases, while the microbial cells remain largely unaffected. The sample after removing the host nucleic acids can be further lysed using a microbial nucleic acid extraction reagent to obtain high-purity DNA or RNA.

[0008] Specifically, in the reagent combination for removing host nucleic acids of this invention, saponins mainly play a lysis role. The inventors have discovered that using 0.001–5% by volume of saponins can gently lyse host cells while avoiding damage to microbial cells. Adding 0.1–100 M MgCl₂ and 0.005–5 M NaCl can improve the lysis / enzymatic digestion effect. Therefore, the reagent combination for removing host nucleic acids of this invention can effectively remove host nucleic acids while avoiding damage to microbial cells. The method is simple to operate, quick, efficient, and highly reproducible, making it suitable for widespread application.

[0009] According to embodiments of the present invention, the above-described reagent combination for removing host nucleic acid may further have the following additional technical features:

[0010] According to embodiments of the present invention, the concentration of the saponin is 0.01 to 2 vol%, for example, 0.01 vol%, 0.012 vol%, 0.037 vol%, 0.05 vol%, 0.1 vol%, 0.111 vol%, 0.333 vol%, 0.5 vol%, 1 vol%, 1.5 vol%, 2 vol%, or a range between any two of these values ​​as endpoints. In some embodiments, the concentration of the saponin is 0.012 to 1 vol%.

[0011] In this document, the term "saponin" refers to a class of glycosides whose aglycone is a triterpenoid or spirostane compound. In some embodiments, saponins may specifically be saponins (CAS No.: 8047-15-2) or digitalis saponins (CAS No.: 11024-24-1). This is to better lyse the host nucleic acid and avoid damaging the microbial cell.

[0012] According to embodiments of the present invention, the concentration of MgCl2 is 1–50 mM, for example, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or any two of these values ​​as a range between endpoints. In some embodiments, the concentration of MgCl2 is 2–40 mM.

[0013] According to embodiments of the present invention, the concentration of NaCl is 0.03–3 M. In some embodiments, the concentration of NaCl is 0.03 M, 0.035 M, 0.07 M, 0.1 M, 0.14 M, 0.5 M, 0.56 M, 1 M, 2 M, 3 M, or a range between any two of these values ​​as endpoints. In some embodiments, the concentration of NaCl is 0.035–2 M.

[0014] According to embodiments of the present invention, the nuclease solution contains 2–20 KU / mL of a totipotent nuclease, binuclease. In some embodiments, the concentration of the totipotent nuclease, binuclease, is 4 KU / mL, 5 KU / mL, 8 KU / mL, 10 KU / mL, 12 KU / mL, 15 KU / mL, 18 KU / mL, 20 KU / mL, or a range between any two of these values ​​as endpoints. In some embodiments, the concentration of the totipotent nuclease, binuclease, is 5–15 KU / mL. This facilitates the efficient degradation of released host nucleic acids.

[0015] According to embodiments of the present invention, the terminating solution contains 1–40 mg / mL proteinase K. In some embodiments, the concentration of proteinase K is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or a range between any two of these values ​​as endpoints. In some embodiments, the concentration of proteinase K is 10–30 mg / mL. This is to better terminate host cell lysis and host nucleic acid degradation.

[0016] According to embodiments of the present invention, the pH value of the lysis buffer is 6 to 10. In some embodiments, the pH value of the lysis buffer is 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any two of these values ​​as a range between endpoints. In some embodiments, the pH value of the lysis buffer is 7 to 8. This allows for better lysis of host cells.

[0017] According to embodiments of the present invention, the lysis buffer further contains Tris, KCl, Na2HPO4, and KH2PO4. Adding these substances to the lysis buffer provides a suitable pH environment for lysis and enzymatic digestion, thereby improving the lysis / enzymatic digestion efficiency.

[0018] According to embodiments of the present invention, the concentration of Tris is 10–100 mM. In some embodiments, the concentration of Tris is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of these values ​​as endpoints. In some embodiments, the concentration of Tris is 10–30 mM.

[0019] According to embodiments of the present invention, the concentration of KCl is 0–5.34 mM. In some embodiments, the concentration of KCl is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or any two of these values ​​as a range between endpoints. In some embodiments, the concentration of KCl is 2–4 mM.

[0020] According to embodiments of the present invention, the concentration of Na2HPO4 is 0–16.2 ​​mM. In some embodiments, the concentration of Na2HPO4 is 1 mM, 3 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 16 mM, or any two of these values ​​as a range between endpoints. In some embodiments, the concentration of Na2HPO4 is 7–9 mM.

[0021] According to embodiments of the present invention, the concentration of KH2PO4 is 0–3.52 mM. In some embodiments, the concentration of KH2PO4 is 1 mM, 2 mM, 3 mM, or any two of these values ​​as a range between endpoints. In some embodiments, the concentration of KH2PO4 is 1–3 mM.

[0022] The inventors obtained the above-mentioned optimal concentrations of Tris, NaCl, KCl, Na2HPO4, and KH2PO4 through numerous experiments, which can further improve the lysis / enzymatic digestion effect.

[0023] According to an embodiment of the present invention, the reagent combination for removing host nucleic acid consists of a lysis buffer, a nuclease solution, and a stop solution. The lysis buffer contains 0.012–1 v / v saponin, 10–30 mM Tris, 2–40 mM MgCl2, 0.035–2 M NaCl, 2–4 mM KCl, 7–9 mM Na2HPO4, and 1–3 mM KH2PO4, with a pH of 7–8. The nuclease solution contains 5–15 KU / mL Binuclease. The stop solution contains 10–30 mg / mL Proteinase K. In some embodiments, the lysis buffer consists of 0.012–1 v / v saponin, 10–30 mM Tris, 2–40 mM MgCl2, 0.035–2 M NaCl, 2–4 mM KCl, 7–9 mM Na2HPO4, and 1–3 mM KH2PO4, with a pH of 7–8; the nuclease solution is 5–15 KU / mL Binuclease; and the stop solution is 10–30 mg / mL Proteinase K. The reagent combination for removing host nucleic acid of the present invention can effectively remove host nucleic acid while avoiding damage to microbial cells. Furthermore, the reagent combination uses only three components—lysis buffer, nuclease solution, and stop solution—greatly simplifying the types of reagents and operation methods. It is time-efficient, highly reproducible, and suitable for widespread application.

[0024] In another aspect, the present invention provides a kit for removing host nucleic acids. According to an embodiment of the present invention, the kit for removing host nucleic acids comprises the reagent combination for removing host nucleic acids described above. As previously stated, the reagent combination for removing host nucleic acids of the present invention can effectively remove host nucleic acids while avoiding damage to microbial cells. The method is simple to operate, quick, efficient, and highly reproducible, making it suitable for widespread application.

[0025] In another aspect, the present invention proposes the application of the reagent combination or kit described above for removing host nucleic acid in the detection of host nucleic acid and / or microbial nucleic acid. As mentioned above, the reagent combination for removing host nucleic acid of the present invention can effectively remove host nucleic acid while avoiding damage to microbial cells. The method is simple to operate, quick, efficient, and highly reproducible, making it suitable for widespread application.

[0026] In another aspect, the present invention provides a method for removing host nucleic acid. According to an embodiment of the present invention, the method includes: (1) centrifuging a sample containing host nucleic acid, removing the supernatant, and collecting the precipitate; (2) mixing and incubating the precipitate, lysis buffer, and nuclease solution to obtain a reaction solution; (3) mixing and incubating the reaction solution with a stop solution; wherein the lysis buffer, nuclease solution, and stop solution are as defined in the reagent combination for removing host nucleic acid as described above or in the kit for removing host nucleic acid as described above. Thus, the method of the present invention can effectively remove host nucleic acid without damaging microbial cells. The method is simple to operate, short in time, highly efficient, and highly reproducible. Furthermore, by configuring only three reaction reagents—lysis buffer, nuclease solution, and stop solution—multiple additions of various reaction reagents are avoided during use, reducing the number of additions, simplifying the operation, and avoiding errors in addition that could affect the results, thus demonstrating high application value.

[0027] According to an embodiment of the present invention, based on 0.2–2 mL of the sample, the amount of lysis buffer added is 150–250 μL, the amount of nuclease solution added is 1–5 μL, and the amount of stop solution added is 10–30 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, in step (2), the incubation temperature is 30–40°C and the time is 5–15 minutes. This is to ensure thorough lysis of the host cell, releasing the host nucleic acid therein, and then lysing the host nucleic acid under the action of nucleases to achieve the purpose of removing the host nucleic acid.

[0029] According to an embodiment of the present invention, in step (3), the incubation temperature is 50–60°C and the time is 5–15 minutes. This is to ensure sufficient lysis of the host nucleic acid and to avoid damaging the microbial cells.

[0030] In another aspect, the present invention provides a method for detecting microbial nucleic acids. According to an embodiment of the present invention, the method includes: treating the sample to be tested using the aforementioned method for removing host nucleic acids, so as to remove host nucleic acids from the sample. Therefore, the method of the present invention can avoid interference from host nucleic acids, and the treated sample can be used for DNA or RNA extraction and purification, achieving the purpose of accurate detection of microbial nucleic acids.

[0031] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0033] Figure 1 The diagram showing the proportion of microbial and human sequences according to Embodiment 2 of the present invention (swab) is displayed;

[0034] Figure 2 The diagram shows a comparison of the abundance of the top ten microorganisms according to Example 2 of the present invention (swabs);

[0035] Figure 3 The diagram shows the proportion of microbial and human sequences (saliva) according to Example 3 of the present invention;

[0036] Figure 4 A comparison chart of the abundance of the top ten microorganisms according to Example 3 of the present invention (saliva) is shown;

[0037] Figure 5 An electrophoresis diagram according to Embodiment 5 of the present invention is shown. Detailed Implementation

[0038] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0039] Example 1: Reagent preparation and efficacy testing for removing host nucleic acid

[0040] 1. Reagent Preparation

[0041] (1) Prepare lysis buffers containing different concentrations of saponins (CAS No.: 8047-15-2), namely 1%, 0.333%, 0.111%, 0.037%, and 0.012%. Other components in the lysis buffers are 20 mM Tris, 2 mM MgCl2, 0.5 M NaCl, 3 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, with a pH of 7.5.

[0042] (2) Prepare lysis buffers with different NaCl concentrations: 0.035M, 0.07M, 0.14M, 0.56M, 1M, and 2M. Other components in the lysis buffers are 0.1% saponin, 20mM Tris, 2mM MgCl2, 3mM KCl, 8mM Na2HPO4, and 2mM KH2PO4, with a pH of 7.5.

[0043] (3) Prepare lysis buffers with different MgCl2 concentrations: 2, 5, 10, 20, and 40 mM. Other components in the lysis buffers are 1% saponin, 20 mM Tris, 0.5 M NaCl, 3 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, with a pH of 7.5.

[0044] (4) Nuclease solution: 10KU / mL Binuclease.

[0045] (5) Termination solution: 20 mg / mL proteinase K.

[0046] 2. Sample Preparation

[0047] Collect oral swab samples: The person being sampled should rinse their mouth briefly with water to remove any remaining food debris. Then, ask them to open their mouth naturally. Using a disposable medical swab, rub the buccal mucosa (both cheeks inside the mouth) up and down with the force of brushing your teeth, while rotating the swab to ensure the swab tip makes full contact with the oral mucosa. After collection, place the swab into a 50mL centrifuge tube. Add 2N mL of PBS according to the number of swabs (N swabs), for example, 2mL of PBS for 1 swab. Vortex the 50mL centrifuge tube for 30 seconds, then transfer the liquid to a new 50mL centrifuge tube as the sample to be tested. Vortex the sample thoroughly before use.

[0048] Note: Swab samples were obtained from voluntary donors.

[0049] 3. Removal of host nucleic acid

[0050] (1) Take 1 mL of sample into a 2 mL centrifuge tube. Place the centrifuge tube in a centrifuge, set the centrifugal force to 10000×g, and continue for 3 minutes to remove the supernatant.

[0051] (2) Add 197 μL of lysis buffer and 3 μL of Binuclease to the centrifuge tube, vortex to mix, and place the centrifuge tube in a constant temperature mixer and incubate at 37°C for 10 minutes.

[0052] (3) Remove the centrifuge tube, add 20 μL of stop solution to the centrifuge tube, and vortex to mix. Place the centrifuge tube in a constant temperature mixer and incubate at 56°C for 10 minutes.

[0053] 4. Nucleic acid extraction

[0054] Nucleic acid extraction was performed using the MGIEAsy microbial DNA extraction kit according to the manufacturer's instructions. The extracted products were stored at 4°C or frozen at -20°C.

[0055] 5QPCR

[0056] According to 2× Configure the reaction buffer according to the Master qPCR Mix (SYBR Green I with UDG) (Qingke, TSE203) instruction manual and perform qPCR.

[0057] The experiment included a control group (no host removal, nucleic acid extraction directly) and a host-removed group (nucleic acid extraction after host removal). The qPCR results are shown in Table 1. Microbial loss rate was represented by the bacterial-specific gene 16S△Ct (experimental group Ct - control group Ct), with a smaller value indicating less loss. Host removal efficiency was represented by the host-specific gene 18S△Ct (experimental group Ct - control group Ct), with a larger value indicating better host removal.

[0058] Lysis buffers containing 0.012%–1% saponins can effectively remove host nucleic acids (18S△Ct≥4.15) while avoiding microbial loss (16S△Ct≤1), and the host removal effect is enhanced with increasing saponin concentration.

[0059] Lysis buffers containing 0.035–2 M NaCl can effectively remove host nucleic acids (18S△Ct≥5.06) while avoiding microbial loss (16S△Ct≤1), and the host removal effect is enhanced with increasing NaCl concentration.

[0060] Lysis buffers containing 2–40 mM MgCl2 can effectively remove host nucleic acids (18S△Ct≥7.41) while avoiding microbial loss (16S△Ct≤1), thus enhancing the host removal effect.

[0061] Table 1. qPCR results

[0062]

[0063]

[0064]

[0065]

[0066] Example 2: Removal of host DNA from swab samples

[0067] 1. Reagent preparation

[0068] The lysis buffer consisted of 1% saponin, 20 mM Tris, 2 mM MgCl2, 0.5 M NaCl, 3 mM KCl, 8 mM Na2HPO4 and 2 mM KH2PO4, with a pH of 7.5.

[0069] Nuclease solution: 10 KU / mL Binuclease.

[0070] Stop solution: 20 mg / mL proteinase K.

[0071] 2. Sample Preparation

[0072] Collect oral swab samples, following the same procedure as step 2 in Example 1.

[0073] 3. Removal of host nucleic acid

[0074] Same as step 3 in Example 1.

[0075] 4. Nucleic acid extraction

[0076] Nucleic acid extraction was performed using the MGIEAsy microbial DNA extraction kit according to the manufacturer's instructions. The extracted products were stored at 4°C or frozen at -20°C.

[0077] 5QPCR, library construction and sequencing:

[0078] According to 2× The reaction buffer was prepared according to the instructions of Master qPCR Mix (SYBR Green I with UDG) (Qingke, TSE203) for qPCR. Libraries were prepared and sequenced using the MGI Easiy DNA Digestion Library Preparation Kit (MGI, 1000005256) and the MGISEQ-2000RS High-Throughput Sequencing Kit (PE100) (MGI, 1000012536).

[0079] The experiment included a control group (no host removal) and the host removal method of this invention (MGI host removal for short).

[0080] The qPCR and sequencing results are shown in Table 2. Figure 1 and Figure 2 As shown, MGI de-hosting resulted in minimal loss of microorganisms and significant removal of host DNA. Even with only 54% of the total sequencing reads of the control group, the number of detected microbial species was still 40 higher than the control group without de-hosting, indicating that MGI de-hosting treatment significantly improves the microbial detection rate. The host DNA removal rate in the MGI de-hosting group reached as high as 99.2%. In summary, MGI reagent demonstrates excellent host DNA removal performance.

[0081] Table 2. qPCR and sequencing results (swabs)

[0082]

[0083] Note: 16S is a bacterial-specific gene, and 18S is a human-specific gene.

[0084] Example 3: Removal of host DNA from saliva samples

[0085] 1. Reagent preparation

[0086] Same as Example 2.

[0087] 2. Sample Preparation

[0088] Collect saliva according to the instructions of the saliva DNA sample collection kit (MGI, 1000025954). Do not mix the collected saliva with the reagents in the kit. Add 4 times the volume of PBS to the saliva and mix well. This mixture is the sample to be tested.

[0089] Note: Saliva samples were obtained from voluntary donors.

[0090] 3. Removal of host nucleic acid

[0091] Same as step 3 in Example 1.

[0092] 4. Nucleic acid extraction

[0093] Nucleic acid extraction was performed using the MGIEAsy microbial DNA extraction kit according to the manufacturer's instructions. The extracted products were stored at 4°C or frozen at -20°C.

[0094] 5QPCR, library construction and sequencing:

[0095] Same as step 5 in Example 2.

[0096] The experiment included a control group (without host removal) and MGI (host removal).

[0097] The qPCR results are shown in Table 3. Figure 3 and Figure 4 As shown, the MGI host-de-host group had a bacterial-specific gene 16ΔCt = -1.55, indicating that removing host gene interference improved bacterial detection sensitivity. Sequencing results showed that the MGI host-de-host group detected 143 species, higher than the 45 species detected in the non-host-de-host control group, indicating a significant increase in species detection. Both qPCR and sequencing results showed that the MGI host removal rate was high, reaching 98.61%.

[0098] Table 3. qPCR and sequencing results (saliva)

[0099]

[0100] Note: 16S is a bacterial-specific gene, and 18S is a human-specific gene.

[0101] Example 4: Removal of host DNA from blood samples

[0102] 1. Reagent preparation

[0103] Same as step 1 in Example 2.

[0104] 2. Sample Preparation

[0105] The blood sample used was a simulated blood sample, specifically 0.2 mL of EDTA-anticoagulated sheep whole blood (Jiangsu Kejing), mixed with 5 × 10⁻⁶ ppm. 6 CFU Escherichia coli (E. coli).

[0106] 3. Red blood cell lysis

[0107] 3.1 Take 0.2 mL of whole blood into a 2 mL centrifuge tube, add 0.8 mL of sterile water, and vortex to mix.

[0108] 3.2 Let stand at room temperature for 5 to 10 minutes until the liquid becomes clear.

[0109] 3.3 Place the centrifuge tubes in the centrifuge, set the centrifugal force to 10000×g, and continue for 3 minutes. Discard the supernatant.

[0110] 3.4 Add 0.8 mL of sterile water to the centrifuge tube and vortex to mix. Place the centrifuge tube in a centrifuge, set the centrifugal force to 10000×g, and centrifuge for 3 minutes. Discard the supernatant.

[0111] 4. Host removal processing

[0112] (1) Add 197 μL of lysis buffer and 3 μL of pluripotent nuclease to the centrifuge tube after the supernatant was removed in step 3.4, vortex to mix, and place the centrifuge tube in a constant temperature mixer and incubate at 37°C for 10 minutes.

[0113] (2) Remove the centrifuge tube, add 20 μL of stop solution to the centrifuge tube, and vortex to mix. Place the centrifuge tube in a constant temperature mixer and incubate at 56°C for 10 minutes.

[0114] 5. Nucleic acid extraction

[0115] Nucleic acid extraction was performed using the MGIEAsy microbial DNA extraction kit according to the manufacturer's instructions. The extracted products were stored at 4°C or frozen at -20°C.

[0116] 6QPCR testing

[0117] According to 2× Configure the reaction buffer according to the Master qPCR Mix (SYBR Green I with UDG) (Qingke TSE203) instruction manual and perform qPCR.

[0118] The qPCR results are shown in Table 4. The host-specific gene (E. coli) in the MGI removal group had a ΔCt of -0.06, indicating that there was almost no significant loss of microorganisms; the sheep-specific gene (18S) had a ΔCt of 6.23, indicating a significant removal effect of host DNA, and the estimated host removal efficiency was as high as 98%.

[0119] Table 4. qPCR Results (Blood)

[0120]

[0121] Example 5: Removal of host DNA from a simulated human sample (obtaining microbial RNA)

[0122] 1. Reagent preparation

[0123] Same as step 1 in Example 2.

[0124] 2. Sample Preparation

[0125] Simulated human-derived samples were prepared using humanized cells (Hacat, 5 × 10⁻⁶). 5 Cells), incorporating easily lysed Gram-negative bacteria Escherichia coli (E. coli, 1×10⁻⁶ cells), 8 CFU was used as the sample to be tested.

[0126] 3. Host removal processing

[0127] Same as step 3 in Example 1.

[0128] 4. Nucleic acid extraction

[0129] Nucleic acid extraction was performed using the MGIEAasy total RNA extraction kit according to the instructions. Extracted products were stored at 4°C or frozen at -80°C.

[0130] 5. Concentration and purity testing:

[0131] Concentration and purity tests were performed according to the NanoDrop 8000 instrument manual. The concentration of the extract after host removal was significantly lower than that of the control group (Group 3), while the purity of the extract after host removal was not significantly different from that of the control group (Table 5).

[0132] Table 5. Concentration and Purity Results

[0133]

[0134] 6. Electrophoresis detection

[0135] Prepare a 1.2% gel using 1xTAE, add 10 μL of dye per 100 mL of 1xTAE; use 1xTAE as electrophoresis buffer; use 3 μL of GoldBandDL2000 DNA Marker, 2 μL of 5x Loading, and 8 μL of sample; incubate at 100V for 30 min.

[0136] Electrophoresis results as follows Figure 5As shown, human and bacterial RNA extraction was normal, with clear 28S, 18S, 23S, and 16S bands, and 28S > 18S, and 23S > 16S. In control group 3, the 18S band was likely relatively weak, showing no clear band. Control groups 1 and 3 showed darker bands above 32000 bp, presumably due to a larger cell quantity and incomplete DNA digestion during RNA extraction. The host-removed group (group 4) showed no human RNA bands (28S, 18S) or DNA bands, indicating that host nucleic acid removal was relatively thorough and avoided excessive host nucleic acid leading to incomplete DNA digestion during RNA extraction.

[0137] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A reagent combination for removing host nucleic acid, characterized in that: include: Lysis solution, nuclease solution and stop solution; The lysate contains saponin, NaCl and MgCl2, wherein the concentration of the saponin is 0.001 to 5% by volume, the concentration of the NaCl is 0.005 to 5M, and the concentration of the MgCl2 is 0.1 to 100mM.

2. The reagent combination according to claim 1, characterized in that The concentration of the saponin is 0.01 to 2% by volume, preferably 0.012 to 1% by volume; The concentration of NaCl is 0.03 to 3 M, preferably 0.035 to 2 M; The concentration of MgCl2 is 1-50 mM, preferably 2-40 mM.

3. The reagent combination according to claim 1, characterized in that The nuclease solution contains 2 to 20 KU / mL of universal nuclease Binuclease, preferably 5 to 15 KU / mL of universal nuclease Binuclease; Optionally, the stop solution contains 1-40 mg / mL proteinase K, preferably 10-30 mg / mL proteinase K; Optionally, the pH value of the lysate is 6-10, preferably 7-8.

4. The reagent combination according to claim 1, characterized in that: The lysate further contains Tris, KCl, Na2HPO4 and KH2PO4; Optionally, the concentration of Tris is 10 to 100 mM, preferably 10 to 30 mM; The concentration of KCl is 0-5.34 mM, preferably 2-4 mM; The concentration of Na2HPO4 is 0-16.2 mM, preferably 7-9 mM; The concentration of KH2PO4 is 0-3.52 mM, preferably 1-3 mM.

5. The reagent combination according to claim 4, characterized in that: The reagent combination for removing host nucleic acid consists of a lysis solution, a nuclease solution and a stop solution; The lysate contains 0.012-1 volume % saponin, 10-30 mM Tris, 2-40 mM MgCl2, 0.035-2 M NaCl, 2-4 mM KCl, 7-9 mM Na2HPO4 and 1-3 mM KH2PO4, and the pH value is 7-8; The nuclease solution contains 5 to 15 KU / mL of universal nuclease Binuclease; The stop solution contains 10-30 mg / mL proteinase K.

6. A kit for removing host nucleic acid, characterized in that include: The reagent combination for removing host nucleic acid according to any one of claims 1 to 5.

7. Use of the reagent combination for removing host nucleic acid according to any one of claims 1 to 5 or the kit for removing host nucleic acid according to claim 6 in removing host nucleic acid and / or detecting microbial nucleic acid.

8. A method for removing host nucleic acid, characterized in that include: (1) centrifuging the sample containing host nucleic acid, removing the supernatant, and collecting the precipitate; (2) mixing and incubating the precipitate, lysate and nuclease solution to obtain a reaction solution; (3) mixing and incubating the reaction solution with a stop solution; The lysis solution, nuclease solution and stop solution are as defined in the reagent combination for removing host nucleic acid according to any one of claims 1 to 5 or the kit for removing host nucleic acid according to claim 6.

9. The method for removing host nucleic acid according to claim 8, characterized in that Based on 0.2-2 mL of the sample, the amount of the lysate added is 150-250 μL, the amount of the nuclease solution added is 1-5 μL, and the amount of the stop solution added is 10-30 μL; Optionally, in step (2), the incubation temperature is 30 to 40° C. and the time is 5 to 15 minutes; Optionally, in step (3), the incubation temperature is 50 to 60° C. and the time is 5 to 15 minutes.

10. A method for detecting microbial nucleic acid, characterized in that: include: The method for removing host nucleic acid according to claim 8 or 9 is used to treat the sample to be tested, so as to remove the host nucleic acid in the sample to be tested.

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