A method and a combination reagent for efficiently releasing, extracting and detecting pathogen DNA from an ex vivo body fluid sample

Abstract

The present application relates to the technical field of in vitro diagnosis sample pretreatment and nucleic acid extraction, and discloses a method and a combined reagent for releasing, extracting and detecting pathogen DNA from an ex vivo body fluid sample. The method sequentially performs host cell transient protection, pre-sensitization of cell wall acting enzymes, subsequent pre-sensitization of proteinase K and non-ionic surfactant, first short pulse lysis of composite lysis beads, instant magnetic bead capture of released DNA, magnetic retention of the first part of magnetic beads, second short pulse lysis of a non-magnetic lysis system, removal of lysis beads, and termination of binding. The first part of magnetic beads is removed from the mechanical action area before the second mechanical lysis, and the lysis beads are removed before the final magnetic separation, thereby reducing magnetic bead damage, DNA continuous shearing, and interference of lysis beads on washing and elution. The obtained DNA can be used for PCR, digital PCR or sequencing detection.

Description

Technical Field

[0001] This invention relates to the fields of in vitro diagnostic sample pretreatment and nucleic acid extraction technology, and in particular to a method and combination reagents for efficiently releasing, extracting and detecting pathogen DNA from ex vivo body fluid samples. Background Technology

[0002] Pathogen DNA detection in bodily fluid samples typically involves sample homogenization, pathogen structure disruption, DNA release, nucleic acid extraction and purification, and amplification or sequencing. The extent to which pathogen DNA is released, and whether the host DNA background is controlled during release, directly affects the detection capability of low-load pathogens and the consistency of test results between different samples. Blood, cerebrospinal fluid, urine, pleural effusion, ascites, bronchoalveolar lavage fluid, sputum, pus, and genital secretions vary in host cell count, protein content, viscosity, and endogenous nuclease activity; therefore, sample lysis and nucleic acid extraction are relatively prone to bias.

[0003] Current methods for lysing bodily fluid samples mainly include chemical lysis, enzymatic lysis, and mechanical lysis. To accommodate pathogens with different structures, such as Gram-negative bacteria, Gram-positive bacteria, fungi, and enveloped viruses, some protocols combine surfactants, proteases, cell wall-acting enzymes, and bead milling for mechanical action, enhancing DNA release by extending reaction time or increasing mechanical strength. While these protocols can improve the lysis rate of some difficult-to-lyse microorganisms, they can also damage host cells and their nuclei, resulting in a large amount of host DNA entering the extraction system.

[0004] For samples with high host cell background, current techniques often employ a host-first lysis and nuclease-removal route. This involves first disrupting host cells using saponins, hypotonic solutions, or other membrane-action reagents, then using nucleases to degrade the host DNA released into the liquid phase, followed by lysis of the pathogen. While this route can reduce some host DNA background, the host lysing agent, nucleases, and their termination components may simultaneously affect the external structure of the pathogen or the released pathogen DNA. Different pathogens have varying tolerances to treatment conditions, which can easily increase recovery bias and may affect subsequent amplification due to residual reagents.

[0005] Another common approach involves continuous mechanical lysis at a fixed intensity, followed by the addition of nucleic acid trapping magnetic beads after lysis is complete. Continuous mechanical treatment treats the pathogen, host cells, and sample matrix as the same mechanical target. When increasing the mechanical intensity to lyse Gram-positive bacteria or fungi with thick cell walls, the host cells and nuclei are easily damaged by non-target forces; continuous oscillation can also easily cause temperature rise and excessive DNA fragmentation.

[0006] If magnetic beads are added only after mechanical lysis is complete, the low-abundance pathogen DNA released initially will be continuously exposed in the complex lysis buffer, potentially subject to degradation by residual nucleases, non-specific adsorption of cell debris, or subsequent mechanical shearing. On the other hand, if the DNA-bound magnetic beads are left directly in the lysis bead system and continue to oscillate at high speed, the magnetic beads and their surface DNA may be impacted by the hard lysis beads. If the lysis beads are not removed during final magnetic separation, they will also hinder resuspension, washing, and elution of the magnetic beads.

[0007] Therefore, there is a need for a processing method that can coordinate the short-term stability of host cells, pre-sensitization of pathogen structures, segmented mechanical energy input, immediate capture of released DNA, and physical separation of lysis beads and magnetic beads, so as to improve the recovery rate of DNA from various pathogens while reducing the interference of host DNA background, continuous DNA shearing, and solid particles on subsequent purification. Summary of the Invention

[0008] The purpose of this invention is to solve the problem of simultaneously achieving high pathogen DNA release efficiency, host DNA background control, released DNA stability, and solid particle separation in the processing of body fluid samples. The invention provides a method for efficiently releasing, extracting, and detecting pathogen DNA from ex vivo body fluid samples, as well as a combination reagent for implementing the method.

[0009] To achieve the above objectives, the first aspect of the present invention provides a method for releasing, extracting, and detecting pathogen DNA from an isolated body fluid sample, comprising the following steps: S1. Mix the ex vivo body fluid sample with the host cell transient preservation solution and incubate at 15–30°C for 2–10 min; the host cell transient preservation solution includes nonionic block polyether, osmotic pressure regulator, divalent metal salt and buffer salt; S2. Add the first presensitization solution to the reaction system obtained in step S1, incubate at 30-40°C for 3-12 min, then add the second presensitization solution prepared separately from the first presensitization solution, and continue incubating at 30-40°C for 2-8 min; the first presensitization solution includes bacterial wall-acting enzymes and fungal wall-acting enzymes, and the second presensitization solution includes proteinase K and nonionic surfactants. S3. Add composite pyrolysis beads to the reaction system obtained in step S2, and perform the first short-pulse mechanical pyrolysis at 4-20°C; the composite pyrolysis beads include small-diameter pyrolysis beads with a particle size of 0.08-0.20 mm and medium-diameter pyrolysis beads with a particle size of 0.30-0.80 mm. S4. After the first short-pulse mechanical lysis, the first part of nucleic acid capture magnetic beads and capture promotion solution are added to the reaction system. The system is allowed to stand for 30–180 s to allow the released pathogen DNA to bind to the first part of the nucleic acid capture magnetic beads. The amount of capture promotion solution added is such that the final concentration of PEG 6000 in the reaction system is 4%–8%, the final concentration of sodium chloride is 0.3–0.8 mol / L, the final concentration of Tris-HCl is 8–20 mmol / L, the pH is 7.8–8.3, and the concentration of the first part of the nucleic acid capture magnetic beads is 0.5–1.5 mg / mL. After standing, the first part of the nucleic acid capture magnetic beads are retained in the first capture container using a magnetic field, and the non-magnetic suspension, including the composite lysis beads, is transferred to the second lysis container. S5. The non-magnetic suspension transferred to the second lysis container is subjected to a second short-pulse mechanical lysis. The oscillation speed of the second short-pulse mechanical lysis is higher than that of the first short-pulse mechanical lysis. The two short-pulse mechanical lysis processes last for 10 to 40 seconds, and the total mechanical action time does not exceed 60 seconds. After the second short-pulse mechanical lysis is completed, the composite lysis beads are removed by using a lysis bead separator with a pore size of 20 to 60 μm or by low-speed centrifugation. The liquid phase without composite lysis beads is collected and added back to the first capture container. S6. Add the termination binding solution and the remaining nucleic acid capturing magnetic beads to the first capture container and mix for 2–10 min. The amount of the termination binding solution added is such that the final concentration of guanidine hydrochloride in the resulting system is 1.5–3.0 mol / L, the final concentration of PEG 8000 is 4%–8%, the final concentration of sodium chloride is 0.3–1.0 mol / L, the final concentration of EDTA is 2–8 mmol / L, and the total concentration of all nucleic acid capturing magnetic beads is 0.7–2.0 mg / mL. The first part of the nucleic acid capturing magnetic beads accounts for 20%–60% of the total mass of the nucleic acid capturing magnetic beads, and no further mechanical lysis is performed after the termination binding solution is added. S7. Perform magnetic separation on the system obtained in step S6, collect the nucleic acid capture magnetic beads bound to DNA, wash and elute the nucleic acid capture magnetic beads to obtain pathogen DNA; use PCR, digital PCR or sequencing methods to detect the in vitro nucleic acid signal of the obtained pathogen DNA.

[0010] The above method is not a simple sequential stacking of enzymatic lysis, mechanical lysis, and magnetic bead extraction. Instead, it revolves around the time process from the release of pathogen DNA from the cell or particle interior to final purification, continuously linking host cell stabilization, pathogen structural weakening, mechanical energy input, DNA capture, and solid particle separation. The host cell transient protective solution first forms a short protective window of several minutes, maintaining relative stability of the host cell membrane and nuclear membrane during subsequent temperature changes, osmotic fluctuations, and mechanical actions, controlling the extent to which host DNA enters the liquid phase from the initial stage of pathogen DNA release.

[0011] The first and second presensitization solutions act sequentially, initially weakening the bacterial peptidoglycan and fungal glucan structures to a limited extent. Then, proteinase K and nonionic surfactants treat the pathogen's surface proteins or membrane structures. This phased approach avoids achieving complete lysis during the presensitization stage, instead gradually introducing pathogens with different external structures into a state more susceptible to mechanical damage. This allows the two-stage particle size lysis beads to complete DNA release within a limited mechanical action time, maintaining a good balance in the treatment of different pathogens.

[0012] The DNA released during the first short-pulse mechanical lysis is promptly bound to the first set of nucleic acid capture magnetic beads in the molecular crowding and ion-shielding environment created by PEG 6000 and sodium chloride. These beads are then magnetically retained within the first capture container. This step essentially traps the released DNA before subjecting the incompletely lysed pathogen to a second mechanical action. The DNA-bound magnetic beads then leave the high-speed movement zone of the composite lysis beads, reducing the continuous impact and shearing of the hard lysis beads on the surface of the magnetic beads and the bound DNA.

[0013] After the second short-pulse mechanical lysis, the composite lysis beads are removed before the termination of binding and final magnetic separation, preventing them from entering the magnetic bead resuspension, washing, and elution processes. The termination of binding solution further terminates the enzymatic reaction and reduces potential nuclease activity, while simultaneously establishing concentrated binding conditions for the remaining free DNA. This forms a two-stage capture process that combines early DNA immediate protection with later complete DNA recovery. The obtained pathogen DNA can be directly used for in vitro amplification or sequencing signal detection, providing a clear and repeatable operational endpoint between sample lysis, DNA purification, and detection.

[0014] Preferably, the nonionic block polyether is poloxamer 188; the osmotic pressure regulator includes trehalose and sorbitol; the divalent metal salt includes magnesium salt and calcium salt; the buffer salt is HEPES; in the reaction system formed in step S1, the final concentration of poloxamer 188 is 0.02%–0.30%, the final concentration of trehalose is 20–120 mmol / L, the final concentration of sorbitol is 20–100 mmol / L, the final concentration of magnesium ions is 0.5–5 mmol / L, the final concentration of calcium ions is 0.1–2 mmol / L, the final concentration of HEPES is 10–50 mmol / L, and the pH is 7.2–8.0.

[0015] Poloxamer 188 forms a mild, non-ionic protective environment at the host cell lipid membrane interface during short-term treatment. Trehalose and sorbitol work together to regulate water activity and osmotic state, while magnesium and calcium ions help maintain the stability of membrane structure and cell junction regions. HEPES keeps the system in a near-neutral buffer zone. The components, when combined within the specified concentration range, can enhance the host cell's short-term tolerance to subsequent pre-sensitization and short-pulse mechanical effects without significantly increasing system viscosity, allowing for a smooth transition between transient host protection and enzymatic treatment.

[0016] Preferably, the bacterial wall-activating enzyme includes lysozyme and mutalysin, the fungal wall-activating enzyme is β-glucanase, and the first pre-sensitization solution further includes EDTA; in the reaction system at the end of step S2, the final concentration of lysozyme is 0.1–2.0 mg / mL, the final concentration of mutalysin is 10–200 U / mL, the final concentration of β-glucanase is 0.05–1.0 mg / mL, the final concentration of proteinase K is 0.02–0.30 mg / mL, the final concentration of nonionic surfactant is 0.005%–0.08%, and the final concentration of EDTA is 0.05–1.0 mmol / L.

[0017] Lysozyme acts on the glycosidic bonds in the peptidoglycan of bacterial cell walls, while mutalycin further acts on the cell wall junctions of some Gram-positive bacteria, and β-glucanase weakens the glucan network in fungal cell walls. Low concentrations of EDTA regulate the local metal ion environment, helping to increase the permeability of the outer structures of some bacteria. The subsequent addition of proteinase K and nonionic surfactants provides limited treatment to pathogen surface proteins and membrane structures, allowing bacteria, fungi, and enveloped DNA viruses to complete presensitization along pathways suitable to their external structures, and preventing the premature release of large amounts of free DNA during the presensitization stage.

[0018] Preferably, the small-diameter pyrolysis beads and the medium-diameter pyrolysis beads are each independently selected from one or more of glass beads, zirconia beads, and ceramic beads; the mass ratio of small-diameter pyrolysis beads to medium-diameter pyrolysis beads is 1:0.25 to 1:1.5, and the packing volume of the composite pyrolysis beads is 0.3 to 1.5 times the volume of the reaction liquid before mechanical pyrolysis; the first short-pulse mechanical pyrolysis is performed for 10 to 30 s at an oscillation speed of 4 to 7 m / s calibrated by the equipment, and the second short-pulse mechanical pyrolysis is performed for 10 to 40 s at an oscillation speed 0.5 to 2.0 m / s higher than that of the first short-pulse mechanical pyrolysis; the mechanical pyrolysis uses a pyrolysis container with a nominal volume of not less than 5 mL, and the total volume of the liquid and the pyrolysis beads does not exceed 60% of the nominal volume of the pyrolysis container.

[0019] Small-diameter lysis beads can generate high-frequency contact, friction, and local shearing per unit time, while medium-diameter lysis beads provide higher single-impact momentum. When the two particle sizes are combined in the aforementioned ratio, the mechanical energy distribution can simultaneously cover both the local weak areas and the overall structure of the pathogen surface. The first short pulse, with a relatively low oscillation speed, opens up the more easily damaged pathogens after pre-sensitization. The second short pulse increases the oscillation speed to treat the remaining, more structurally stable pathogens. Maintaining at least 40% volume space in the lysis container allows the lysis beads to form sufficient reciprocating motion paths, reducing bead accumulation and ineffective vibration caused by overloading.

[0020] Preferably, the nucleic acid capturing magnetic beads are carboxyl magnetic beads and / or silicon-based magnetic beads with an average particle size of 0.5 to 2.0 μm; the first part of the nucleic acid capturing magnetic beads accounts for 35% to 45% of the total mass of the nucleic acid capturing magnetic beads; after the first part of the nucleic acid capturing magnetic beads has completed static binding, it is attached to the side wall of the first capture container under the action of a magnetic field, and 30 to 100 μL of liquid is retained on the surface of the first part of the nucleic acid capturing magnetic beads to prevent the magnetic beads from drying out.

[0021] Carboxyl-based or silicon-based magnetic beads with an average particle size of 0.5–2.0 μm possess a large binding surface area per unit mass and can complete the magnetic response in a short time. Controlling the first batch of nucleic acid capture beads to 35%–45% of the total bead mass provides sufficient immediate binding sites for the DNA released in the first short pulse and retains adequate binding capacity for the DNA released after the second lysis. Maintaining a small amount of covering solution during magnetic retention helps maintain the hydration state of the bead surface, reducing irreversible aggregation of the beads and preventing local concentration or desorption of bound DNA during the waiting period for the second lysis.

[0022] Preferably, after the second short-pulse mechanical lysis, the reaction system is centrifuged at 300–800 × g for 20–60 s through a lysis bead separator with a pore size of 40 μm, and the lysis bead separator is rinsed with 30–100 μL of protective carrier solution. In step S7, the nucleic acid capture magnetic beads are washed sequentially with a first washing solution containing ethanol and a second washing solution containing low-salt ethanol, and then eluted with 10 mmol / L Tris-HCl buffer or nuclease-free water at 50–70 °C for 3–10 min. The ex vivo body fluid sample is selected from one of blood, cerebrospinal fluid, urine, pleural effusion, ascites, bronchoalveolar lavage fluid, sputum, pus, and genital tract secretions. The pathogen is selected from one or more of bacteria, fungi, and enveloped DNA viruses.

[0023] The 40 μm lysis bead separator can retain composite lysis beads with a particle size of not less than 0.08 mm, while allowing liquid phase containing free DNA and components other than micron-sized magnetic beads to pass through. Combined with low-speed centrifugation and a small amount of protective carrier solution rinsing, liquid phase retention and DNA loss in the gaps between lysis beads can be reduced. Two-stage ethanol washing gradually removes guanidine salts, polyethylene glycol, proteins, and sample matrix residues. Subsequently, gentle heating reduces the binding strength of DNA to the surface of the magnetic beads, ensuring that the eluted DNA maintains a purity and concentration suitable for PCR, digital PCR, and sequencing reactions.

[0024] A second aspect of the present invention provides a pathogen DNA release and extraction combination reagent for carrying out the method described above, comprising a host cell transient protection solution, a first presensitization solution, a second presensitization solution, a capture-promoting base solution, a binding termination base solution, nucleic acid capture magnetic beads, composite lysis beads, and a lysis bead separation element with a pore size of 20–60 μm; the host cell transient protection solution comprises poloxamer 188, trehalose, sorbitol, magnesium salt, calcium salt, and HEPES; the first presensitization solution comprises lysozyme, mutalysin, β-glucanase, and EDTA; the second presensitization solution comprises proteinase K and a nonionic surfactant; the capture-promoting base solution comprises PEG 6000, sodium chloride, and Tris-HCl; and the binding termination base solution comprises guanidine hydrochloride, PEG 8000, sodium chloride, EDTA, and Tris-HCl.

[0025] The combined reagents are formulated separately according to the action stages, ensuring that the transient protection of host cells, the two-stage pre-sensitization of pathogens, the immediate capture after the first lysis, the solid-liquid separation after the second lysis, and the termination binding each have independent yet interconnected component bases. The first and second pre-sensitization solutions are stored separately to reduce the impact of proteinase K on the storage period of cell wall-acting enzymes; the separate formulation of the capture-promoting base solution and the termination binding base solution ensures that the early mild capture environment and the later highly efficient termination binding environment remain stable. The inclusion of composite lysis beads and lysis bead separators in the combined reagents facilitates a coordinated relationship between mechanical processing parameters and subsequent solid separation conditions.

[0026] Preferably, the host cell transient preservation solution is a triple-concentrated solution comprising 0.30% poloxamer 188, 180 mmol / L trehalose, 120 mmol / L sorbitol, 6 mmol / L magnesium chloride, 1.5 mmol / L calcium chloride, and 60 mmol / L HEPES, with a pH of 7.6; the host cell transient preservation solution is used at a ratio of 250 μL per 500 μL of ex vivo body fluid sample.

[0027] After adding the triple-concentrated solution according to the stated ratio, 0.10% poloxamer 188, 60 mmol / L trehalose, 40 mmol / L sorbitol, 2 mmol / L magnesium chloride, 0.5 mmol / L calcium chloride, and 20 mmol / L HEPES can be directly formed in the sample system. This setup reduces concentration fluctuations caused by on-site preparation of each solution, while maintaining suitable system fluidity and mixing space after the protective solution is added, facilitating the subsequent addition of the two pre-sensitization solutions at fixed volumes.

[0028] Preferably, the first presensitization solution comprises 4.5 mg / mL lysozyme, 450 U / mL mutalycin, 1.8 mg / mL β-glucanase, and 1.8 mmol / L EDTA; the second presensitization solution comprises 1.44 mg / mL proteinase K and 0.36% polysorbate 20; both the first and second presensitization solutions use a protective carrier solution as a solvent, which comprises 0.10% poloxamer 188, 60 mmol / L trehalose, 40 mmol / L sorbitol, 2 mmol / L magnesium chloride, 0.5 mmol / L calcium chloride, and 20 mmol / L HEPES, with a pH of 7.6; the addition volumes of the first and second presensitization solutions are 100 μL and 50 μL, respectively, per 500 μL of ex vivo body fluid sample.

[0029] The first and second presensitization solutions use protective carrier solutions that are compatible with the composition of the host protection system, which can avoid weakening the already established transient host protection state due to sudden changes in solvent composition when adding presensitization components. Adding them sequentially in fixed volumes of 100 μL and 50 μL allows for well-defined working concentrations of cell wall-acting enzymes and subsequent membrane protein-acting components, and establishes a reproducible treatment sequence of cell wall structure first, followed by membrane protein structure, while maintaining controllable total volume.

[0030] Preferably, the capture-enhancing base solution comprises 25.64% PEG 6000, 1.923 mol / L sodium chloride, and 42.74 mmol / L Tris-HCl, with a pH of 8.0; based on every 500 μL of ex vivo body fluid sample, 280.8 μL of the capture-enhancing base solution is mixed with 19.2 μL of a concentrated suspension of carboxylated magnetic beads with a magnetic solid concentration of 50 mg / mL to obtain 300 μL of the first part of the magnetic bead capture working solution; the binding termination base solution comprises 4.10 mol / L guanidine hydrochloride, 10.25% PEG 8000, 0.768 mol / L sodium chloride, 10.09 mmol / L EDTA, and 20.49 mmol / L Tris-HCl, with a pH of 7.8; 1171.2 μL of the binding termination base solution is mixed with 28.8 μL of the concentrated suspension of carboxylated magnetic beads to obtain 1200 μL of the binding termination working solution.

[0031] The base solution and the concentrated magnetic bead suspension are pre-mixed at a fixed volume, ensuring that the magnetic bead mass, PEG concentration, and salt concentration in the first part of the magnetic bead capture working solution accurately correspond to the sample processing volume. After the first lysis, 300 μL of the first part of the magnetic bead capture working solution is added, allowing for timely capture of released DNA without introducing strong denaturing conditions. Subsequent addition of 1200 μL of the termination binding working solution converts the system into a termination binding environment containing guanidine hydrochloride, PEG 8000, sodium chloride, and EDTA, terminating the enzymatic reaction, reducing potential nuclease activity, and concentrating the binding of remaining DNA. Maintaining a fixed volume and magnetic bead mass distribution between the two working solutions ensures stable batch-to-batch consistency in both immediate capture and final recovery.

[0032] The present invention has the following beneficial effects: 1. This invention utilizes poloxamer 188, trehalose, sorbitol, magnesium ions, calcium ions, and HEPES to construct a short-term transient protective environment for host cells, maintaining relative stability of the host cell membrane and nuclear membrane during pre-sensitization, temperature transitions, and short-pulse mechanical actions. This allows for control over the extent to which host DNA enters the liquid phase at the sample processing stage, while preserving suitable reaction fluidity for subsequent enzymatic processing and pathogen mechanical lysis.

[0033] 2. This invention employs a two-stage pre-sensitization method, first using bacterial and fungal wall-acting enzymes, followed by proteinase K and nonionic surfactants, to sequentially and selectively weaken the membrane protein structures of bacterial peptidoglycan, fungal glucan networks, and enveloped DNA viruses. This process lowers the mechanical cleavage threshold of various pathogens and maintains a good balance in the DNA release process of pathogens with different structures.

[0034] 3. This invention combines high-frequency local friction and shearing with high-momentum instantaneous collisions through two-stage particle size composite lysis beads and two short-pulse mechanical lysis processes with increasing oscillation intensity. The first short pulse preferentially treats pathogens that are more easily destroyed after pre-sensitization, while the second short pulse treats the remaining pathogens with more stable structures, balancing DNA release efficiency and fragment integrity under the condition that the total mechanical action time does not exceed 60 s.

[0035] 4. In this invention, the released DNA is collected by a first set of nucleic acid capture magnetic beads immediately after the first short pulse, and the magnetic beads are retained in the first capture container by a magnetic field before the second short pulse. This shortens the release time of low-load pathogen DNA in the complex liquid phase and also allows the DNA-bound magnetic beads to avoid the high-speed impact area of ​​the subsequent hard lysis beads, thereby maintaining the stability of the magnetic bead structure, magnetic responsiveness, and DNA-bound state.

[0036] 5. In this invention, after the second short pulse, the composite lysed beads are first removed using a lysed bead separator or by low-speed centrifugation, and then the liquid phase is added back to the first capture container to terminate the binding. Therefore, the lysed beads do not enter the final magnetic separation, magnetic bead resuspension, ethanol washing, and heating elution processes, which reduces the likelihood of lysed beads trapping magnetic beads, retaining liquid phase, or hindering washing mass transfer, making the solid particle separation and nucleic acid purification processes more seamless.

[0037] 6. This invention sets up a two-stage DNA binding process with the immediate capture of the first part of the magnetic beads and the termination of binding of the remaining magnetic beads. Guanidine hydrochloride and EDTA in the termination solution terminate the enzymatic reaction and reduce the potential nuclease activity, while PEG8000 and sodium chloride promote the concentration and binding of the remaining free DNA to the surface of the magnetic beads. This allows for a clear processing endpoint and a stable operating rhythm for pathogen DNA release, early protection, final recovery and purification.

[0038] 7. This invention comprises a host cell transient protection solution, two pre-sensitization solutions, a capture-promoting base solution, a binding termination base solution, nucleic acid capture magnetic beads, composite lysis beads, and a lysis bead separator, all of which are separately formulated and used in combination. The concentration, volume, magnetic bead mass distribution, and solid separation conditions of each component can be predetermined, ensuring good consistency in the processing of different batches. The resulting pathogen DNA can be used for in vitro nucleic acid signal detection such as PCR, digital PCR, and sequencing. Attached Figure Description

[0039] Figure 1 This is a process flow diagram for Example 1. Detailed Implementation

[0040] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0041] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] The present invention will now be described in detail with reference to specific embodiments, which are intended to understand rather than limit the invention.

[0045] General description of test materials and testing: The low-cell, low-protein simulated body fluid matrix consisted of 140 mmol / L sodium chloride, 5 mmol / L potassium chloride, 1 mmol / L calcium chloride, 0.5 mmol / L magnesium chloride, 10 mmol / L HEPES, and 0.5 g / L bovine serum albumin, with a pH of 7.4.

[0046] The high-host-cell matrix used the same basic matrix, but with a bovine serum albumin concentration of 5.0 g / L. The high-protein, high-viscosity sputum-mimicking matrix consisted of isotonic HEPES buffer, 3.0% mucin, and 5.0% bovine serum albumin, at a pH of 7.4.

[0047] Bacterial and fungal materials were prepared using inactivated standards that maintained intact cell structure, specifically inactivated standards for *Escherichia coli* ATCC25922, *Staphylococcus aureus* ATCC 25923, and *Candida albicans* ATCC 10231. Viral materials were prepared using non-replicating virus-like particles with a particle size of 80–120 nm, a lipid envelope, and encapsulated double-stranded DNA target fragments. The target nucleic acid copy number of each standard was assigned using digital PCR. Before use, the standards were treated with nucleases and purified to remove free DNA from the outer layer.

[0048] Pathogen DNA recovery rate was calculated as the ratio of the total copy number of pathogen target DNA in the elution buffer (corrected for volume and dilution) to the theoretically added target DNA copy number in 500 μL of sample. Host DNA was determined using digital PCR of the RPP30 gene; based on the assumption that each diploid human cell contains 2 RPP30 copies and 6.6 pg of DNA, the amount of host DNA released was calculated as "total RPP30 copy number ÷ 2 × 6.6 pg ÷ original sample volume".

[0049] Both digital PCR and qPCR employed target detection systems validated for specificity, linearity, and amplification efficiency. Each batch of tests included a negative extraction control, a blank amplification control, a positive standard, and an exogenous amplification internal standard. Pathogen DNA signal detection was used only to evaluate target nucleic acids in ex vivo samples and did not directly lead to disease diagnoses. Example 1

[0050] Reference Figure 1 This embodiment discloses a method for releasing, extracting, and detecting pathogen DNA from a low-cell, low-protein ex vivo simulated body fluid sample.

[0051] Preparation of a triple-concentrated transient host cell preservation solution: Weigh 0.300 g of poloxamer 188, 6.810 g of trehalose dihydrate, 2.186 g of sorbitol, 0.122 g of magnesium chloride hexahydrate, 0.022 g of calcium chloride dihydrate, and 1.430 g of HEPES. Dissolve in approximately 80 mL of nuclease-free water, adjust the pH to 7.6, and then bring the volume to 100 mL. The resulting solution contains 0.30% poloxamer 188, 180 mmol / L trehalose, 120 mmol / L sorbitol, 6 mmol / L magnesium chloride, 1.5 mmol / L calcium chloride, and 60 mmol / L HEPES. Filter through a 0.22 μm filter and store at 2–8 °C.

[0052] Preparation of protective carrier solution: The triple-concentrated host cell transient protection solution was diluted three times with nuclease-free water to obtain a protective carrier solution containing 0.10% poloxamer 188, 60 mmol / L trehalose, 40 mmol / L sorbitol, 2 mmol / L magnesium chloride, 0.5 mmol / L calcium chloride and 20 mmol / L HEPES, with a pH of 7.6.

[0053] Preparation of the first presensitization solution: Add 45.0 mg of lysozyme, 4500 U of mutalysin, 18.0 mg of β-glucanase, and 6.7 mg of disodium EDTA dihydrate to every 10 mL of the first presensitization solution. Adjust the volume to 10 mL with protective carrier fluid to achieve the following concentrations: lysozyme 4.5 mg / mL, mutalysin 450 U / mL, β-glucanase 1.8 mg / mL, and EDTA 1.8 mmol / L. Aliquot and store at -20°C.

[0054] Preparation of the second presensitization solution: Add 14.4 mg of proteinase K and 36.0 mg of polysorbate 20 to every 10 mL of the second presensitization solution, and bring the volume to 10 mL with protective carrier fluid to achieve a proteinase K concentration of 1.44 mg / mL and a polysorbate 20 concentration of 0.36%. After aliquoting, store at -20℃.

[0055] The capture-enhancing base solution contained 25.64% PEG 6000, 1.923 mol / L sodium chloride, and 42.74 mmol / L Tris-HCl, with a pH of 8.0. 280.8 μL of the capture-enhancing base solution was mixed with 19.2 μL of a concentrated suspension of carboxyl magnetic beads with a magnetic solids concentration of 50 mg / mL and an average particle size of 0.8–1.2 μm to obtain 300 μL of the first part of the magnetic bead capture working solution. This working solution contained 24.0% PEG 6000, 1.8 mol / L sodium chloride, 40 mmol / L Tris-HCl, and 3.2 mg / mL carboxyl magnetic beads. The solution was equilibrated to 20–25 °C and fully resuspended before use.

[0056] The termination solution contained 4.10 mol / L guanidine hydrochloride, 10.25% PEG 8000, 0.768 mol / L sodium chloride, 10.09 mmol / L EDTA, and 20.49 mmol / L Tris-HCl, with a pH of 7.8. 1171.2 μL of the termination solution was mixed with 28.8 μL of the concentrated suspension of the carboxyl magnetic beads to obtain 1200 μL of the termination working solution. This working solution contained 4.0 mol / L guanidine hydrochloride, 10.0% PEG 8000, 0.75 mol / L sodium chloride, 9.85 mmol / L EDTA, 20 mmol / L Tris-HCl, and 1.2 mg / mL carboxyl magnetic beads.

[0057] The first wash buffer was an 80% ethanol aqueous solution containing 10 mmol / L Tris-HCl and 100 mmol / L sodium chloride, with a pH of 7.5; the second wash buffer was a 75% ethanol aqueous solution containing 10 mmol / L Tris-HCl, with a pH of 7.5; and the elution buffer was a 10 mmol / L Tris-HCl buffer with a pH of 8.0.

[0058] S1. Take 500 μL of low-cell, low-protein simulated body fluid sample, add human leukocytes to a concentration of 2.0 × 10^5 cells / mL, and add inactivated standard substances for Escherichia coli, Staphylococcus aureus, and Candida albicans, as well as enveloped DNA virus-like particles, to make the target DNA concentration of each pathogen 1.0 × 10^4 copies / mL. Use a wide-mouth pipette to pipette five times, add 250 μL of triple-concentrated host cell transient protection solution, vortex at low speed for 5 s, and incubate at 25°C for 5 min. In the resulting 750 μL system, the final concentrations of poloxamer 188, trehalose, sorbitol, magnesium chloride, calcium chloride, and HEPES are 0.10%, 60 mmol / L, 40 mmol / L, 2 mmol / L, 0.5 mmol / L, and 20 mmol / L, respectively, with a pH of 7.6.

[0059] S2. Add 100 μL of the first presensitization solution and incubate at 37℃ and 300 r / min for 6 min; then add 50 μL of the second presensitization solution and continue incubating at 37℃ and 300 r / min for 4 min. At the end of presensitization, the reaction volume is 900 μL, with final concentrations of lysozyme, mutalysin, β-glucanase, proteinase K, polysorbate 20, and EDTA of 0.50 mg / mL, 50 U / mL, 0.20 mg / mL, 0.08 mg / mL, 0.02%, and 0.20 mmol / L, respectively.

[0060] S3. Add glass pyrolysis beads with particle sizes of 0.10 mm and 0.50 mm in a mass ratio of 1:0.75 to a 5 mL screw-cap polypropylene pyrolysis tube. The bulk volume of the pyrolysis beads should be 0.8 times the volume of the 900 μL reaction liquid. Use a bead mill with reciprocating oscillation along the tube axis at a calibrated speed of 5.5 m / s for 20 s at 4–10 °C to complete the first short-pulse mechanical pyrolysis. The total volume of the liquid and the pyrolysis beads should be less than 40% of the nominal volume of the pyrolysis tube.

[0061] S4. Immediately after the first short pulse, add 300 μL of the first part of the magnetic bead capture working solution, gently invert and mix 8 times, and let stand for 60 s. After addition, the total liquid volume is 1200 μL, with the final concentrations of PEG 6000, sodium chloride, Tris-HCl, and the first part of carboxyl magnetic beads being 6.0%, 0.45 mol / L, 10 mmol / L, and 0.80 mg / mL, respectively.

[0062] S5. Place the lysis tube on the side wall magnetic rack for 2 min to allow the first portion of carboxyl magnetic beads to adhere to the tube wall. Keep the lysis tube in the magnetic field and use a wide-mouth low-adsorption pipette tip to transfer all non-magnetic suspension, including the glass lysis beads, to another lysis tube with a nominal volume of 5 mL; retain approximately 50 μL of liquid in the first capture tube to cover the first portion of magnetic beads.

[0063] S6. Place the second lysis tube in a 4–10°C temperature control module for equilibration for 2 min, then oscillate at 6.5 m / s for 25 s to complete the second short-pulse mechanical lysis. The total time for the two mechanical actions is 45 s. Transfer the contents of the second lysis tube into a nylon lysis bead separator with a pore size of 40 μm, centrifuge at 500 × g for 30 s; rinse the separator with 50 μL of protective carrier liquid, and centrifuge again for 30 s. Adjust the obtained filtrate to 1150 μL with protective carrier liquid, and add it back to the first capture tube retaining the first portion of magnetic beads, so that the total liquid volume before termination of binding is restored to 1200 μL.

[0064] S7. Add 1200 μL of the termination binding working solution and mix at 800 r / min for 5 min. No further mechanical lysis is performed after this addition. In the resulting 2400 μL system, the concentrations of guanidine hydrochloride, total polyethylene glycol, sodium chloride, EDTA, Tris-HCl, and the total concentration of nucleic acid capturing magnetic beads were 2.0 mol / L, 8.0%, 0.60 mol / L, approximately 5.0 mmol / L, 15 mmol / L, and 1.0 mg / mL, respectively. The first batch of magnetic beads weighed 0.96 mg, and the remaining magnetic beads weighed 1.44 mg, representing 40% and 60% of the total bead mass, respectively.

[0065] S8. Place the first capture tube on a magnetic rack and let it stand for 2 min, then discard the supernatant. Add 800 μL of the first washing buffer to resuspend the magnetic beads, perform magnetic separation, and discard the washing buffer. Then add 800 μL of the second washing buffer and wash again. After the magnetic beads are left uncapped and dried for 2 min, add 60 μL of elution buffer, incubate at 60°C for 5 min, perform magnetic separation, and collect the elution buffer. The obtained DNA can be used for digital PCR, qPCR, or sequencing detection. Example 2

[0066] This embodiment discloses a method for the release, extraction, and detection of pathogen DNA in body fluid samples with a high host cell background. This embodiment uses a high host cell matrix, with a human leukocyte concentration of 1.0 × 10^6 cells / mL and a target DNA concentration of 1.0 × 10^4 copies / mL for each pathogen.

[0067] Add 300 μL of triple-concentrated host cell transient protection solution to 500 μL of sample and incubate at 25°C for 7 min; add 106.7 μL of first presensitization solution and incubate at 37°C for 6 min; then add 53.3 μL of second presensitization solution and incubate at 37°C for 4 min. The total volume of liquid at the end of presensitization is 960 μL, and the final concentration of each presensitization component is consistent with that in Example 1.

[0068] The stacked volume of the composite lysis beads was 0.8 times the volume of the pre-sensitized liquid. The first short pulse was applied with oscillation at 5.8 m / s for 20 s; 320 μL of the first portion of the magnetic bead capture working solution was added and allowed to stand for 60 s. The final concentrations of PEG 6000, sodium chloride, and the first portion of the magnetic beads in the resulting 1280 μL liquid phase were 6.0%, 0.45 mol / L, and 0.80 mg / mL, respectively. After magnetically retaining the first portion of the magnetic beads, the non-magnetic suspension was transferred to the second lysis tube and oscillated at 6.8 m / s for 25 s at 4–10 °C.

[0069] After the second lysis, the lysed beads were removed as in Example 1. Approximately 50 μL of liquid was retained in the first capture tube to cover the first portion of magnetic beads. The filtrate was adjusted to 1230 μL with a protective carrier solution and then added back, bringing the total volume before termination of binding to 1280 μL. 1280 μL of the termination binding working solution was added and mixed for 5 min. The masses of the first portion of magnetic beads and the remaining magnetic beads were 1.024 mg and 1.536 mg, respectively, accounting for 40% and 60% of the total mass of the magnetic beads. The remaining magnetic separation, washing, elution, and detection operations were the same as in Example 1. Example 3

[0070] This embodiment discloses a method for the release, extraction, and detection of pathogen DNA in high-protein, high-viscosity body fluid samples. 500 μL of simulated sputum sample containing 3.0% mucin and 5.0% bovine serum albumin was taken, and 500 μL of isotonic buffer containing 150 mmol / L sodium chloride and 10 mmol / L HEPES (pH 7.4) was added. The mixture was then slowly pipetted 8 times using a wide-mouth pipette tip. A 500 μL diluted sample was taken as the processing unit.

[0071] Host transient protection and two-stage presensitization were performed according to Example 1. The stacked volume of the composite lysis beads was adjusted to 1.0 times the volume of the presensitized liquid at the end of the process. The first short pulse was applied with oscillation at 5.8 m / s for 25 s, followed by the addition of the first portion of magnetic beads to capture the working fluid and allowing it to stand for 90 s. After the first portion of magnetic beads was magnetically retained, the non-magnetic suspension was transferred. The second short pulse was applied with oscillation at 6.8 m / s for 30 s. The total time for the two mechanical actions was 55 s.

[0072] After the second lysis, the sample was centrifuged at 600×g for 45 s using a lysis bead separator with a pore size of 40 μm, and then washed with 50 μL of protective carrier liquid. The filtrate adjustment and refill, termination of binding, magnetic separation, washing, elution, and detection procedures were the same as in Example 1.

[0073] Comparative Example 1 The only difference between this comparative example and Example 1 is that the host cell transient protection solution is not added, and 250 μL of isotonic buffer containing 20 mmol / L HEPES and 150 mmol / L sodium chloride is used instead of the three-fold concentrated host cell transient protection solution to maintain a consistent reaction volume; the remaining steps are the same as in Example 1.

[0074] Comparative Example 2 The only difference between this comparative example and Example 1 is that pathogen structure pre-sensitization is not performed, and 100 μL and 50 μL of protective carrier solution are used instead of the first and second pre-sensitization solutions, respectively; the remaining steps are the same as in Example 1.

[0075] Comparative Example 3 The only difference between this comparative example and Example 1 is that the two-stage presensitization is not used. The first presensitization solution and the second presensitization solution are added to the system obtained in step S1 at the same amount as in Example 1, and incubated together at 37°C and 300 r / min for 10 min. The remaining steps are the same as in Example 1.

[0076] Comparative Example 4 This comparative example employed continuous mechanical lysis followed by magnetic bead capture. After host transient protection and two-stage pre-sensitization, the system containing composite lysis beads was continuously oscillated at 6.1 m / s for 45 s without intermediate settling or immediate capture. After continuous lysis, the lysis beads were first removed using a 40 μm lysis bead separator, then 300 μL of the first portion of the magnetic bead capture working solution was added and allowed to stand for 180 s, followed by the addition of the binding termination working solution; the remaining operations were the same as in Example 1.

[0077] Comparative Example 5 The only difference between this comparative example and Example 1 is that the first batch of magnetic beads is not captured immediately after the first short pulse. After the two short pulses and the separation of the lysed beads are completed, all 2.40 mg of carboxyl magnetic beads are added to the liquid phase without lysed beads along with the termination binding solution; the rest of the operation is the same as in Example 1.

[0078] Comparative Example 6 This comparative example employs a host-first lysis and nuclease-removal route. 500 μL of the same simulated body fluid sample as in Example 1 was taken, and saponins were added to a final concentration of 0.05%, incubated at 25°C for 10 min. DNase I was added to a final concentration of 20 U / mL, and magnesium chloride was added to bring the magnesium ion concentration to a final concentration of 2 mmol / L, reacting at 37°C for 10 min. EDTA was added to a final concentration of 5 mmol / L to terminate the DNase I reaction. Subsequently, the process was performed according to the two-stage pre-sensitization, two short pulses, first-part magnetic bead retention, lysis bead removal, termination of binding, washing, and elution steps of Example 1.

[0079] Performance testing Unless otherwise stated, Examples 1-3 and Comparative Examples 1-6 were each performed in 3 independent replicates, and the continuous data in the table are average values. Low load detection tests were performed in 20 independent replicates for each concentration.

[0080] 1. Part One: Pipetting Performance of the Magnetic Bead Capture Working Fluid The first part of the magnetic bead capture working solution was equilibrated to 4℃, 15℃, 20℃, 25℃ and 32℃ respectively. 300 μL of working solution was taken with a pipette with a range of 20 to 1000 μL and a wide-mouth pipette tip. The volume recovery rate was calculated by weighing. The solution was pipetted 10 times at each temperature and the coefficient of variation was calculated based on the measured volume.

[0081] Table 1. Pipetting performance of the magnetic bead trapping working fluid (Part 1) 4℃ 95.1 4.2 The viscosity is high, and the suction cups leave obvious residue on the sides. 15℃ 97.4 2.5 For pipetting, reverse pipetting is recommended. 20℃ 98.6 1.7 Uniform flow, meeting operational requirements 25℃ 99.1 1.3 Stable operation, optimal temperature 32℃ 99.4 1.1 Good liquidity Table 1 shows that the working solution exhibits good pipetting accuracy and repeatability at 20–25°C. Therefore, in this embodiment, the first portion of the magnetic bead-captured working solution is pre-equilibrated to 20–25°C, and quantitative addition is performed using a wide-mouth pipette tip. After magnetically retaining the first portion of magnetic beads and transferring the non-magnetic suspension, the second pyrolysis tube is re-equilibrated at 4–10°C and subjected to a second short pulse, thus balancing the pipetting capability of the PEG-containing working solution with mechanical pyrolysis temperature control.

[0082] 2. Bonding properties of different magnetic bead surfaces by chemical properties Carboxyl magnetic beads, silicon magnetic beads, zwitterionic magnetic beads, and amino magnetic beads with an average particle size of 0.8–1.2 μm and a nominal nucleic acid binding capacity of 8–12 μg / mg were selected. The amount added was calculated based on the equivalent nominal nucleic acid binding capacity, while maintaining the same PEG, sodium chloride, pH, and treatment steps. The relative value of host nucleic acid co-capture was set to 1.00 for the carboxyl magnetic bead group.

[0083] Table 2. Bonding properties of different magnetic bead surfaces by chemical properties Carboxyl magnetic beads 91.2 1.00 Stable binding and thorough elution Silicon-based magnetic beads 86.7 1.08 With a high recovery rate, it can be used as an alternative. zwitterionic magnetic beads 82.3 0.94 Low host nonspecific adsorption Amino magnetic beads 75.8 1.17 The recovery rate is low under these combined conditions. Under conditions of equal particle size and nominal binding capacity, both carboxyl magnetic beads and silicon-based magnetic beads exhibited good DNA binding ability in the PEG-sodium chloride binding environment of the present invention, with carboxyl magnetic beads showing more stable recovery and elution performance.

[0084] 3. Pathogen DNA recovery rate and host DNA background Digital PCR was used to detect the copy number of target DNA from four types of pathogens, and the average pathogen DNA recovery rate was calculated. RPP30 digital PCR was used to convert the amount of host DNA released. The relative ratio of pathogen to host DNA was calculated as "average pathogen recovery rate of each group ÷ amount of host DNA released in each group", and then normalized to 1.00 using Example 1.

[0085] Table 3 Pathogen DNA Recovery Rate and Host DNA Background Example 1 88.3 126.4 1.00 9.4 Example 2 84.9 153.7 0.79 10.6 Example 3 81.8 142.9 0.82 12.1 Comparative Example 1 85.7 388.2 0.32 10.8 Comparative Example 2 61.5 119.1 0.74 19.6 Comparative Example 3 80.4 137.3 0.84 14.1 Comparative Example 4 81.9 347.4 0.34 16.7 Comparative Example 5 75.8 139.2 0.78 21.4 Comparative Example 6 78.1 74.3 1.50 24.8 Example 1 achieved a good overall balance between high pathogen DNA recovery rate, low host DNA release, and low recovery rate variability. Comparative Example 1, without transient host protection, showed a significantly increased host DNA release; Comparative Example 2, without pre-sensitization, showed a significantly decreased pathogen DNA recovery rate; Comparative Example 4, after continuous lysis before capture, showed increased host DNA release and coefficient of variation; Comparative Example 5, after canceling immediate capture, showed a decreased recovery rate and increased variability. Comparative Example 6 had the lowest host DNA release, resulting in a higher normalized pathogen / host DNA ratio, but its average pathogen recovery rate, inter-pathogen homogeneity, and reproducibility were all lower than in Example 1, and the amplification inhibition described later was more pronounced, indicating that a single ratio cannot replace a comprehensive evaluation of recovery rate, bias, reproducibility, and inhibition.

[0086] 4. Recovery bias of different pathogen structures DNA recoveries were calculated for Escherichia coli, Staphylococcus aureus, Candida albicans, and enveloped DNA virus-like particles, and structure-related recovery bias was characterized by the ratio of the highest to the lowest recoveries in the same group.

[0087] Table 4 DNA recovery rates of different pathogen structures Example 1 90.8 86.4 82.7 93.3 1.13 Example 2 87.2 82.3 78.4 91.7 1.17 Example 3 84.1 79.2 74.6 89.3 1.20 Comparative Example 2 70.6 48.3 35.7 91.4 2.56 Comparative Example 3 87.9 75.6 68.8 89.3 1.30 Comparative Example 6 84.7 69.8 58.3 99.6 1.71 Examples 1-3 showed relatively balanced recovery for the four types of pathogens with significant structural differences. Comparative Example 2 lacked pre-sensitization, resulting in a significant decrease in the recovery rate of Gram-positive bacteria and fungi; Comparative Example 3 added all pre-sensitization components simultaneously, and the recovery rate of fungi and Gram-positive bacteria was lower than that of Example 1; Comparative Example 6 showed high recovery of virus-like particles, but significantly low recovery of Candida albicans.

[0088] 5. Total DNA fragment integrity and amplification inhibition Capillary electrophoresis was used to detect the total DNA fragment length distribution in the elution buffer, and the N50 of the total DNA fragments and the proportion of fragments greater than 10 kb were calculated. This indicator is used to evaluate the overall degree of mechanical shearing and is not used alone as evidence of pathogen DNA specificity and integrity. An equal copy number of exogenous plasmid internal standard was added to equal volumes of elution buffer and nuclease-free water. The amplification inhibition ΔCt was calculated by subtracting the Ct value of the water control group from the Ct value of the elution buffer group.

[0089] Table 5 Total DNA fragment integrity and amplification inhibition Example 1 16.8 69.4 0.31 Example 2 14.1 61.8 0.44 Example 3 12.7 56.3 0.63 Comparative Example 1 11.2 47.5 0.35 Comparative Example 4 6.9 28.7 0.58 Comparative Example 5 8.4 37.2 0.71 Comparative Example 6 9.7 43.8 1.26 In Examples 1-3, the total DNA fragment integrity was high, and the amplification inhibition ΔCt was less than 0.7. In Comparative Example 4, the total DNA fragment N50 decreased significantly after continuous mechanical lysis; Comparative Example 6 showed high amplification inhibition, indicating that the residues of host lysis, nuclease treatment, and termination systems are more likely to affect subsequent amplification.

[0090] 6. Detection capability of low loading in different sample matrices Spiking gradients of 100, 50, 20, and 10 copies / mL were set up in low-cell, low-protein, high-host-cell, and high-protein, high-viscosity matrices, respectively. Each concentration point was tested 20 times independently, and the detection rate was calculated based on the number of positive pathogen-specific signals. The lowest spiked concentration with at least 19 / 20 positive results was defined as the lowest concentration required to achieve an empirical detection rate of at least 95% for this group.

[0091] Table 6. Detection capability of low loading in different sample matrices Low cell and low protein matrix 20 / 20 20 / 20 19 / 20 14 / 20 20 copies / mL High host cell matrix 20 / 20 20 / 20 18 / 20 11 / 20 50 copies / mL High-protein, high-viscosity matrix 20 / 20 19 / 20 17 / 20 9 / 20 50 copies / mL The core processing sequence of this invention can be used with different matrices. The minimum spiking concentration for achieving an empirical detection rate of at least 95% in low-cell, low-protein matrices is 20 copies / mL; for high-host-cell and high-protein, high-viscosity matrices, it is 50 copies / mL. This expression is based on directly observed repeated detection results and does not rely on an undisclosed probabilistic regression model.

[0092] 7. Batch repeatability Three batches of key reagents were prepared independently according to Example 1, and each batch was used to treat simulated body fluid samples of the same composition. The pathogen / host DNA ratio was calculated according to the formulas described in Table 3.

[0093] Table 7. Repeatability of different reagent batches Batch 1 88.3 126.4 1.00 Batch 2 86.9 132.7 0.94 Batch 3 89.1 123.8 1.03 The average pathogen DNA recovery rate, host DNA release amount, and relative ratio obtained from the three batches of reagents showed little difference, indicating that the processing procedure had good batch consistency after clarifying the stock solution concentration, added volume, magnetic bead mass distribution, and solid separation steps.

[0094] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for releasing, extracting, and detecting pathogen DNA from isolated body fluid samples, characterized in that, Includes the following steps: S1. Mix the ex vivo body fluid sample with the host cell transient preservation solution and incubate at 15–30°C for 2–10 min; the host cell transient preservation solution includes nonionic block polyether, osmotic pressure regulator, divalent metal salt and buffer salt; S2. Add the first presensitization solution to the reaction system obtained in step S1, incubate at 30-40°C for 3-12 min, then add the second presensitization solution prepared separately from the first presensitization solution, and continue incubating at 30-40°C for 2-8 min; the first presensitization solution includes bacterial wall-acting enzymes and fungal wall-acting enzymes, and the second presensitization solution includes proteinase K and nonionic surfactants. S3. Add composite pyrolysis beads to the reaction system obtained in step S2, and perform the first short-pulse mechanical pyrolysis at 4-20°C; the composite pyrolysis beads include small-diameter pyrolysis beads with a particle size of 0.08-0.20 mm and medium-diameter pyrolysis beads with a particle size of 0.30-0.80 mm. S4. After the first short-pulse mechanical lysis, the first part of nucleic acid capture magnetic beads and capture promotion solution are added to the reaction system. The system is allowed to stand for 30–180 s to allow the released pathogen DNA to bind to the first part of the nucleic acid capture magnetic beads. The amount of capture promotion solution added is such that the final concentration of PEG 6000 in the reaction system is 4%–8%, the final concentration of sodium chloride is 0.3–0.8 mol / L, the final concentration of Tris-HCl is 8–20 mmol / L, the pH is 7.8–8.3, and the concentration of the first part of the nucleic acid capture magnetic beads is 0.5–1.5 mg / mL. After standing, the first part of the nucleic acid capture magnetic beads are retained in the first capture container using a magnetic field, and the non-magnetic suspension, including the composite lysis beads, is transferred to the second lysis container. S5. The non-magnetic suspension transferred to the second lysis container is subjected to a second short-pulse mechanical lysis. The oscillation speed of the second short-pulse mechanical lysis is higher than that of the first short-pulse mechanical lysis. The two short-pulse mechanical lysis processes last for 10 to 40 seconds, and the total mechanical action time does not exceed 60 seconds. After the second short-pulse mechanical lysis is completed, the composite lysis beads are removed by using a lysis bead separator with a pore size of 20 to 60 μm or by low-speed centrifugation. The liquid phase without composite lysis beads is collected and added back to the first capture container. S6. Add the termination binding solution and the remaining nucleic acid capturing magnetic beads to the first capture container and mix for 2–10 min. The amount of the termination binding solution added is such that the final concentration of guanidine hydrochloride in the resulting system is 1.5–3.0 mol / L, the final concentration of PEG 8000 is 4%–8%, the final concentration of sodium chloride is 0.3–1.0 mol / L, the final concentration of EDTA is 2–8 mmol / L, and the total concentration of all nucleic acid capturing magnetic beads is 0.7–2.0 mg / mL. The first part of the nucleic acid capturing magnetic beads accounts for 20%–60% of the total mass of the nucleic acid capturing magnetic beads, and no further mechanical lysis is performed after the termination binding solution is added. S7. Perform magnetic separation on the system obtained in step S6, collect nucleic acid capture magnetic beads bound with DNA, wash and elute the nucleic acid capture magnetic beads to obtain pathogen DNA. The obtained pathogen DNA was subjected to in vitro nucleic acid signal detection using PCR, digital PCR, or sequencing methods.

2. The method according to claim 1, characterized in that, The nonionic block polyether is poloxamer 188; the osmotic pressure regulator includes trehalose and sorbitol; the divalent metal salt includes magnesium salt and calcium salt; the buffer salt is HEPES; in the reaction system formed in step S1, the final concentration of poloxamer 188 is 0.02%–0.30%, the final concentration of trehalose is 20–120 mmol / L, the final concentration of sorbitol is 20–100 mmol / L, the final concentration of magnesium ions is 0.5–5 mmol / L, the final concentration of calcium ions is 0.1–2 mmol / L, the final concentration of HEPES is 10–50 mmol / L, and the pH is 7.2–8.

0.

3. The method according to claim 1 or 2, characterized in that, The bacterial wall-activating enzymes include lysozyme and mutalysin, and the fungal wall-activating enzyme is β-glucanase. The first pre-sensitization solution also includes EDTA. In the reaction system at the end of step S2, the final concentration of lysozyme is 0.1–2.0 mg / mL, the final concentration of mutalysin is 10–200 U / mL, the final concentration of β-glucanase is 0.05–1.0 mg / mL, the final concentration of proteinase K is 0.02–0.30 mg / mL, the final concentration of nonionic surfactant is 0.005%–0.08%, and the final concentration of EDTA is 0.05–1.0 mmol / L.

4. The method according to any one of claims 1 to 3, characterized in that, The small-diameter pyrolysis beads and medium-diameter pyrolysis beads are each independently selected from one or more of glass beads, zirconia beads, and ceramic beads; the mass ratio of small-diameter pyrolysis beads to medium-diameter pyrolysis beads is 1:0.25 to 1:1.5, and the packing volume of the composite pyrolysis beads is 0.3 to 1.5 times the volume of the reaction liquid before mechanical pyrolysis; the first short-pulse mechanical pyrolysis is performed at an oscillation speed of 4 to 7 m / s calibrated by the equipment for 10 to 30 s, and the second short-pulse mechanical pyrolysis is performed at an oscillation speed 0.5 to 2.0 m / s higher than that of the first short-pulse mechanical pyrolysis for 10 to 40 s; Mechanical pyrolysis uses a pyrolysis vessel with a nominal volume of not less than 5 mL, and the total volume of the liquid and pyrolysis beads does not exceed 60% of the nominal volume of the pyrolysis vessel.

5. The method according to any one of claims 1 to 4, characterized in that, The nucleic acid capturing magnetic beads are carboxyl magnetic beads and / or silicon-based magnetic beads with an average particle size of 0.5 to 2.0 μm; the first part of the nucleic acid capturing magnetic beads accounts for 35% to 45% of the total mass of the nucleic acid capturing magnetic beads; after the first part of the nucleic acid capturing magnetic beads has completed static binding, it is attached to the side wall of the first capture container under the action of a magnetic field, and 30 to 100 μL of liquid is retained on the surface of the first part of the nucleic acid capturing magnetic beads to prevent the magnetic beads from drying out.

6. The method according to any one of claims 1 to 5, characterized in that, After the second short-pulse mechanical lysis, the reaction system is centrifuged at 300–800 × g for 20–60 s through a lysis bead separator with a pore size of 40 μm, and then rinsed with 30–100 μL of protective carrier solution. In step S7, the nucleic acid capture magnetic beads are washed sequentially with a first washing buffer containing ethanol and a second washing buffer containing low-salt ethanol, and then eluted with 10 mmol / L Tris-HCl buffer or nuclease-free water at 50–70 °C for 3–10 min. The ex vivo body fluid sample is selected from one of the following: blood, cerebrospinal fluid, urine, pleural effusion, ascites, bronchoalveolar lavage fluid, sputum, pus, and genital tract secretions. The pathogen is selected from one or more of bacteria, fungi, and enveloped DNA viruses.

7. A pathogen DNA release and extraction combination reagent for carrying out the method according to any one of claims 1 to 6, characterized in that, The device includes separately configured host cell transient protection solution, first presensitization solution, second presensitization solution, capture-promoting base solution, termination binding base solution, nucleic acid capture magnetic beads, composite lysis beads, and lysis bead separation element with a pore size of 20–60 μm. The host cell transient protection solution includes poloxamer 188, trehalose, sorbitol, magnesium salt, calcium salt, and HEPES. The first presensitization solution includes lysozyme, mutalysin, β-glucanase, and EDTA. The second presensitization solution includes proteinase K and a nonionic surfactant. The capture-promoting base solution includes PEG 6000, sodium chloride, and Tris-HCl. The termination binding base solution includes guanidine hydrochloride, PEG 8000, sodium chloride, EDTA, and Tris-HCl.

8. The combined reagent according to claim 7, characterized in that, The host cell transient preservation solution is a triple-concentrated solution comprising 0.30% poloxamer 188, 180 mmol / L trehalose, 120 mmol / L sorbitol, 6 mmol / L magnesium chloride, 1.5 mmol / L calcium chloride, and 60 mmol / L HEPES, with a pH of 7.

6. The host cell transient preservation solution is used at a ratio of 250 μL per 500 μL of ex vivo body fluid sample.

9. The combined reagent according to claim 7 or 8, characterized in that, The first presensitization solution comprises 4.5 mg / mL lysozyme, 450 U / mL mutalycin, 1.8 mg / mL β-glucanase, and 1.8 mmol / L EDTA; the second presensitization solution comprises 1.44 mg / mL proteinase K and 0.36% polysorbate 20; both the first and second presensitization solutions use a protective carrier solution as a solvent, which comprises 0.10% poloxamer 188, 60 mmol / L trehalose, 40 mmol / L sorbitol, 2 mmol / L magnesium chloride, 0.5 mmol / L calcium chloride, and 20 mmol / L HEPES, with a pH of 7.6; the addition volumes of the first and second presensitization solutions are 100 μL and 50 μL, respectively, per 500 μL of ex vivo body fluid sample.

10. The combined reagent according to any one of claims 7 to 9, characterized in that, The capture-enhancing base solution comprises 25.64% PEG 6000, 1.923 mol / L sodium chloride, and 42.74 mmol / L Tris-HCl, with a pH of 8.

0. Based on 500 μL of ex vivo body fluid sample, 280.8 μL of the capture-enhancing base solution is mixed with 19.2 μL of a concentrated suspension of carboxylated magnetic beads with a magnetic solid concentration of 50 mg / mL to obtain 300 μL of the first portion of the magnetic bead capture working solution. The binding termination base solution comprises 4.10 mol / L guanidine hydrochloride, 10.25% PEG 8000, 0.768 mol / L sodium chloride, 10.09 mmol / L EDTA, and 20.49 mmol / L Tris-HCl, with a pH of 7.

8. 1171.2 μL of the binding termination base solution is mixed with 28.8 μL of the concentrated suspension of carboxylated magnetic beads to obtain 1200 μL of the binding termination working solution.

Images