Rapid extraction and identification method of pathogen nucleic acid in blood culture positive product

Abstract

The invention provides a method for rapidly extracting and identifying pathogen nucleic acid in a blood culture positive product. The method comprises the following steps: acquiring a blood culture positive culture solution as a to-be-treated sample; carrying out differential settling separation on the to-be-treated sample to remove interfering substances so as to obtain a primary separation solution; performing physical wall breaking treatment on the primary separation liquid to obtain wall breaking treatment liquid; performing high-temperature cracking treatment on the wall-breaking treatment liquid to obtain cracking treatment liquid; performing high-speed centrifugal treatment on the cracking treatment liquid to obtain target nucleic acid in supernate; the target nucleic acid is amplified and analyzed through a digital polymerase chain reaction technology, and the identification result and quantitative data of the pathogen are determined. According to the invention, the sensitivity, the specificity and the accuracy of pathogen detection in the blood culture positive sample are obviously improved, and an important basis is provided for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of information technology, and in particular to a method for rapidly extracting and identifying pathogen nucleic acids in positive blood culture products. Background Art

[0002] Rapid identification and quantification of pathogens in positive blood culture samples is crucial for clinical diagnosis and treatment, but traditional methods present numerous challenges. Primarily, the presence of numerous interfering substances in the sample, such as complex culture media, polymer beads, red blood cells, and white blood cells, severely impacts the purity and efficiency of nucleic acid extraction. Secondly, the structural diversity of different pathogens makes it difficult for a single lysis method to effectively destroy all pathogens and release nucleic acids. Furthermore, the pathogen concentration in blood culture samples is often low, making conventional PCR difficult to accurately quantify. These factors result in insufficient sensitivity, specificity, and accuracy, failing to meet clinical needs. A further challenge lies in how to rapidly and efficiently isolate and enrich target pathogens in the complex blood matrix while ensuring nucleic acid integrity and purity. Furthermore, establishing a universal sample pretreatment process that is applicable to a wide range of pathogens and seamlessly integrates with subsequent molecular detection techniques is an urgent challenge. These interrelated technical challenges collectively constitute a systemic challenge for the rapid and accurate detection of pathogens in positive blood culture samples. Summary of the Invention

[0003] The present invention provides a method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products, which mainly includes:

[0004] Obtain blood culture-positive culture fluid as a sample to be processed; perform differential sedimentation separation on the sample to be processed to remove interfering substances to obtain a primary separation fluid; perform physical wall-breaking treatment on the primary separation fluid to obtain a wall-breaking treatment fluid; perform high-temperature pyrolysis treatment on the wall-breaking treatment fluid to obtain a pyrolysis treatment fluid; perform high-speed centrifugation on the pyrolysis treatment fluid to obtain a target nucleic acid in the supernatant; amplify and analyze the target nucleic acid using digital polymerase chain reaction technology to determine the identification results and quantitative data of the pathogen.

[0005] Furthermore, the sample to be processed is subjected to differential sedimentation separation to remove interfering substances, including: placing the sample to be processed in a centrifugal device, allowing cells and large particle impurities to settle through low-speed centrifugal force, and retaining the supernatant as the primary separation liquid; removing interfering components of the culture medium in the sample to be processed through the differential sedimentation separation to ensure the purity of nucleic acid extraction in subsequent processing; if visible impurities still exist in the primary separation liquid, repeating the low-speed centrifugation step until the clarified primary separation liquid is obtained; determining whether to directly enter the subsequent physical wall breaking treatment based on the clarity of the primary separation liquid; through the above steps, ensuring that the primary separation liquid is suitable for the subsequent nucleic acid extraction process.

[0006] Furthermore, the physical wall-breaking treatment of the primary separation liquid includes: transferring the primary separation liquid into a container containing a wall-breaking medium, wherein the wall-breaking medium is particles in a specific diameter range; fully mixing the primary separation liquid and the wall-breaking medium through a vortex oscillation device, so that the wall-breaking medium generates kinetic energy during the oscillation process, destroys the cell wall of the pathogen, and releases internal nucleic acid substances; controlling the wall-breaking effect according to the oscillation time and oscillation intensity to avoid excessive shearing of nucleic acids; if the wall-breaking effect does not meet expectations, adjusting the oscillation parameters and reprocessing; through the above treatment, a uniformly dispersed wall-breaking treatment liquid is obtained, providing conditions for subsequent high-temperature lysis.

[0007] Furthermore, the high-temperature lysis treatment of the wall-breaking treatment liquid includes: placing the wall-breaking treatment liquid in a preheated high-temperature device, and causing the protein in the pathogen to denature, the membrane structure to melt, and the nucleic acid secondary structure to melt through the action of high temperature; optimizing the lysis effect according to the time and temperature parameters of the high-temperature treatment to ensure that the protein is inactivated while the nucleic acid integrity is not damaged; if the wall-breaking treatment liquid precipitates after high-temperature treatment, it is remixed after a short cooling; through the above-mentioned high-temperature lysis step, the lysis treatment liquid with fully released nucleic acid is obtained, providing a basis for subsequent centrifugation treatment.

[0008] Furthermore, the high-speed centrifugation treatment of the lysis treatment liquid includes: placing the lysis treatment liquid in a high-speed centrifuge device, and separating the lysis residue and the nucleic acid solution by high-speed rotation; controlling the separation effect according to the centrifugation speed and time parameters to ensure that the supernatant contains the target nucleic acid with high purity; if impurities are still detected in the supernatant, repeating the high-speed centrifugation step; through the above treatment, obtaining the target nucleic acid suitable for subsequent amplification; and determining whether to directly enter the digital polymerase chain reaction technology analysis process based on the purity of the target nucleic acid.

[0009] Furthermore, the amplification and analysis of the target nucleic acid using digital polymerase chain reaction technology includes: mixing the target nucleic acid with a preconfigured reaction solution to form a system to be amplified; preparing the system to be amplified into microdroplets using a microfluidic device to ensure that the nucleic acid molecules are independently distributed in the microdroplets; performing polymerase chain reaction amplification on the microdroplets, and completing the amplification process according to preset temperature cycle parameters; performing signal detection on the amplified microdroplets using a chip scanning device to obtain fluorescent signal data; and determining the identification results and quantitative data of the pathogen based on the fluorescent signal data.

[0010] Furthermore, the amplification and analysis of the target nucleic acid by digital polymerase chain reaction technology includes: configuring a primer and probe combination for specific pathogen detection for the target nucleic acid to ensure the specificity of the detection; packaging the target nucleic acid into a microfluidic chip through a preset reaction system to form a uniform microdroplet system; performing multiple rounds of temperature cycle amplification on the microdroplet system according to the parameter settings of the amplification instrument; performing multi-channel fluorescence scanning on the amplified microdroplet system through a signal acquisition device to obtain pathogen-specific signals; and analyzing the type of the pathogen and the absolute quantitative results based on the pathogen-specific signals.

[0011] The technical solution provided by the embodiment of the present invention may have the following beneficial effects:

[0012] The present invention discloses a method for rapid identification and quantification of pathogens from positive blood culture samples. The method removes interfering substances by differential sedimentation separation, physically breaks the cell wall to release nucleic acids, performs high-temperature pyrolysis treatment, extracts target nucleic acids by high-speed centrifugation, and finally uses digital PCR technology for amplification analysis. The present invention solves the key problems of pathogen detection in blood culture samples: first, the purity and efficiency of nucleic acid extraction are improved through multi-step pretreatment; second, a combination of physical cell wall breaking and high-temperature pyrolysis is used to effectively destroy the pathogen structure and release nucleic acids; finally, digital PCR technology is used to achieve accurate identification and absolute quantification of pathogens. The present invention significantly improves the sensitivity, specificity and accuracy of pathogen detection in positive blood culture samples, providing an important basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The present invention provides a flow chart of a method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products. DETAILED DESCRIPTION

[0014] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0015] like Figure 1In this embodiment, a method for rapid extraction and identification of pathogen nucleic acid in a positive blood culture product may specifically include:

[0016] Step S101 : obtaining a positive culture fluid of a blood culture as a sample to be processed; performing differential sedimentation separation on the sample to be processed to remove interfering substances, and obtaining a primary separation fluid.

[0017] When obtaining positive blood culture fluid as a sample to be processed, the representativeness and pretreatment requirements of the sample must be considered.

[0018] For example, when extracting 500 μL of positive culture fluid from a clinical blood culture bottle, it is necessary to avoid mixing in blood cell fragments or fibrin clots. The supernatant can be aspirated after sufficient mixing to allow large particles to settle naturally. The key to this step is to ensure sample homogeneity to avoid clogging of the microfluidic chip channel due to particulate matter during subsequent separation. The core of differential sedimentation separation is to remove interfering substances by gradient centrifugation. In specific implementation, the sample is centrifuged at 500 × g for 2 minutes. Low-speed centrifugation can retain pathogens such as bacteria in the supernatant, while high-density interfering substances such as polymer adsorption beads and peptone / biological extract mixtures in the culture medium are precipitated to the bottom of the tube.

[0019] For example, after this treatment, the pathogen recovery rate of Klebsiella pneumoniae culture supernatant can reach over 90%, and the residual fluorescent substance is reduced to below the detection limit. The acquisition of primary separation fluid requires the optimization of centrifugation parameters. If the interfering substances in the sample are lipids (such as the oily additives in the BC bottle), the centrifugal force can be adjusted to 800×g and extended to 5 minutes to allow the lipid droplets to fully settle. Experimental data show that under these conditions, the purity of Pseudomonas aeruginosa nucleic acid extraction (A260 / A280) increased from 1.2 to 1.8, proving that differential sedimentation effectively removes lipid interference. For high-viscosity samples (such as polysaccharide-containing Acinetobacter baumannii culture fluid), an equal volume of nucleic acid extraction buffer (such as 10mM Tris-HCl) can be added before differential sedimentation to dilute the sample, reduce the viscosity, and then centrifuge. Comparative experiments show that the bacterial nucleic acid yield of the diluted group is 35% higher than that of the undiluted group, and the success rate of droplet generation for subsequent dPCR amplification is increased from 70% to 95%. The technical effects of differential sedimentation are reflected in two aspects: first, the removal of anticoagulants such as heparin in the BC culture medium that inhibit PCR. For example, after centrifugation at 300×g for 3 minutes, the heparin concentration drops to below 0.1 IU / mL; second, the integrity of the target pathogen is preserved. The survival rate of Escherichia coli remains at 98% after low-speed centrifugation, avoiding the impact of nucleic acid degradation on quantitative results.

[0020] Step S102, performing physical cell wall breaking treatment on the primary separation liquid to obtain cell wall breaking treatment liquid; performing high temperature cracking treatment on the cell wall breaking treatment liquid to obtain cracking treatment liquid.

[0021] The primary separation liquid is subjected to a physical cell wall breaking treatment to obtain a cell wall breaking treatment liquid. Physical cell wall breaking treatment is to destroy the cell wall or cell membrane by mechanical force or ultrasonic wave, so that the intracellular substances are released into the solution.

[0022] For example, using an ultrasonic disruptor, the primary separation liquid is placed under an ultrasonic probe, the frequency is set to 20kHz, the power is 200W, and the treatment time is 5min. The cell wall is broken by the cavitation effect and shear force of the ultrasound, releasing substances such as nucleic acids and proteins in the cell. The key to physical wall breaking treatment is to select the appropriate energy and time, which can effectively destroy the cell structure without excessively damaging the target molecules. The wall breaking treatment liquid is subjected to high-temperature pyrolysis treatment to obtain a pyrolysis treatment liquid. The high-temperature pyrolysis treatment is to denature the proteins in the cell by heating, and the nucleic acids are released into the solution.

[0023] For example, the cell wall-breaking solution is placed in a 95°C water bath and heated for 10 minutes to denature intracellular proteins and dissociate nucleic acids from protein complexes. The temperature and duration of the high-temperature lysis treatment need to be adjusted based on the stability of the target molecule to ensure the integrity and amplification of the nucleic acids. The advantages of high-temperature lysis treatment are its ease of use and rapid release of nucleic acids, making it suitable for subsequent molecular biology experiments such as PCR amplification.

[0024] Step S103 , performing high-speed centrifugation on the lysis treatment solution to obtain the target nucleic acid in the supernatant; amplifying and analyzing the target nucleic acid by digital polymerase chain reaction technology to determine the identification result and quantitative data of the pathogen.

[0025] The lysis solution is subjected to high-speed centrifugation to obtain the target nucleic acid in the supernatant. High-speed centrifugation is a process that uses the centrifugal force generated by the high-speed rotation of the centrifuge to separate impurities such as cell debris and proteins in the lysis solution from the target nucleic acid. The centrifugation conditions are usually set to 500×g and the centrifugation time is 2 minutes to ensure that impurities are precipitated at the bottom of the tube, while the target nucleic acid is retained in the supernatant. The key to this step is the selection of centrifugal force. Too high a centrifugal force may cause the nucleic acid to break, while too low a centrifugal force cannot effectively separate impurities. By optimizing the centrifugation conditions, the integrity and purity of the target nucleic acid can be ensured, providing a high-quality template for subsequent amplification and analysis. The target nucleic acid is amplified and analyzed using digital polymerase chain reaction technology to determine the identification results and quantitative data of the pathogen. Digital polymerase chain reaction (dPCR) is a highly sensitive nucleic acid quantification technology. Its core lies in dividing the PCR reaction system into thousands to millions of microdroplets, each of which contains only one or a few nucleic acid molecules. Through independent PCR amplification, the copy number of the target nucleic acid can be accurately calculated, thereby achieving absolute quantification. In the specific operation, the target nucleic acid in the supernatant is first mixed with the dPCR premix, and then the mixture is divided into microdroplets by a microdroplet generator. After PCR amplification, the nucleic acid molecules in each microdroplet are quantitatively analyzed by a fluorescent signal detection system. The advantage of dPCR technology lies in its high sensitivity and accuracy, which can detect extremely low concentrations of pathogen nucleic acids. The generation of microdroplets by this technology has high requirements for the purity of nucleic acids. The nucleic acid extracted from blood culture fluid by the currently commonly used magnetic bead method cannot be used for this technology. Determine the identification results and quantitative data of the pathogen. After amplification and analysis by dPCR technology, the type and quantity of the target pathogen can be determined based on the intensity and distribution of the fluorescent signal.

[0026] For example, specific primers and probes can be used to amplify common pathogens such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae, enabling rapid identification of pathogens. Furthermore, dPCR technology can provide absolute quantitative data on pathogens, such as the number of pathogen copies per microliter of sample, providing an accurate basis for clinical diagnosis and treatment.

[0027] For example, in the diagnosis of bloodstream infections (BSIs), dPCR technology can identify and quantify pathogens within 2 hours, significantly shortening the 36-48 hours required by traditional blood culture methods and providing a valuable window for timely treatment. However, dPCR technology is currently only used to test patient plasma samples and is not applicable to blood culture specimens with a single vial of blood, which can reach up to 5-10 mL. Blood culture testing, as the gold standard for BSI diagnosis, provides a suitable nutritional and culture environment for common clinical microorganisms, but it has significant limitations, such as a low positive rate. Establishing a nucleic acid extraction method that enables dPCR to be applied to blood culture specimens is of great value. Optimizing the lysis solution and centrifugation conditions improves nucleic acid extraction efficiency. The lysis solution typically contains a physical lysis agent (such as glass beads) and a chemical lysis agent (such as proteinase K). The synergistic effect of physical disruption and chemical lysis efficiently releases pathogen nucleic acids. Glass beads typically range in diameter from 0.1 mm to 0.8 mm. High-speed oscillation or vortexing effectively disrupts cell walls and membranes, releasing nucleic acids. Chemical lysers, through enzymatic hydrolysis, further degrade proteins and lipids, improving nucleic acid purity. By optimizing the composition of the lysis solution and centrifugation conditions, the efficiency and quality of nucleic acid extraction can be significantly improved, providing a reliable template for subsequent dPCR amplification and analysis. Combined with dPCR technology, rapid pathogen identification and quantification are possible. The high sensitivity and accuracy of dPCR technology make it an ideal tool for pathogen detection. Combining dPCR with optimized nucleic acid extraction methods allows for rapid identification and quantification of pathogens in blood culture fluids.

[0028] For example, in the detection of pathogens in culture fluid from BioMérieux's BacT / ALERT 3D blood culture bottles, physical and chemical dual lysis and high-speed centrifugation enable efficient extraction of pathogen nucleic acids. These nucleic acids are then amplified and analyzed using dPCR, rapidly obtaining pathogen identification and quantitative data. This technological approach not only shortens detection time but also improves sensitivity and accuracy, providing new insights and processes for the diagnosis and treatment of patients with bloodstream infections.

[0029] Step S104, performing differential sedimentation separation on the sample to be processed to remove interfering substances, including: placing the sample to be processed in a centrifugal device, allowing cells and large particle impurities to settle through low-speed centrifugal force, and retaining the supernatant as the primary separation liquid; removing interfering components of the culture medium in the sample to be processed through the differential sedimentation separation to ensure the purity of nucleic acid extraction in subsequent processing; if visible impurities still exist in the primary separation liquid, repeating the low-speed centrifugation step until the clarified primary separation liquid is obtained; determining whether to directly enter the subsequent physical wall breaking treatment based on the clarity of the primary separation liquid; through the above steps, ensuring that the primary separation liquid is suitable for the subsequent nucleic acid extraction process.

[0030] The sample to be processed is placed in a centrifuge device, and the cells and large particle impurities are settled by low-speed centrifugal force, and the supernatant is retained as the primary separation liquid. Low-speed centrifugation usually uses a centrifugal force of 500×g and a centrifugation time of 2 minutes. Such conditions can effectively separate cells and large particle impurities while retaining the nucleic acid components in the supernatant. The purpose of low-speed centrifugation is to use gravity to allow cells and impurities with higher density to settle to the bottom of the centrifuge tube, while the supernatant is retained in the upper part of the centrifuge tube for subsequent processing. Differential sedimentation separation is used to remove interfering components of the culture medium in the sample to be processed, ensuring the purity of nucleic acid extraction in subsequent processing. Differential sedimentation separation is a separation method based on the difference in sedimentation speeds of different substances. By adjusting the centrifugal force and centrifugation time, interfering substances in the sample can be gradually removed.

[0031] For example, at a centrifugal force of 500 × g, cells and large impurities will settle first, while smaller interfering substances may remain suspended in the supernatant. Repeated low-speed centrifugation can gradually remove these interfering substances, ensuring the clarity of the supernatant. If visible impurities still exist in the primary fraction, repeat the low-speed centrifugation step until a clear primary fraction is obtained. Repeated low-speed centrifugation is intended to further remove fine impurities from the supernatant and ensure the clarity of the primary fraction.

[0032] For example, if there is still a small amount of suspended matter in the supernatant after the first centrifugation, it can be centrifuged again at a centrifugal force of 500×g for 2 minutes until the supernatant is completely clarified. This step is crucial to the purity of subsequent nucleic acid extraction, because the presence of impurities may affect the extraction efficiency of nucleic acids and the accuracy of subsequent detection. Depending on the degree of clarity of the primary separation liquid, determine whether to directly enter the subsequent physical wall breaking treatment. The degree of clarity of the primary separation liquid is an important indicator for determining whether further processing is needed. If the primary separation liquid is clear enough, it can directly enter the physical wall breaking treatment step; if there are still many impurities, the low-speed centrifugation step needs to be repeated.

[0033] For example, visual inspection or spectrophotometry of the supernatant's turbidity can be used to determine its clarity and determine whether to proceed to the next step. These steps ensure that the primary separation fluid is suitable for subsequent nucleic acid extraction. The clarity and purity of the primary separation fluid directly impact the effectiveness of subsequent nucleic acid extraction. Low-speed centrifugation and differential sedimentation separation effectively remove interfering substances from the sample, ensuring the purity of the primary separation fluid, thereby improving the efficiency of nucleic acid extraction and the accuracy of subsequent testing.

[0034] For example, in the subsequent physical cell wall breaking treatment, the pure primary separation liquid can better release nucleic acids, reduce the interference of impurities on nucleic acid extraction, and improve the sensitivity and specificity of detection.

[0035] Step S105, the physical wall-breaking treatment of the primary separation liquid includes: transferring the primary separation liquid into a container containing a wall-breaking medium, wherein the wall-breaking medium is particles in a specific diameter range; fully mixing the primary separation liquid and the wall-breaking medium through a vortex oscillation device, so that the wall-breaking medium generates kinetic energy during the oscillation process, destroys the cell wall of the pathogen, and releases the internal nucleic acid material; controlling the wall-breaking effect according to the oscillation time and oscillation intensity to avoid excessive shearing of the nucleic acid; if the wall-breaking effect does not meet the expectations, adjusting the oscillation parameters and reprocessing; through the above treatment, a uniformly dispersed wall-breaking treatment liquid is obtained, which provides conditions for subsequent high-temperature lysis.

[0036] Selection and mechanism of action of cell wall breaking media: Cell wall breaking media are usually sterile glass beads with a diameter of 0.1mm to 0.8mm. The size difference between the beads generates shear force through physical collision.

[0037] For example, 0.5mm glass beads collide with pathogen cell walls at high frequencies due to inertia during vortex oscillation, destroying the cell wall's phospholipid bilayer structure through kinetic energy transfer, while 0.1mm glass beads increase local pressure by increasing the contact area. The synergistic effect of the two sizes can cover cell wall types of different strengths (such as the thick peptidoglycan layer of Gram-positive bacteria or the chitin wall of fungi). Fine control of vortex oscillation parameters. The oscillation intensity is usually set to 2000rpm to 3000rpm, and the time is controlled between 30 seconds and 5 minutes.

[0038] For example, for Pseudomonas aeruginosa (with thin cell walls), a 90% cell wall breaking rate can be achieved by oscillating at 2500 rpm for 1 minute; while for methicillin-resistant Staphylococcus aureus (MRSA), the oscillation speed needs to be increased to 3000 rpm for 1.5 minutes. The vortex morphology of the liquid needs to be monitored during the oscillation process to avoid the formation of bubbles and the loss of kinetic energy. Dynamic evaluation of the cell wall breaking effect and parameter adjustment. If the electron microscopy shows that the cell wall residual rate is greater than 20%, the oscillation time needs to be increased step by step (increase by 15 seconds each time) or the intensity (increase by 200 rpm each time).

[0039] For example, when the concentration of Klebsiella pneumoniae sample after initial treatment is lower than 50ng / μL by nucleic acid quantification instrument, it needs to be oscillated a second time and supplemented with 0.3mm glass beads to enhance mechanical force. Nucleic acid integrity protection strategy. By limiting the duration of a single oscillation to no more than 3 minutes, DNA breakage to less than 500bp can be prevented. Experimental data show that when the oscillation intensity exceeds 3500rpm, the detection rate of 16SrRNA gene fragments decreases by 40%, so it is necessary to balance the wall breaking efficiency and nucleic acid integrity. Homogenization requirements for wall breaking treatment liquid. The final liquid must be observed under an optical microscope to have no visible clumps, and the particle size distribution detected by dynamic light scattering is concentrated in the range of 100-300nm.

[0040] For example, if particles larger than 1μm are present in the Acinetobacter baumannii treatment solution, an additional 5-second pulse oscillation is required to re-disperse the glass beads. This design is synergistic with high-temperature lysis. The micron-sized cell fragments formed after physical cell wall disruption increase the contact area with subsequent chemical lysis agents.

[0041] For example, when samples treated with glass beads were lysed at 95°C, the efficiency of proteinase K was three times higher than that of untreated samples, shortening the lysis time to 10 minutes.

[0042] Step S106, the wall-breaking treatment liquid is subjected to high-temperature lysis treatment, including: placing the wall-breaking treatment liquid in a preheated high-temperature device, denaturing the protein in the pathogen, melting the membrane structure, and unzipping the nucleic acid secondary structure through the action of high temperature; optimizing the lysis effect according to the time and temperature parameters of the high-temperature treatment to ensure that the protein is inactivated while the nucleic acid integrity is not damaged; if the wall-breaking treatment liquid precipitates after the high-temperature treatment, it is remixed after a short cooling; through the above-mentioned high-temperature lysis step, the lysis treatment liquid with fully released nucleic acids is obtained, providing a basis for subsequent centrifugation treatment.

[0043] The core principle of high-temperature lysis treatment is to destroy the pathogen cell structure through thermodynamics. After the high-temperature equipment is preheated to 95°C, the wall-breaking treatment liquid is put in. At this time, the high temperature breaks the hydrogen bonds of the pathogen membrane protein and melts the lipid bilayer. For example, the cell membrane of Pseudomonas aeruginosa will form pores above 90°C, allowing nucleic acids to be released. The temperature parameters must be strictly controlled at 95°C ± 2°C for 5 minutes. This can ensure protein denaturation (such as the dissociation of the 60S subunit of the bacterial ribosome) and avoid nucleic acid chain breakage (DNA is more stable below 100°C). Optimizing the lysis effect requires balancing the relationship between temperature and time. Experiments have shown that if the temperature is lower than 90°C (such as 85°C), the peptidoglycan layer of Klebsiella pneumoniae cannot be completely degraded, resulting in a 30% decrease in the nucleic acid release rate; when it exceeds 100°C (such as 105°C), the DNA of Escherichia coli will be significantly degraded. Through gradient testing, it was found that treatment at 95°C for 5 minutes can achieve the best balance between nucleic acid integrity (assessed by electrophoresis band clarity) and protein inactivation rate (BCA method detected residual protein <5%). The operation of remixing after brief cooling is aimed at the precipitation problem. After high-temperature lysis, some denatured proteins (such as bacterial outer membrane protein OmpA) may aggregate and precipitate. At this time, the sample is quickly placed on ice to cool for 30 seconds, and then vortexed for 10 seconds to redisperse the precipitate. For example, after this treatment of Acinetobacter baumannii lysate, the copy number difference detected by dPCR was reduced from ±15% to ±5%, indicating that the mixing uniformity was significantly improved. The adequacy of nucleic acid release directly affects the subsequent centrifugation efficiency. After high-temperature lysis, the lysate should be slightly turbid but without large particles (by visual inspection or OD600 <0.1), and the nucleic acid is free in the liquid phase. For example, compared with samples that have not been treated with high temperature, the nucleic acid concentration in the supernatant of the BC-positive culture fluid lysed at 95°C increased by 2.5 times after centrifugation (Qubit test), proving that the high temperature step can effectively destroy the bacterial capsule (such as the K antigen of Klebsiella pneumoniae) that encapsulates nucleic acids. The synergistic effect of high temperature lysis and physical wall breaking. Glass bead crushing (0.5mm diameter) combined with 95°C lysis can increase the nucleic acid yield of Staphylococcus aureus by 40% compared to a single physical method. This is because after high temperature softens the cell wall, the glass beads have higher shearing efficiency, and high temperature can inactivate nucleases (such as the Nuc enzyme of Staphylococcus aureus), doubly protecting the integrity of nucleic acids.

[0044] Step S107, performing high-speed centrifugation on the lysis treatment liquid, includes: placing the lysis treatment liquid in a high-speed centrifuge device, and separating the lysis residue and the nucleic acid solution by high-speed rotation; controlling the separation effect according to centrifugation speed and time parameters to ensure that the supernatant contains the target nucleic acid with high purity; if impurities are still detected in the supernatant, repeating the high-speed centrifugation step; through the above treatment, obtaining the target nucleic acid suitable for subsequent amplification; and determining whether to directly enter the digital polymerase chain reaction technology analysis process based on the purity of the target nucleic acid.

[0045] The core of high-speed centrifugation is to separate substances of different densities by utilizing differences in centrifugal force. The lysis solution contains impurities such as cell debris and proteins, which have a higher density than the nucleic acid solution. By setting the centrifugal force parameter (e.g., 12,000 × g), the impurities can be sedimented to the bottom of the tube, while the nucleic acids remain in the supernatant.

[0046] For example, for Gram-negative bacterial lysates, centrifugation at 12,000 × g for 10 minutes can effectively separate interfering substances such as lipopolysaccharides, ensuring a supernatant transmittance of OD260 / 280 of 1.8-2.0, meeting dPCR purity requirements. Optimization of centrifugation speed and time should be tailored to the sample type. For BC culture media containing high polysaccharide concentrations, the centrifugal force should be increased to 15,000 × g and the time should be extended to 15 minutes to fully remove viscous impurities. Experimental data show that when the speed is lower than 10,000 × g, the supernatant turbidity increases by 30%, leading to an increased failure rate of dPCR droplet generation. Speeds exceeding 20,000 × g may cause mechanical shearing of nucleic acids, necessitating electrophoresis to verify integrity. The decision to repeat the centrifugation step is based on absorbance or fluorescence detection. If the OD260 / 230 after the first centrifugation is less than 1.5, it indicates the presence of phenolic residues, and a second centrifugation and replacement of the centrifuge tube are required to avoid cross-contamination.

[0047] For example, after two centrifugations at 12,000 × g, the impurity peak area of ​​the Pseudomonas aeruginosa sample was reduced by 92%, and the stability of the CT value was improved to ±0.3 cycles. The correlation between nucleic acid purity and subsequent processes is reflected in the amplification efficiency. When OD260 / 280 is between 1.7-2.1 and there is no protein residue, dPCR can be performed directly; if inhibitors (such as heparin) are detected, a silica gel membrane purification step is required. Clinical comparisons show that the uniformity of droplet generation in samples with qualified purity is 95%, while that of untreated samples is only 65%. Verification of the suitability of the target nucleic acid needs to be combined with end-point detection. When the fragment length is confirmed to be >200bp and there is no tailing phenomenon by electrophoresis, it indicates that the nucleic acid integrity meets the amplification requirements.

[0048] For example, after Klebsiella pneumoniae DNA was treated with the above process, the dPCR copy number variation coefficient (CV) was reduced from 12% to less than 5%.

[0049] Step S108, amplifying and analyzing the target nucleic acid using digital polymerase chain reaction technology, includes: mixing the target nucleic acid with a preconfigured reaction solution to form a system to be amplified; preparing the system to be amplified into microdroplets using a microfluidic device to ensure that the nucleic acid molecules are independently distributed in the microdroplets; performing polymerase chain reaction amplification on the microdroplets, and completing the amplification process according to preset temperature cycle parameters; performing signal detection on the amplified microdroplets using a chip scanning device to obtain fluorescence signal data; and determining the identification result and quantitative data of the pathogen based on the fluorescence signal data.

[0050] The target nucleic acid is mixed with a pre-configured reaction solution to form the amplification system. The key to this step lies in the preparation of the reaction solution, which typically includes components such as primers, probes, DNA polymerase, dNTPs, and buffer. Primers and probes must be designed to target the specific sequence of the target nucleic acid to ensure specific amplification.

[0051] For example, designing primers and probes for the 16S rRNA gene of Pseudomonas aeruginosa can avoid cross-reactions with other bacteria. The pH value and ion concentration of the reaction solution also need to be optimized to ensure the activity of the DNA polymerase. By precisely controlling the composition and proportion of the reaction solution, the efficiency and accuracy of subsequent amplification can be ensured. The system to be amplified is prepared into microdroplets using microfluidic equipment to ensure that the nucleic acid molecules are independently distributed in the microdroplets. Microfluidic technology is the core of digital PCR. Its principle is to use microfluidic chips and oil phases to divide the reaction solution into tens of thousands of microdroplets, and the volume of each microdroplet is usually at the picoliter level.

[0052] For example, the interfacial tension between the oil and water phases is used to disperse the reaction solution into microdroplets with a diameter of approximately 100 microns. Each microdroplet may contain one or more nucleic acid molecules, or may even contain no nucleic acid molecules. In this way, the target nucleic acid molecules can be independently distributed in the microdroplets, thereby achieving amplification and quantification at the single-molecule level. The microdroplets are subjected to polymerase chain reaction amplification, and the amplification process is completed according to preset temperature cycle parameters. The temperature cycle of PCR amplification generally includes three steps: denaturation, annealing, and extension.

[0053] For example, for the gyrB gene of Escherichia coli, the denaturation temperature can be set at 95°C, the annealing temperature at 60°C, and the extension temperature at 72°C, with each step taking 30 seconds, 30 seconds, and 60 seconds, respectively, for a total of 40 cycles. When performing PCR amplification in microdroplets, the extremely small size of each microdroplet and its high thermal conductivity shorten the time for each step and improve amplification efficiency. Precise control of temperature cycling parameters ensures efficient amplification of the target nucleic acid while avoiding nonspecific amplification. A chip scanner detects the signal of the amplified microdroplets and acquires fluorescence signal data. Chip scanners typically use the principles of fluorescence microscopy or flow cytometry to detect the fluorescence signal of each microdroplet.

[0054] For example, a fluorescent probe is used to label the target nucleic acid. During the amplification process, the probe binds to the target sequence and releases a fluorescent signal. By measuring the fluorescence intensity of each microdroplet, it is possible to determine whether it contains the target nucleic acid. The intensity of the fluorescent signal is proportional to the copy number of the target nucleic acid. Therefore, quantitative analysis of the fluorescent signal can determine the absolute number of target nucleic acids. Based on the fluorescent signal data, the pathogen identification results and quantitative data can be determined. Statistical analysis of the fluorescent signal data can also be used to calculate the copy number of the target nucleic acid.

[0055] For example, if 5,000 out of 10,000 microdroplets show a positive fluorescent signal, the concentration of the target nucleic acid can be inferred to be 5,000 copies / μL. Combined with the specific sequence information of the target nucleic acid, the type of pathogen can be further identified.

[0056] For example, detecting the gyrB gene of Pseudomonas aeruginosa can confirm the presence of Pseudomonas aeruginosa in the sample. In this way, not only can the pathogen be quickly identified, but its absolute quantification can also be achieved, providing accurate data support for clinical diagnosis and treatment.

[0057] Step S109, amplifying and analyzing the target nucleic acid using digital polymerase chain reaction technology, includes: configuring a primer and probe combination for specific pathogen detection for the target nucleic acid to ensure the specificity of the detection; packaging the target nucleic acid into a microfluidic chip through a preset reaction system to form a uniform microdroplet system; performing multiple rounds of temperature cycle amplification on the microdroplet system according to the parameter settings of the amplification instrument; performing multi-channel fluorescence scanning on the amplified microdroplet system through a signal acquisition device to obtain pathogen-specific signals; and analyzing the type of the pathogen and the absolute quantitative result based on the pathogen-specific signals.

[0058] 1. Targeting the target nucleic acid to detect specific pathogens requires designing highly specific primers based on conserved pathogen gene sequences. For example, when detecting Pseudomonas aeruginosa, the conserved region of the outer membrane protein OprL gene is selected, with the forward primer 5'-GACGGGTGAGTAATGCCTA-3' and the reverse primer 5'-CACTGGTGTTCCTTCCTATA-3' designed. These primers are paired with a FAM-labeled TaqMan probe 5'-FAM-CCGTCGCCGTAGAGGAAAT-BHQ1-3'. Primer Tm values ​​are controlled between 58-62°C, and product length is set between 80-150 bp to ensure amplification efficiency. This design avoids cross-reactions with the human genome or commensal bacteria, ensuring detection specificity exceeding 99%. 2. Microfluidic chip packaging requires optimizing the oil-water phase ratio to achieve uniform microdroplets. When using the self-developed chip of Linghang Company, 20μL PCR premix (containing target nucleic acid, primer probe, dNTPs) is mixed with 80μL fluorinated oil (containing 0.5% surfactant) through a microfluidic channel at a flow rate ratio of 3:1 to generate droplets with a diameter of approximately 50μm and a coefficient of variation (CV) of less than 5%. The chip temperature must be kept constant at 4°C to prevent pre-amplification. Each droplet contains an average of 0.5-1 nucleic acid molecules, which meets the requirements of Poisson distribution. This process must be operated under negative pressure to avoid bubbles interfering with droplet generation. 3. The temperature cycle amplification parameter settings must match the nucleic acid characteristics of the pathogen. Taking Klebsiella pneumoniae as an example: pre-denaturation at 95°C for 3 minutes, followed by 45 cycles of 95°C for 15 seconds (denaturation) → 60°C for 30 seconds (annealing / extension), and finally a final extension at 72°C for 5 minutes. The annealing temperature is adjusted according to the primer Tm value ±2°C. More than 40 cycles can detect pathogens as low as 1 copy / μL. The thermal amplification instrument must have a heating and cooling rate of 0.5°C / second to ensure the synchronization of reactions within the microdroplets, and the temperature uniformity error must be less than 0.3°C. 4. Multi-channel fluorescence scanning uses dual laser excitation (488nm / 640nm) and a four-color filter system. For example, when detecting Acinetobacter baumannii, the FAM channel collects target gene signals, the HEX channel synchronously detects internal standard genes (such as human β-actin), and the Cy5 channel monitors droplet integrity. Each droplet must scan 20 optical sections to eliminate debris interference, and the signal threshold is set to 3 times the standard deviation of the background fluorescence. The device resolution must reach 10μm / pixel to ensure accurate capture of droplet edge signals. 5. Absolute quantitative analysis is achieved by counting the proportion of positive droplets. If 500 FAM-positive signals are detected out of a total of 20,000 droplets, the pathogen concentration is converted to 250 copies / μL according to the Poisson correction formula (assuming the droplet volume is 0.5nL). The software automatically excludes HEX-negative droplets (possibly containing PCR inhibitors) and double-positive droplets (excessive nucleic acid encapsulation), resulting in a final result with an error range of ±5%. The lower limit of quantification can reach 0.1 copies / μL, and the linear range spans six orders of magnitude (1-10^6 copies / μL).

[0059] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. In addition, the various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the concept of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products, characterized in that: include: Obtain positive blood culture fluid as a sample to be processed; performing differential sedimentation separation on the sample to be processed to remove interfering substances to obtain a primary separation liquid; Performing physical cell wall breaking treatment on the primary separation liquid to obtain a cell wall breaking treatment liquid; performing high-temperature cracking treatment on the cell wall-breaking treatment liquid to obtain a cracking treatment liquid; Performing high-speed centrifugation on the lysis solution to obtain the target nucleic acid in the supernatant; The target nucleic acid is amplified and analyzed by digital polymerase chain reaction technology to determine the identification results and quantitative data of the pathogen.

2. The method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products according to claim 1, characterized in that: The step of performing differential sedimentation separation on the sample to be processed to remove interfering substances comprises: Placing the sample to be processed in a centrifugal device, allowing cells and large particles to settle under the action of low-speed centrifugal force, and retaining the supernatant as the primary separation liquid; Removing interfering components of the culture medium in the sample to be processed by the differential sedimentation separation to ensure the purity of nucleic acid extraction in subsequent processing; If visible impurities still exist in the primary separation liquid, repeat the low-speed centrifugation step until the primary separation liquid is clarified; Determining whether to directly proceed to subsequent physical cell wall breaking treatment based on the clarity of the primary separation liquid; Through the above steps, it is ensured that the primary separation solution is suitable for the subsequent nucleic acid extraction process.

3. The method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products according to claim 1, characterized in that: The physical wall breaking treatment of the primary separation liquid comprises: Transferring the primary separation solution into a container containing a cell wall disruption medium, wherein the cell wall disruption medium is particles of a specific diameter range; The primary separation solution and the cell wall breaking medium are fully mixed by a vortex oscillation device, so that the cell wall breaking medium generates kinetic energy during the oscillation process, destroys the cell wall of the pathogen, and releases the nucleic acid substance inside; Control the cell wall breaking effect by adjusting the shaking time and intensity to avoid excessive shearing of nucleic acids; If the cell wall breaking effect does not meet expectations, adjust the oscillation parameters and reprocess; Through the above treatment, the uniformly dispersed wall-breaking treatment liquid is obtained, which provides conditions for subsequent high-temperature cracking.

4. The method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products according to claim 1, characterized in that: The high-temperature cracking treatment of the wall-breaking treatment liquid comprises: The cell wall breaking treatment solution is placed in a preheated high-temperature device to denature proteins, melt membrane structures, and dissociate nucleic acid secondary structures in the pathogens through the action of high temperature; Optimize the lysis effect according to the time and temperature parameters of high temperature treatment to ensure protein inactivation while maintaining nucleic acid integrity; If the cell wall breaking treatment liquid precipitates after high temperature treatment, it is remixed after short cooling; Through the above-mentioned high-temperature lysis step, the lysis treatment liquid with fully released nucleic acids is obtained, providing a basis for subsequent centrifugation treatment.

5. The method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products according to claim 1, characterized in that: The high-speed centrifugation treatment of the lysis treatment solution comprises: placing the lysis treatment solution in a high-speed centrifugal device to separate the lysis residue and the nucleic acid solution by high-speed rotation; Controlling the separation effect according to centrifugation speed and time parameters to ensure that the supernatant contains the target nucleic acid with high purity; If impurities are still detected in the supernatant, repeat the high-speed centrifugation step; Through the above treatment, the target nucleic acid suitable for subsequent amplification is obtained; According to the purity of the target nucleic acid, determine whether to directly enter the digital polymerase chain reaction technology analysis process.

6. The method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products according to claim 1, characterized in that: The amplification and analysis of the target nucleic acid by digital polymerase chain reaction technology includes: Mixing the target nucleic acid with a pre-configured reaction solution to form a system to be amplified; The system to be amplified is prepared into microdroplets by a microfluidic device to ensure that the nucleic acid molecules are independently distributed in the microdroplets; Performing polymerase chain reaction amplification on the microdroplets, and completing the amplification process according to preset temperature cycle parameters; The amplified microdroplets are subjected to signal detection by a chip scanning device to obtain fluorescence signal data; The identification result and quantitative data of the pathogen are determined based on the fluorescence signal data.

7. The method for rapid extraction and identification of pathogen nucleic acid in positive blood culture products according to claim 1, characterized in that: The amplification and analysis of the target nucleic acid by digital polymerase chain reaction technology includes: Targeting the target nucleic acid, configuring a primer and probe combination for specific pathogen detection to ensure the specificity of the detection; The target nucleic acid is dispensed into a microfluidic chip through a preset reaction system to form a uniform micro-droplet system; According to the parameter settings of the amplification instrument, the micro-droplet system is subjected to multiple rounds of temperature cycle amplification; Performing multi-channel fluorescence scanning on the amplified micro-droplet system using a signal acquisition device to obtain pathogen-specific signals; The type of the pathogen and the absolute quantitative result are analyzed based on the pathogen-specific signal.

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