A method for absolute quantification of nucleic acid based on nucleic acid isothermal amplification

Abstract

The application discloses a nucleic acid absolute quantification method based on nucleic acid constant temperature amplification, which comprises the following specific steps: step one, sample pretreatment, blood samples and saliva samples are pretreated respectively; step two, reaction system configuration and assembly; step three, digital micro-reaction unit preparation and amplification; and step four, multiple signal analysis and absolute quantification. In the application, multiple primer design and a double-probe system are adopted, so that the mutation detection rate is improved, and the detection limit can be reduced to 0.1% variation through Cas12a-sgRNA auxiliary verification. The double-emulsification technology of the micro-fluidic chip is adopted, so that the droplet diameter variation coefficient is controlled within 5%, and the clogged micropore rate is reduced by real-time analysis of the micropore array image through the ImageJ software, automatic marking and exclusion of abnormal units.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection and quantification technology, specifically to an absolute quantification method for nucleic acids based on isothermal amplification. Background Technology

[0002] Nucleic acid amplification technology is a core tool in modern molecular diagnostics. While polymerase chain reaction (PCR) is widely used as the gold standard, its reliance on sophisticated thermal cycling equipment, complex operation, and long processing time limit its application in resource-constrained areas and point-of-care testing (POCT) scenarios. To overcome this limitation, isothermal amplification technology has emerged. By achieving rapid nucleic acid amplification at a single constant temperature, it significantly reduces reliance on equipment and has become a crucial technical support for pathogen detection, clinical diagnosis, and field monitoring.

[0003] Current mainstream isothermal amplification technologies include loop-mediated isothermal amplification (LAMP), sequence-dependent amplification (NASBA), strand displacement amplification (SDA), and recombinase polymerase amplification (RPA). These technologies achieve nucleic acid amplification under isothermal conditions through different mechanisms: LAMP utilizes the strand displacement activity of Bst DNA polymerase and specially designed primers to generate stem-loop structures for efficient amplification; NASBA and transcription-mediated amplification (TMA) amplify target sequences through RNA polymerase-driven transcription; and RPA achieves room-temperature amplification through rapid primer invasion mediated by recombinase. These technologies have collectively driven the development of molecular diagnostics towards speed and portability, especially during the COVID-19 pandemic, where LAMP and RPA-based detection kits have enabled viral nucleic acid detection within 30 minutes.

[0004] Despite significant progress in isothermal amplification techniques, existing methods still face several key challenges: 1. Insufficient specificity and anti-mutation ability: Traditional isothermal amplification relies on a single primer-probe system. When the target sequence mutates (such as viral gene drift or tumor mutation), false negatives are easily caused by decreased primer / probe binding efficiency. For example, although LAMP improves specificity through multi-primer design, its detection sensitivity for highly variable viruses (such as influenza virus H5N1) is still less than 100%. In addition, the risk of non-specific amplification is high under isothermal conditions, especially in complex clinical samples (such as whole blood and saliva), where homologous sequences or inhibitors can easily cause cross-reactions.

[0005] 2. Limited Quantitative Accuracy: Digital isothermal amplification techniques (such as digital LAMP) achieve absolute quantification through microreaction unit segmentation, but their accuracy is limited by unit uniformity and the Poisson distribution assumption. In existing microdroplet generation technologies, the droplet volume variation coefficient (CV) is typically >5%, leading to quantification errors of up to ±15% for low copy number samples. Furthermore, physical microwell chips are prone to clogging with prolonged use, further undermining the assumption of random nucleic acid distribution and affecting quantitative reliability.

[0006] In summary, a method for absolute quantification of nucleic acids based on isothermal amplification was designed. Summary of the Invention

[0007] To overcome the above-mentioned shortcomings, this invention provides a method for absolute quantification of nucleic acids based on isothermal amplification.

[0008] The present invention achieves the above objectives through the following technical solutions: An absolute quantification method for nucleic acids based on isothermal amplification includes the following specific steps: Step 1: Sample preprocessing, preprocessing blood samples and saliva samples separately; Step 2: Prepare and assemble the reaction system. The reaction system includes primer combination, enzyme mixture and buffer. First mix primer combination and buffer, preheat at 55°C for 5 minutes, add blood sample and saliva sample, denature at 90°C for 2 minutes and then immediately put on ice, finally add enzyme mixture and gently pipette to mix. Step 3: Digital microreaction unit preparation and amplification. A 20 μL reaction system was divided into 20,000 1 nL droplets using a 10 μm pore size microfluidic chip. The micropore array image was analyzed in real time using ImageJ software. Units with fluorescence intensity less than 1.5 times the background value were marked as blocked and abnormal units were eliminated. The generated droplets were amplified at 42℃ for 40 minutes, and fluorescence signals were collected every 2 minutes. Step 4: Multiple signal analysis and absolute quantification. After amplification, the Cas12a-sgRNA complex is added and reacted at 37°C for 10 minutes to target and cleave the wild-type sequence, leaving only the mutant sequence that can be detected. The fluorescence of the mutant and conserved regions is detected simultaneously by dual probe signal acquisition to detect the mutation ratio and absolute copy number.

[0009] Preferably, in step two, the primer combination includes an outer primer with a concentration of 0.2 μM, a nested inner primer with a concentration of 0.4 μM, and a dual probe with a concentration of 0.3 μM.

[0010] Preferably, in step two, the total reaction volume is 25 μL, the amount of blood and saliva samples added does not exceed 5 μL, and the volume of the enzyme mixture does not exceed 1 / 10 of the total volume.

[0011] Preferably, the enzyme mixture comprises M-MLV reverse transcriptase and T7 RNA polymerase in an enzyme ratio of 1:2.5, prepared with a 42°C preheated buffer, the buffer comprising inhibitors with added 5% betaine and 0.01% BSA.

[0012] Preferably, in step three, the droplet volume is 5 ± 0.25 nL and the generation rate is 10,000 drops / second.

[0013] Preferably, step three also includes a quality control step, in which each batch of chips is set up with a positive control and a negative control, and if the coefficient of variation is >15%, it is determined to be invalid.

[0014] Preferably, in step four, the formula for calculating the mutation ratio is as follows: Zscore = (FAM signal / HEX signal) - Threshold Where Zscore represents the mutation ratio, FAM signal represents the mutation type signal, and HEX signal represents the conserved region signal.

[0015] Preferably, in step four, the formula for calculating the absolute copy number is as follows: N = -V × ln(1-P) + / N t ) Where N represents the absolute copy number, V represents the actual average droplet volume, and P... + N represents the proportion of positive droplets. t This is expressed as the total number of droplets.

[0016] The beneficial effects of this invention are: in this method for absolute quantification of nucleic acids based on isothermal amplification, 1. The use of multiple primer design and dual probe system improves the mutation detection rate. With the assistance of Cas12a-sgRNA, the detection limit can be reduced to 0.1% of the mutation. 2. The dual emulsification technology using microfluidic chips is adopted to control the droplet diameter variation coefficient to within 5%. At the same time, the image of the micropore array is analyzed in real time using ImageJ software. Image processing is used to identify blocked micropores, reduce the pore blockage rate, and automatically mark and eliminate abnormal units. Attached Figure Description

[0017] The present invention will be described by way of example and with reference to the accompanying drawings, wherein: Figure 1 This is a method diagram of the present invention. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic diagrams, illustrating only the basic structure of the invention, and therefore only show the components relevant to the invention.

[0019] like Figure 1 As shown, a method for absolute quantification of nucleic acids based on isothermal amplification includes the following specific steps: Step 1: Sample preprocessing, preprocessing blood samples and saliva samples separately; Step 2: Prepare and assemble the reaction system. The reaction system includes primer combination, enzyme mixture and buffer. First mix primer combination and buffer, preheat at 55°C for 5 minutes, add blood sample and saliva sample, denature at 90°C for 2 minutes and then immediately put on ice, finally add enzyme mixture and gently pipette to mix. Step 3: Digital microreaction unit preparation and amplification. A 20 μL reaction system was divided into 20,000 1 nL droplets using a 10 μm pore size microfluidic chip. The micropore array image was analyzed in real time using ImageJ software. Units with fluorescence intensity less than 1.5 times the background value were marked as blocked and abnormal units were eliminated. The generated droplets were amplified at 42℃ for 40 minutes, and fluorescence signals were collected every 2 minutes. Step 4: Multiple signal analysis and absolute quantification. After amplification, the Cas12a-sgRNA complex is added and reacted at 37°C for 10 minutes to target and cleave the wild-type sequence, leaving only the mutant sequence that can be detected. The fluorescence of the mutant and conserved regions is detected simultaneously by dual probe signal acquisition to detect the mutation ratio and absolute copy number.

[0020] Specifically, in step two, the primer combination includes an outer primer with a concentration of 0.2 μM, a nested inner primer with a concentration of 0.4 μM, and a dual probe with a concentration of 0.3 μM.

[0021] Specifically, in step two, the total reaction volume is 25 μL, the amount of blood and saliva samples added does not exceed 5 μL, and the volume of the enzyme mixture does not exceed 1 / 10 of the total volume.

[0022] Specifically, the enzyme mixture comprises M-MLV reverse transcriptase and T7 RNA polymerase in an enzyme ratio of 1:2.5, prepared with a 42°C preheated buffer, which includes inhibitors with added 5% betaine and 0.01% BSA.

[0023] Specifically, in step three, the droplet volume is 5 ± 0.25 nL, and the generation rate is 10,000 drops / second.

[0024] Specifically, step three also includes a quality control step, in which positive and negative controls are set for each batch of chips, and the chip is deemed invalid if the coefficient of variation is greater than 15%.

[0025] Specifically, in step four, the formula for calculating the mutation ratio is as follows: Zscore = (FAM signal / HEX signal) - Threshold Where Zscore represents the mutation ratio, FAM signal represents the mutation type signal, and HEX signal represents the conserved region signal.

[0026] Specifically, in step four, the formula for calculating the absolute copy number is as follows: N = -V × ln(1-P) + / N t ) Where N represents the absolute copy number, V represents the actual average droplet volume, and P... + N represents the proportion of positive droplets. t This is expressed as the total number of droplets.

[0027] The specific implementation of step one is as follows: Sample preprocessing: Blood samples and saliva samples were preprocessed separately.

[0028] 1.1 Blood Sample Preprocessing 1.1.1 Sample Collection and Anticoagulation Treatment Use the venous blood collection method, and use blood collection tubes containing EDTA-K2 anticoagulant (1.5-2.0 mg EDTA / ml blood). Collect 2-5 ml of blood and gently invert 8-10 times to mix. Avoid using heparin anticoagulant, as it will inhibit subsequent enzymatic reactions. Complete subsequent processing within 30 minutes after blood collection, and store at 4°C for no more than 2 hours.

[0029] 1.1.2 Serum / Plasma Separation Centrifuge whole blood samples at 1000×g for 15 minutes at 4°C; carefully aspirate the upper plasma layer, avoiding contact with the middle leukocyte layer; aliquot into 500μl tubes and store at -80°C, avoiding repeated freeze-thaw cycles.

[0030] 1.1.3 Anti-interference processing Add 0.5% β-mercaptoethanol (final concentration) to the plasma and mix gently. For high-lipid samples, add 1% PEG8000 (final concentration) to precipitate lipoproteins, let stand at 4°C for 10 minutes, and then centrifuge again.

[0031] 1.1.4 Nucleic acid extraction Use the silica-membrane centrifugation column method, following the kit instructions; elute with 50 μL of RNase-free water, and proceed immediately to the next step or store at -80°C.

[0032] 1.2 Saliva Sample Pretreatment 1.2.1 Preparation before sample collection Do not eat, drink, smoke, or chew gum for 30 minutes before sampling; rinse your mouth with water and gently massage your cheeks for 30 seconds to promote saliva secretion.

[0033] 1.2.2 Saliva Collection and Preservation Using a saliva collection tube containing a stabilizer (such as the MGIEAsy saliva collection kit), collect 1 ml of liquid saliva (avoid foam); add an equal volume of preservation solution, vortex to mix for 10 seconds, and it can be stored at room temperature for 1 month or at 4°C for 3 months.

[0034] 1.2.3 Sample Clarification Process Centrifuge at 4000×g for 10 minutes at 4℃; take the supernatant and dilute it with PBS buffer (pH 7.4) at a ratio of 1:1.

[0035] 1.2.4 Nucleic acid extraction Extraction was performed using magnetic beads, with 5% betaine (final concentration) added to increase RNA yield; the elution volume was 30 μl, and the RNA was used immediately for the reaction or stored at -80°C.

[0036] The specific implementation of step two is as follows: 2.1 Primer and probe design and optimization 2.1.1 Principles of Multiple Primer Design The outer primer length is 20-25 nt, the inner primer is 18-22 nt, the GC content is 40-60%, the Tm value is 58-62℃, the difference in Tm value between the upstream and downstream primers is ≤2℃, the 3' end of the primer should avoid continuous G / C, and the last position should preferably be C or G.

[0037] 2.1.2 Dual-probe system configuration Calibration probe (conservative region): 5' end HEX labeled, 3' end BHQ1 quenched; Indicator probe (variable region): 5' end FAM labeled, 3' end BHQ1 quenched. The probe concentration is 0.3 μM to avoid the formation of secondary structures (ΔG ≥ -4.5 kcal / mol).

[0038] 2.1.3 Primer and probe verification OligoAnalyzer software was used to examine dimers and hairpin structures, and BLAST alignment was used to ensure primer specificity and avoid non-specific amplification.

[0039] 2.2 Enzyme Mixture Preparation 2.2.1 Optimization of enzyme combination ratio M-MLV reverse transcriptase: 200U / reaction, T7 RNA polymerase: 500U / reaction, enzyme ratio 1:2.5 (reverse transcriptase: T7 polymerase), optimizing enzyme activity balance.

[0040] 2.2.2 Enzyme Storage and Reconstitution The enzyme storage solution contains 50% glycerol and should be aliquoted and stored at -80°C. Before use, dilute with a buffer solution preheated to 42°C and avoid repeated freeze-thaw cycles.

[0041] 2.2.3 Additive Optimization Adding 0.01% BSA (final concentration) protects enzyme activity, and adding 5% betaine (final concentration) enhances GC-rich sequence amplification efficiency.

[0042] 2.3 Optimization of Buffer System 2.3.1 Preparation of basic buffer solution 20mM Tris-HCl (pH 8.3), 10mM MgCl2, 50mM KCl (TE buffer preparation and function - Thermo Fisher Scientific), 1mM DTT, 0.1mM EDTA, to protect enzyme stability.

[0043] 2.3.2 Optimization of Anti-inhibitors Adding 5% DMSO (final concentration) reduces secondary structure, and 1% Tween-20 (final concentration) reduces sample adhesion loss.

[0044] 2.3.3 dNTPs Configuration The concentrations of dATP, dCTP, and dGTP were each 0.4 mM, dUTP was 0.8 mM, and the Mg²⁺ concentration was optimized to 6 mM.

[0045] 2.4 Assembly of the reaction system 2.4.1 Optimization of Mixed Sequence First, mix the primers, probes, and buffer, preheat at 55°C for 5 minutes, add the template RNA / DNA, denature at 90°C for 2 minutes, then immediately place on ice, and finally add the enzyme mixture and gently mix by pipetting.

[0046] 2.4.2 Normalization of reaction volume The total reaction volume is 25 μl, of which the amount of template RNA / DNA added does not exceed 5 μl, and the enzyme mixture is added last, occupying no more than 1 / 10 of the total volume.

[0047] 2.4.3 Quality Control Template-free control and positive control were set up, and the internal reference gene was detected simultaneously in each batch of reaction.

[0048] The specific implementation of step three is as follows: 3.1 Microfluidic Chip Design and Manufacturing 3.1.1 Selection of Chip Materials Material type: Polydimethylsiloxane (PDMS) was used as the chip substrate, which has good biocompatibility and breathability. A PDMS substrate with a thickness of 5 mm was prepared by mixing PDMS prepolymer with a curing agent at a mass ratio of 10:1, vacuum degassing, and curing in an oven at 80°C for 2 hours.

[0049] Surface treatment: The surface of the chip channel needs to be hydrophobically treated by vapor deposition of trimethylchlorosilane (TMCS) or coating with fluorinated silane (such as Rain-X) to achieve a water contact angle of 110°±5° and prevent droplets from adhering to the channel wall.

[0050] 3.1.2 Microchannel Structure Design T-structure parameters: The droplet generation region adopts a T-junction channel design, with a continuous phase (oil phase) channel width of 100μm and a dispersed phase (aqueous phase) channel width of 50μm, both with a depth of 40μm. Droplet breakage is achieved through a stepped outlet design (height difference of 20μm) utilizing the Laplace pressure difference.

[0051] Storage area design: The droplet storage area adopts a honeycomb array structure with a single chamber volume of 5nL, which can accommodate approximately 50,000 independent reaction units. Adjacent units are separated by a 5μm high isolation wall to prevent cross-contamination.

[0052] 3.1.3 3D Printing-Aided Manufacturing High-precision printing: For complex chip structures (such as helical hybrid channels), PolyJet Matrix 3D printing technology was used, with VeroClear RGD810 photosensitive resin as the structural material and TangoBlackPlus FLX980 as the sealing layer material. The printing resolution was set to a layer thickness of 25μm, and the surface roughness Ra≤3.2μm.

[0053] Post-processing: After printing, the surface is cleaned with isopropanol, cured with UV light for 10 minutes, and then treated with oxygen plasma (60W power, 30 seconds) to activate the surface hydroxyl groups and improve the bonding strength.

[0054] 3.2 Droplet Generation and Loading 3.2.1 Two-phase fluid configuration Aqueous phase system: Contains an optimized nucleic acid amplification reaction mixture, with 5% glycerol (final concentration) added to adjust the viscosity to 8 cP to ensure droplet formation stability.

[0055] Oil phase system: Fluorinated oil containing 2% surfactant (such as 3M™ Fluorinert FC-40) is used. The surfactant is a mixture of polyglycerol ricinoleate (PGPR 415) and perfluoropolyether (PFPE) at a mass ratio of 1:3 to reduce the interfacial tension to 12mN / m.

[0056] 3.2.2 Pressure Driven System Pressure parameter settings: Using the Elveflow OB1 pressure controller, the oil phase drive pressure is set to 4.0 kPa and the water phase drive pressure is set to 3.8 kPa. The droplet generation frequency is controlled to 12 Hz by adjusting the pressure ratio.

[0057] Automated operation: The autonomous transport mechanism of the Degassed PDMS chip drives the fluid through air permeation in the outlet storage tank, generating 51,000 uniform droplets within 80 minutes with a volume variation coefficient (CV) ≤3%.

[0058] 3.2.3 Droplet quality control Diameter monitoring: The droplet formation process was observed in real time using an optical microscope (40× objective lens), and the droplet diameter was analyzed using ImageJ software. The target range was 5.0±0.25μm. 200 droplets were randomly sampled from each batch for statistical analysis.

[0059] Stability test: The generated droplets were left to stand at a constant temperature of 37°C for 30 minutes. Only after the absence of fusion phenomenon (fusion rate < 0.1%) was verified by fluorescence imaging could subsequent amplification be carried out.

[0060] 3.3 Alternative solutions for micro-pore array chips 3.3.1 Fabrication of silicon-based microporous chips Etching process: A hexagonal micropore array with a pore diameter of 50 μm, a depth of 40 μm, and a pore density of 250 pores / mm² was fabricated on a 4-inch silicon wafer using deep reactive ion etching (DRIE) technology. A 100 nm SiO2 insulating layer was grown by thermal oxidation, followed by sputtering a 50 nm Ti / Au electrode layer for impedance monitoring.

[0061] Bonding and encapsulation: The Pyrex glass cover is anodicly bonded (400°C, 1000V) to achieve a seal, forming a closed reaction unit.

[0062] 3.3.2 Inkjet Printing Droplet Array Printing parameters: Use a piezoelectric inkjet printer (such as the FUJIFILM Dimatix DMP-2800), nozzle diameter 21μm, drive voltage 35V, temperature 40℃. Droplet volume can be adjusted from 1-100pL via waveform adjustment.

[0063] Immobilization treatment: Coat the surface of the glass substrate with a 0.1 mm thick UV-curable adhesive (such as NOA 63), and expose it to UV light immediately after printing (365 nm, 10 mW / cm², 30 seconds) to fix the droplets and prevent fusion during amplification.

[0064] 3.4 Optimization and Loading of the Amplification System 3.4.1 Preparation of enzyme mixture Enzyme ratio optimization: M-MLV reverse transcriptase (200 U / reaction) and T7 RNA polymerase (500 U / reaction) were mixed at a ratio of 1:2.5, and 0.01% BSA and 1 mM DTT were added to protect enzyme activity. The mixture was prepared using preheated buffer (20 mM Tris-HCl pH 8.3, 10 mM MgCl2) at 42°C, aliquoted into 10 μL tubes, and stored at -80°C.

[0065] Inhibitor addition: For complex samples (such as whole blood), 5% betaine (final concentration) and 0.5% β-mercaptoethanol should be added to the system to enhance GC-rich sequence amplification and inhibit hemoglobin interference, respectively.

[0066] 3.4.2 Sample Loading Method Microfluidic chip loading: The reaction mixture is injected into the chip at a flow rate of 1 μL / min using a high-precision injection pump. At the same time, the inlet pressure is monitored. When the pressure rises sharply (>10 kPa), it is determined that the channel is blocked and the chip needs to be replaced.

[0067] Microporous chip loading: Using the centrifugal coating method, 2μL of reaction solution was dropped onto the center of the chip and centrifuged at 2000×g for 2 minutes to ensure that the liquid uniformly filled the micropores. Residual air bubbles were removed by vacuum degassing treatment (-80kPa, 5 minutes).

[0068] 3.5 Control of isothermal amplification conditions 3.5.1 Temperature Parameter Settings Temperature range selection: Based on the optimal activity temperature of the amplification enzyme, the LAMP reaction was set to 65℃±0.5℃, and the RPA reaction was set to 37℃±0.5℃. A Peltier temperature control module (TEC1-12706) was used in conjunction with a K-type thermocouple (accuracy ±0.1℃), and closed-loop temperature control was achieved through a PID algorithm.

[0069] Thermal uniformity is ensured: A 0.5mm thick aluminum heat spreader is laid under the chip, and the surface temperature gradient is controlled within ±0.3℃ (verified by an infrared thermal imager).

[0070] 3.5.2 Optimization of Reaction Time Kinetic monitoring: The amplification plateau time was determined through preliminary experiments for 10... 4 For samples of copies / μL, LAMP reactions typically reach a plateau at 30 minutes, and RPA reactions at 20 minutes. In formal experiments, the reaction time is set to plateau time + 10 minutes to ensure sufficient amplification.

[0071] Real-time monitoring: Fluorescence signals are collected every 2 minutes to plot amplification kinetics curves and the plateau time point is automatically determined by the second derivative method.

[0072] 3.5.3 Pressure and Environmental Control Sealing pressure regulation: For microporous array chips, a constant pressure of 0.2 MPa is applied using a pneumatic pressurization device to prevent sample evaporation. The pressure is adjusted in real time via feedback from a pressure sensor (accuracy ±2 kPa).

[0073] Humidity control: A saturated dipotassium hydrogen phosphate solution is placed in the reaction chamber to maintain a relative humidity of 70%±5% to avoid localized drying caused by chip edge effects.

[0074] 3.6 Construction of Dynamic Monitoring System 3.6.1 Fluorescence Imaging Module Optical configuration: An inverted fluorescence microscope (Olympus IX73) was used, equipped with 488nm (FAM) and 532nm (HEX) laser sources, and bandpass filters (FAM: 525 / 50nm, HEX: 570 / 50nm). An Andor iXon Ultra 897EMCCD camera was used, with an exposure time set to 200ms.

[0075] Scanning method: Automatic stage (accuracy ±1μm) is used for stitching imaging. Single field imaging time ≤1 second, full chip (20×20mm²) imaging time ≤5 minutes.

[0076] 3.6.2 Image Processing and Analysis Droplet recognition algorithm: Based on Otsu threshold segmentation and morphological operations, the droplet recognition accuracy is ≥99%. Abnormal droplets are filtered out by roundness factor (≥0.85) and area range (4-6μm²).

[0077] Fluorescence intensity quantification: The average fluorescence intensity (MFI) of each droplet was extracted, and a negative threshold was set (MFI of NTC + 3SD). The criteria for judging a positive droplet were MFI > threshold and the amplification curve showing a typical S-shape.

[0078] 3.6.3 Abnormal Unit Exclusion Mechanism Physical anomaly detection: Through bright-field image analysis, identify and eliminate physically anomalous units such as bubbles (area < 2μm²) and channel blockages (no signal in 3 consecutive units).

[0079] Fluorescence anomaly filtering: Units with sudden changes in fluorescence signal (>50% / 2 minutes) are judged as non-specific amplification and automatically marked as invalid units.

[0080] Case 1: Absolute Quantitative Detection of EGFR L858R Mutation in Lung Cancer in Clinical Samples Sample preprocessing Five ml of blood was collected from patients with non-small cell lung cancer using EDTA-K2 anticoagulant tubes. Plasma was separated by centrifugation at 1000×g for 15 minutes at 4°C. 200 μl of the supernatant plasma was collected, and 0.5% β-mercaptoethanol (final concentration) was added to remove PCR inhibitors. Circulating tumor DNA (ctDNA) was extracted using magnetic beads, with an elution volume of 30 μl. For tumor tissue samples, paraffin-embedded samples were fixed in formaldehyde (FFPE), and DNA was extracted using the QIAamp DNA FFPE Tissue Kit. High-lipid interference was removed by treatment with 1% PEG8000.

[0081] Reaction system configuration Primer combination: Designed targeting the L858R mutation site in exon 21 of the EGFR gene: Outer primers (0.2 μM): upstream 5'-GCAATGAGCTGCGTGATG-3', downstream 5'-TCATGCGTCTTCATGGC-3' Nested inner primers (0.4 μM): upstream 5'-GGTGAGGTGAGTGTGAG-3', downstream 5'-TGGATCCAGAGGAGCTG-3' Dual probes (0.3 μM each): HEX-labeled conserved region probe (wild type): 5'-HEX-CGTGATGGTGAGGTG-MGB-3' FAM-labeled mutant region probe (L858R): 5'-FAM-AGCTGCGTGATGGTGA-MGB-3' Enzyme mixture: M-MLV reverse transcriptase (200 U / reaction) and T7 RNA polymerase (500 U / reaction) were mixed at a ratio of 1:2.5 in an optimized buffer (20 mM Tris-HCl pH 8.3, 10 mM MgCl2) containing 5% betaine and 0.01% BSA.

[0082] Reaction assembly: Add the following to a 25 μL system in sequence: 15 μL primer-buffer mixture, 5 μL sample DNA, denature at 90 °C for 2 minutes, incubate on ice for 5 minutes, and finally add 5 μL enzyme mixture and mix thoroughly by pipetting.

[0083] Digital Microreaction Unit Preparation and Amplification A 20 μL reaction system was divided into 20,000 1 nL droplets using a 10 μm pore size PDMS microfluidic chip (honeycomb array structure). The oil phase consisted of FC-40 containing 2% PFPE surfactant. The microarray was monitored in real time using ImageJ software, and blocking cells with fluorescence intensity < 1.5 times the background value were excluded (approximately 3.2% of abnormal cells were automatically marked and excluded). Amplification was performed at 42℃ for 40 minutes, with FAM / HEX dual-channel fluorescence signals acquired every 2 minutes.

[0084] Multiple signal analysis and absolute quantification After amplification, the Cas12a-sgRNA complex (targeting the wild-type sequence) was added, and the reaction was carried out at 37°C for 10 minutes. The absolute copy number was calculated using the formula N = -V × ln(1 - P+ / Nt). Actual average droplet volume V = 1 nL The proportion of positive droplets P+ = (number of FAM positive droplets / total number of effective droplets) = 125 / 19360 ≈ 0.646% Total number of droplets Nt = 19360 The calculated value is N = -1 × ln(1 - 0.00646) = 0.00648 copies / droplet. Total sample copy number = 0.00648 × 19360 = 125 copies / reaction The mutation rate was calculated as Zscore = (FAM signal / HEX signal) - threshold. When FAM / HEX = 0.62 and the threshold was set to 0.2, the Zscore was 0.42, corresponding to a mutation rate of 21.3%, which was in good agreement with the NGS detection result (20.8%).

[0085] Quality control Each batch included a positive control (a plasmid standard containing the L858R mutation, 100 copies / μL) and a negative control (plasma DNA from healthy individuals). The coefficient of variation (CV) was 3.8% < 15%, meeting quality control requirements. This method has a detection limit of 0.1% for the L858R mutation and can be used for guiding targeted therapy and monitoring the efficacy of treatment in lung cancer patients.

[0086] Case Study 2: Rapid Quantitative Detection of Escherichia coli O157:H7 in Food Sample preprocessing Take 25g of beef sample and add 225mL of BPW enrichment broth. Incubate at 37℃ with shaking for 6 hours. Centrifuge 1mL of culture at 4000×g for 10 minutes. Wash the precipitate twice with PBS buffer, add 300μL of lysis buffer containing lysozyme (20mg / mL), and incubate at 65℃ for 30 minutes. Extract genomic DNA using a silica gel membrane centrifugation column method. The elution volume is 50μL. The A260 / A280 ratio is determined by NanoDrop to be 1.82, and the DNA concentration is 35ng / μL.

[0087] Reaction system optimization Target gene selection: hlyA virulence gene of Escherichia coli O157:H7 (GenBank accession number AF078743), 327bp in length.

[0088] Primer and probe design: Outer primers (0.2 μM): upstream 5'-GCTGGTGCTGTATGGTG-3', downstream 5'-CGTTGGTGAGCAAATGAG-3' Inner primers (0.4 μM): upstream 5'-GGTGAGCAAATGAGTGG-3', downstream 5'-CTGGTGCTGTATGGTGA-3' FAM-labeled probe (0.3 μM): 5'-FAM-TGGTGAGCAAATGAGTG-MGB-3' HEX-labeled internal control probe (0.3 μM): 5'-HEX-CGTTGGTGAGCAAATGA-BHQ1-3' (targeting the E. coli 16S rRNA gene) Enzyme mixture: Bst DNA polymerase (8 U / reaction) and Nfo endonuclease (2 U / reaction), SDA buffer containing 15% trehalose (10 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgSO4).

[0089] Digital microfluidic amplification and detection A 20 μL reaction system (containing 5 μL of sample DNA) was used to generate 20,000 1 nL droplets via a microfluidic chip and amplified at 42 °C for 40 minutes. Amplification kinetics showed that positive samples reached the fluorescence threshold at 18 minutes, while negative controls showed no amplification signal. Targeted cleavage of the hlyA homologous sequence of non-O157:H7 strains by Cas12a-sgRNA showed a 100% detection rate for E. coli O157:H7 and no cross-reactivity with other serotypes (such as O26 and O111).

[0090] Absolute quantitative results Count of positive droplets: 287 Total effective droplet count: 19,650 P+=287 / 19650≈1.46% The absolute copy number N = -1 × ln(1 - 0.0146) = 0.0147 copies / droplet Initial sample concentration = 0.0147 × 19650 / 5 μL = 57.8 copies / μL Corresponding colony forming units (CFU): Based on the standard curve established in the preliminary experiment (1 copy = 0.8 CFU), the result was 46.2 CFU / g, with a relative deviation of <5% from the plate count result (48 CFU / g).

[0091] This method takes only 4 hours from sample processing to result output, which significantly shortens the detection cycle compared to the traditional culture method (3-5 days). The detection limit is 10 CFU / g, which meets the limit requirements of GB 29921-2021 for pathogenic bacteria in ready-to-eat foods.

[0092] Case 3: Absolute Quantitative Detection of Norovirus in Environmental Water Bodies Sample collection and preprocessing A 1L sample of water from the inlet of a municipal wastewater treatment plant was collected and filtered through a 0.45μm filter. Viral particles were then concentrated using polyethylene glycol (PEG) precipitation: 8% PEG 8000 and 0.3M NaCl were added, and the mixture was incubated at 4°C for 12 hours, centrifuged at 8000×g for 30 minutes, and the precipitate was resuspended in 1mL PBS. RNA was extracted using the QIAamp Viral RNA Mini Kit, with an elution volume of 50μL. An RNase inhibitor (40U / μL) was added, and the mixture was stored at -80°C for later use.

[0093] Construction of isothermal amplification system Multiple amplification strategy: designed targeting the conserved regions (ORF1-ORF2 linker regions) of norovirus GI and GII types: GI primer and probe set: outer primer 0.2 μM, inner primer 0.4 μM, Cy5-labeled probe 0.3 μM (5'-Cy5-AGATGCGGTGCTGATGCA-BHQ2-3'). GII type primer and probe set: outer primer 0.2 μM, inner primer 0.4 μM, Texas Red labeled probe 0.3 μM (5'-Texas Red-TGATGCGGTGCTGATGCA-BHQ2-3') Internal reference gene (MS2 phage RNA): 0.3 μM FAM-labeled probe, used to monitor RNA extraction efficiency. Integrated reverse transcription-isothermal amplification reaction: A 25 μL system contained 5 μL RNA sample, M-MLV reverse transcriptase (200 U), T7 RNA polymerase (500 U), 5% betaine, 0.01% BSA, and 5 mM DTT. Preheating was performed at 55℃ for 5 minutes, followed by isothermal amplification at 42℃ for 60 minutes, with multi-channel fluorescence collected every 5 minutes.

[0094] Digital droplet detection and data analysis Droplets were generated using a 10μm pore size microfluidic chip, and approximately 2.8% of anomalous units were eliminated through ImageJ software analysis. After amplification: 156 positive droplets of type GI and 89 positive droplets of type GII Number of phage-positive droplets in internal control MS2: 213 (extraction efficiency = 213 / theoretical value 230 ≈ 92.6%) Calculate using the formula: GI copy number = -1 × ln(1 - 156 / 19440) × 19440 / 5 = 312 copies / μL GII type copy number = -1×ln(1-89 / 19440)×19440 / 5 = 178 copies / μL Viral titer = (312 + 178) × 50 μL × 1000 mL / L = 2.45 × 10 7 copies / L Method Validation Norovirus RNA standards were serially diluted 10-fold (10¹) 0 Validated at ~10¹ copies / μL, the method showed linearity over nine orders of magnitude (R²=0.997) and a detection limit of 0.5 copies / reaction. Compared with RT-qPCR, the quantitative results of the two methods showed good correlation (R²=0.986), but this method had higher detection accuracy for low-copy samples (<100 copies / μL) (CV=4.2% vs 11.8%).

[0095] Based on the above description, those skilled in the art can make various changes and modifications without departing from the technical concept of this invention. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for absolute quantification of nucleic acids based on isothermal amplification, characterized in that: The specific steps include the following: Step 1: Sample preprocessing, preprocessing blood samples and saliva samples separately; Step 2: Prepare and assemble the reaction system. The reaction system includes primer combination, enzyme mixture and buffer. First mix primer combination and buffer, preheat at 55°C for 5 minutes, add blood sample and saliva sample, denature at 90°C for 2 minutes and then immediately put on ice, finally add enzyme mixture and gently pipette to mix. Step 3: Digital microreaction unit preparation and amplification. A 20 μL reaction system was divided into 20,000 1 nL droplets using a 10 μm pore size microfluidic chip. The micropore array image was analyzed in real time using ImageJ software. Units with fluorescence intensity less than 1.5 times the background value were marked as blocked and abnormal units were eliminated. The generated droplets were amplified at 42℃ for 40 minutes, and fluorescence signals were collected every 2 minutes. Step 4: Multiple signal analysis and absolute quantification. After amplification, the Cas12a-sgRNA complex is added and reacted at 37°C for 10 minutes to target and cleave the wild-type sequence, leaving only the mutant sequence that can be detected. The fluorescence of the mutant and conserved regions is detected simultaneously by dual probe signal acquisition to detect the mutation ratio and absolute copy number.

2. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: In step two, the primer combination includes an outer primer with a concentration of 0.2 μM, a nested inner primer with a concentration of 0.4 μM, and a dual probe with a concentration of 0.3 μM.

3. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: In step two, the total reaction volume is 25 μL, the amount of blood and saliva samples added does not exceed 5 μL, and the volume of the enzyme mixture does not exceed 1 / 10 of the total volume.

4. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: The enzyme mixture comprises M-MLV reverse transcriptase and T7 RNA polymerase in an enzyme ratio of 1:2.5, prepared with a 42°C preheated buffer containing 5% betaine and 0.01% BSA added to the inhibitor.

5. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: In step three, the droplet volume is 5 ± 0.25 nL, and the generation rate is 10,000 drops / second.

6. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: Step three also includes a quality control step, in which positive and negative controls are set for each batch of chips, and the chips are deemed invalid if the coefficient of variation is greater than 15%.

7. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: In step four, the formula for calculating the mutation ratio is as follows: Zscore = (FAM signal / HEX signal) - Threshold Where Zscore represents the mutation ratio, FAM signal represents the mutation type signal, and HEX signal represents the conserved region signal.

8. The method for absolute quantification of nucleic acids based on isothermal amplification according to claim 1, characterized in that: In step four, the formula for calculating the absolute copy number is as follows: N=-V×ln(1-P + / N t ) Where N represents the absolute copy number, V represents the actual average droplet volume, and P... + N represents the proportion of positive droplets. t This is expressed as the total number of droplets.

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