Method for evaluating copy number of specific nucleic acid in quantitative pseudovirus antigen standard substance
By combining DNase enzyme treatment and hydroxysilane magnetic bead extraction with mismatch probe design, the error problem of nucleic acid quantification in pseudovirus particles was solved, achieving high-precision evaluation of nucleic acid copy number and improving the accuracy and comparability of pseudovirus standards.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies for quantifying target nucleic acids in pseudovirus particles suffer from problems such as interference from free DNA, unstable nucleic acid extraction efficiency, and insufficient specificity in the design of amplification primers and probes, leading to quantitative errors and inaccurate determination of standard values.
DNase enzyme treatment was used to remove free DNA, hydroxysilane-coated magnetic beads were used to extract nucleic acids, and specific primers and mismatched fluorescent probes were designed. Microdroplet digital PCR reaction and signal correction were performed using a Poisson distribution model.
This improved the accuracy and consistency of the number of specific nucleic acid copies in pseudovirus antigen standards, and enhanced the standardization and traceability of nucleic acid detection reagent systems.
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Figure CN121653233A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the technical field of pseudovirus evaluation, specifically relating to a method for evaluating the copy number of specific nucleic acids in quantitative pseudovirus antigen standards. Background Technology
[0002] Pseudovirus antigen standards, as important reference materials in molecular diagnostics, vaccine evaluation, and quality control, possess excellent safety and reproducibility due to their structural mimicry of real viruses and lack of infectivity, and are widely used in infectious disease detection. With the development of absolute quantitative technologies such as digital PCR, accurately assessing the target nucleic acid copy number carried in pseudovirus standards has become an indispensable core task in the research and clinical evaluation of nucleic acid detection reagents. This evaluation not only affects the quality consistency of pseudovirus preparations but also influences the standardized control of downstream diagnostic sensitivity.
[0003] Currently, the quantification of target nucleic acids in pseudovirus particles mainly employs two technical routes: one is the relative quantification method based on quantitative real-time PCR (qPCR), which requires the construction of a standard curve and is easily affected by template purity and reaction system fluctuations; the other is the absolute quantification method represented by droplet digital PCR (ddPCR), which utilizes droplet blocking and a Poisson statistical model to achieve direct counting at the molecular level, possessing advantages such as independence from standard curves, high precision, and strong repeatability. Although ddPCR technology performs excellently in nucleic acid quantification, it still faces several challenges in pseudovirus samples, such as: interference from free DNA leading to overestimation of total nucleic acid, unstable nucleic acid extraction efficiency causing quantitative errors, insufficient specificity in amplification primer and probe design, and a lack of unified calculation standards in the statistical modeling process of droplet readings, which limits its widespread application in standard determination scenarios.
[0004] Therefore, there is an urgent need to propose a copy number evaluation method that is structurally closed-loop and has clearly defined indicators applicable to pseudovirus standards, in order to improve the accuracy and comparability of standard values and promote the standardization and traceability of viral nucleic acid detection reagent systems. Summary of the Invention
[0005] The purpose of this invention is to provide a method for evaluating the copy number of specific nucleic acids in quantitative pseudovirus antigen standards, comprising the following steps: S1. Provision of Standards and Gene Sequence Localization: Providing pseudovirus antigen standards containing the target gene sequence to be tested, wherein the target gene sequence is shown in SEQ ID NO:1; S2. Free DNA enzyme treatment and termination: The pseudovirus antigen standard is digested with DNase enzyme to remove non-encapsulated free DNA from the standard, and the enzyme reaction is terminated by high-temperature inactivation at 65°C. S3. Extraction and purification of target nucleic acid: The processed pseudovirus nucleic acid was extracted using magnetic beads with a hydroxysilane coating. Binding, washing and temperature-controlled elution were performed at pH 6.5-7.2, with the elution temperature controlled between 55-60°C, to obtain the target nucleic acid for subsequent amplification reactions. S4. Construction of digital PCR reaction system: The reaction system contains specific primer pairs and fluorescent probes designed for the target sequence. The primer pairs are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively, with a length of 20–24 bases and a GC content controlled at 45–55%. The probe, as shown in SEQ ID NO:4, contains a central C–T mismatched base, with its 5' end labeled with a FAM or HEX fluorescent group and its 3' end labeled with a BHQ1 or MGB quenching group. S5. Microdroplet formation and amplification: The reaction system is mixed with the oil phase to form microdroplets, generating 100,000–200,000 isolated independent reaction units, and the reverse transcription and PCR amplification reactions are completed under the set temperature control curve. S6. Fluorescence Reading and Poisson Modeling Calculation: Collect droplet fluorescence signals and calculate the proportion of positive droplets based on a Poisson distribution model, where the Poisson distribution model describes the random distribution of target nucleic acids within the droplets; satisfying:
[0006] in, This represents the number of target nucleic acid molecules in a unit droplet; k is a non-negative integer, representing the actual number of target nucleic acid molecules contained in a certain droplet; Here, is the Poisson distribution parameter, representing the average number of target nucleic acid molecules per unit droplet volume; The base of the natural logarithm The factorial of k; The minimum number of positive droplets was set at 500, and the relative standard deviation of the detection results was controlled within 5% to obtain the absolute copy number of specific nucleic acid in the pseudovirus antigen standard.
[0007] As a preferred technical solution, in step S3, during the magnetic bead extraction operation, the volume ratio of lysis buffer to magnetic beads is 3-5:1, and the binding reaction time is 5-12 minutes. The magnetic beads are monodisperse hydroxysilicone-coated magnetic beads with a particle size of 1.2~1.5 μm, and their surfaces are modified with charge neutralization to enhance the selective adsorption capacity of the target nucleic acid. The washing buffer was 80% ethanol, and the washing was performed twice for 30 seconds each time. The elution buffer was RNase-free water, and the elution volume was 60–80 μL.
[0008] As a preferred technical solution, in step S4, the design of the specific primers satisfies the following conditions: The primer binding site spacing is 90–110 bp, the primer dimer free energy ΔG is higher than −6 kcal / mol, the 3' end does not contain G bases, and during the design process, a sequence exclusion algorithm is used to remove regions that are homologous to the host genome or packaging vector sequence.
[0009] As a preferred technical solution, in step S4, the fluorescent probe SEQ ID NO:4 is a TaqMan structure probe with a C-T mismatched base introduced at position 9–12 of its center. The total length of the probe is 20–22 bases. The 5' fluorescent group is selected from FAM, HEX or TET, the 3' quenching group is BHQ1, and the probe is purified by HPLC.
[0010] As a preferred technical solution, in step S4, the droplet digital PCR reaction system includes the following components: PCR reaction buffer 1×, MgCl2 concentration of 3.0 mM, total concentration of dNTP mixture of 200 μM, final concentration of primers of 500 nM, final concentration of probe of 250 nM, reverse transcriptase 1 μL, high-fidelity DNA polymerase of 0.5 U, and total reaction volume of 20 μL.
[0011] As a preferred technical solution, in step S5, the emulsification conditions for forming microdroplets are a rotation speed of 2300 rpm and a time of 90 seconds. The oil phase used is biocompatible silicone oil containing 2% surfactant. The number of microdroplets formed is not less than 130,000, and the average volume is 0.9–1.2 nL.
[0012] As a preferred technical solution, in step S6, the fluorescence signal is read in a dual-channel mode. The droplets corresponding to different fluorescence channels are classified and counted, and the non-uniformity of droplet distribution is corrected based on the compensation calculation model of Poisson distribution to obtain the corrected nucleic acid copy number result.
[0013] As a preferred technical solution, in step S3, the extracted nucleic acid sample is tested by absorbance, and its A260 / A280 ratio is controlled within the range of 1.9–2.0. Furthermore, agarose gel electrophoresis confirms that there is no obvious degradation.
[0014] As a preferred technical solution, in step S6, the parameter λ in the Poisson distribution model represents the average number of target nucleic acid molecules per unit volume of the reaction system. This parameter is calculated through the ratio of the number of positive droplets to the total number of droplets, satisfying the following:
[0015] in, is the average number of target molecules per microliter of reaction system; k is the number of positive droplets detected; n is the total number of droplets; This is the operation for the natural logarithm.
[0016] As a preferred technical solution, the method is applicable to the quantitative evaluation of various pseudovirus antigen standards, which include pseudovirus particles encoding at least one of the viral gene sequences such as EV71 VP1, HBV preS1, HCV Core, or HIV gag.
[0017] The beneficial effects of this invention are: This invention introduces an enzyme treatment step targeting the characteristics of pseudovirus particles in sample pretreatment. It utilizes DNase to selectively remove free nucleic acid components and blocks enzyme activity through high-temperature heat inactivation, thus fundamentally solving the problem of "false high values" interference from free DNA in subsequent quantification. Compared to traditional physical purification methods, this treatment strategy is more specific and consistent in removing non-encapsulated target sequences, and is particularly suitable for pseudovirus antigen standard systems with complex sample structures and a high probability of free contamination.
[0018] In terms of nucleic acid extraction and reaction construction, this invention employs magnetic beads coated with a hydroxysilane layer, combined with acid-base window control and temperature-controlled elution mechanisms, to achieve efficient release and enrichment of encapsulated nucleic acids. Simultaneously, by introducing mismatched base regulation and binding region sequence decrossing strategies in primer and probe design, the amplification system maintains strong discrimination ability even in high background and low copy number scenarios, significantly improving the separation of specific amplification signals. This primer-probe co-design not only enhances analytical sensitivity but also effectively avoids the risk of false amplification from residual vector sequences.
[0019] At the data modeling level, this invention establishes a droplet statistical correction mechanism based on Poisson distribution and sets limiting conditions for the lower threshold and droplet number density in the calculation of the positive droplet proportion, making the final copy number estimation closer to the actual sample structure characteristics. By linking the modeling function parameter λ with the reaction system volume and dilution factor, a mathematical closed loop from single-droplet qualitative signal to the absolute value of the sample population is completed, effectively improving the consistency and traceability of the method across different batches of standards. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the method flow of the present invention; Figure 2 This is a schematic diagram of the comparative experimental results (λ value) of the present invention; Figure 3 This is a schematic diagram showing the comparative experimental results (estimated copy number) of the present invention. Detailed Implementation
[0021] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.
[0022] Example 1
[0023] This embodiment provides a method for evaluating the copy number of specific nucleic acids in quantitative pseudovirus antigen standards, the steps of which are as follows: Figure 1 As shown.
[0024] In step S1, pseudovirus particles containing the target gene sequence shown in SEQ ID NO:1 were constructed. The target sequence was inserted into the lentiviral expression vector pLVX-IRES-Puro, and co-transfected with helper plasmids psPAX2 and pMD2.G into HEK293T cells. After transfection and culture for 48 hours, the cell supernatant was collected, filtered through a 0.22 μm filter membrane, and ultracentrifuged at 80,000 g for 4 hours. The recovered pseudovirus particles were resuspended in Virus Storage Buffer and stored at −80 °C for later use.
[0025] In step S2, 100 μL of pseudovirus antigen standard was taken and 2 μL (1 U / μL) of DNase I working solution was added. The mixture was incubated at 37°C for 30 minutes to degrade free nucleic acid. Then, 2 μL of 50 mM EDTA was added and the mixture was incubated at 65°C for 10 minutes to inactivate the enzyme, ensuring that only encapsulated target nucleic acid was retained.
[0026] In step S3, nucleic acid was extracted from pseudovirus particles using magnetic beads. The DNase-treated sample was added to 350 μL of lysis buffer (containing GuSCN and β-mercaptoethanol). After complete lysis, 30 μL of magnetic beads with a diameter of approximately 1.3 μm and a surface coated with hydroxysilyl groups were added, and the binding reaction was carried out at pH 6.8–7.0 for 12 minutes. After binding, the sample was washed twice with 80% ethanol for 30 seconds each time, and the magnetic beads were magnetically separated. Finally, 60 μL of preheated (65°C) RNase-free water was added for elution to obtain the target RNA solution.
[0027] In step S4, a droplet digital PCR reaction system with a total volume of 20 μL was constructed, comprising: 10 μL of ddPCRSupermix for Probes (dUTP-free), 1 μL of forward primer SEQ ID NO:2 (10 μM), 1 μL of reverse primer SEQ ID NO:3 (10 μM), 0.5 μL of fluorescent probe SEQ ID NO:4 (10 μM), 1 μL of reverse transcriptase (200 U / μL), 5 μL of template RNA, and RNase-free water to a final volume of 20 μL. After preparation, the reaction mixture was incubated on ice for 5 minutes and immediately used for droplet generation.
[0028] In step S5, 20 μL of reaction mixture was mixed with 40 μL of oil phase to form microdroplets with an average volume of approximately 1 nanoliter in a droplet generation chip at 2200 rpm for 90 seconds. The emulsified droplets were then transferred to a PCR instrument and subjected to the following program: reverse transcription was performed at 50°C for 30 minutes, followed by pre-denaturation at 95°C for 10 minutes, and then 45 PCR cycles were performed, each cycle including denaturation at 94°C for 30 seconds and annealing extension at 60°C for 1 minute, and finally extension at 98°C for 10 minutes.
[0029] In step S6, the QX200 digital PCR reading system was used to collect droplet fluorescence signals, and the FAM channel was used for the detection of target nucleic acids. After the positive and negative droplets were separated, the average number of target molecules λ per unit reaction volume was estimated using the Poisson distribution model, and the number of target nucleic acid copies in the pseudovirus standard was calculated accordingly.
[0030] Example 2
[0031] Based on Example 1, this embodiment constructs a set of absolute quantification procedures for pseudovirus nucleic acid under different experimental conditions by adjusting enzyme treatment conditions, nucleic acid extraction parameters, and primer design strategies, thereby verifying the applicability and robustness of the method.
[0032] In step S1, lentiviral pseudovirus particles containing the sequence SEQ ID NO:1 were constructed. HEK293T cells were co-transfected with the target fragment, helper packaging plasmids psPAX2 and pMD2.G using the pLenti-CMV vector expression system. After culturing for 72 hours, the supernatant was collected, filtered through a 0.45 μm filter, and the viral particles were enriched by ultracentrifugation at 60,000 g for 3 hours. The cells were then resuspended in Tris buffer for storage.
[0033] In step S2, take 150 μL of pseudovirus standard, add 3 μL (2 U / μL) of DNase I solution, treat at 37°C for 20 minutes, then add 3 μL of 50 mM EDTA to terminate the reaction, and place in a 65°C water bath for 10 minutes for complete inactivation.
[0034] In step S3, 400 μL of lysis buffer was added to completely lyse the viral structure, followed by 100 μL of magnetic bead solution (lysis buffer to magnetic beads volume ratio of 4:1). The magnetic beads were 1.5 μm hydroxysilane-coated structures, and the binding reaction time was set to 10 minutes. After binding, the sample was washed twice with 80% ethanol for 30 seconds each time, and finally eluted with 65 μL of RNase-free water at 58°C to obtain the RNA extract. The extracted nucleic acid was analyzed by NanoDrop, and the A260 / A280 ratio was approximately 1.94, indicating good purity; agarose gel electrophoresis confirmed that no obvious degradation tailing was observed.
[0035] In step S4, a digital PCR reaction system with a total volume of 25 μL was constructed, including 1× reaction buffer, 4.0 mM MgCl2, 300 μM dNTP mixture, 1 μL each of primers (10 μM), 0.6 μL of probe (10 μM), 2 μL of reverse transcriptase, 0.5 U of high-fidelity DNA polymerase, 6 μL of RNA template, and the remainder to be made up with RNase-free water. The forward primer SEQ ID NO:2 binds to positions 15–38 of the target sequence, and the reverse primer SEQ ID NO:3 binds to positions 125–148, with an interval of approximately 105 bp. The probe SEQ ID NO:4 is 21 bases long, with a C–T mismatched base introduced at position 10. After HPLC purification, the 5' end is labeled HEX, and the 3' end is labeled BHQ1.
[0036] In step S5, the PCR reaction system was mixed with biocompatible silicone oil (containing 2% surfactant), and droplets were generated in a dedicated microdroplet chip at 2300 rpm for 90 seconds, resulting in approximately 140,000 droplets with an average volume of 1.1 nanoliters. The droplets were then transferred to a thermal cycling system and the following program was performed: reverse transcription at 50°C for 30 minutes, denaturation at 95°C for 10 minutes, followed by 45 PCR cycles (94°C for 30 seconds, 60°C for 1 minute), and a final extension at 98°C for 10 minutes.
[0037] In step S6, a dual-channel digital PCR system was used to read droplet fluorescence signals, with the HEX channel selected as the positive droplet discrimination channel. After recording the number of positive droplets and the total number of droplets, a Poisson distribution model was used for modeling. Comparative Example 1: Nucleic acid evaluation method for pseudovirus standards that have not undergone free DNAse treatment This comparative example provides a pseudovirus nucleic acid quantification method that does not include a cell-free DNA removal step, verifying the impact of step S2 on the specific nucleic acid copy number assessment procedure. This method is largely consistent with the procedure in Example 1, except that the DNase treatment step is omitted.
[0038] In step S1, a pseudoviral antigen standard containing the target gene sequence shown in SEQ ID NO:1 was provided and co-transfected in HEK293T cells using a lentiviral three-plasmid system (vector pLVX-IRES-Puro, helper plasmids psPAX2 and pMD2.G). After 48 hours, the supernatant was collected, filtered, purified by centrifugation at 80,000 g, and resuspended for storage.
[0039] This comparative example omits step S2, does not treat the samples with DNase I, and does not perform the 65℃ enzyme inactivation step, directly proceeding to the nucleic acid extraction process.
[0040] In step S3, 400 μL of lysis buffer containing GuSCN was added to 150 μL of untreated pseudovirus sample. After complete lysis, 80 μL of hydroxysilane-coated magnetic beads with a particle size of 1.5 μm were added (lysis buffer to magnetic beads volume ratio of 5:1), and the mixture was reacted for 12 minutes at a pH of 6.7. After two washes with 80% ethanol, 70 μL of nucleic acid solution was eluted with RNase-free water at 58°C.
[0041] In step S4, a digital PCR reaction system with a total volume of 25 μL was constructed, including: 1× PCR reaction buffer, 4.0 mM MgCl2, 300 μM total concentration of dNTP mixture, 500 nM concentrations of both forward primer SEQ ID NO:2 and reverse primer SEQ ID NO:3, 250 nM concentration of probe SEQ ID NO:4, 2 μL reverse transcriptase, 0.5 U high-fidelity DNA polymerase, 5 μL RNA template, and the remainder being RNase-free water.
[0042] In step S5, the above reaction system was mixed with the oil phase and emulsified at 2300 rpm for 90 seconds to generate microdroplets with an average volume of approximately 1.0 nanoliter and a total number of no less than 140,000. PCR amplification was then performed using the following temperature control program: 50℃ for 30 minutes, 95℃ for 10 minutes, for 45 cycles (94℃ for 30 seconds, 60℃ for 1 minute), followed by a final extension at 98℃ for 10 minutes.
[0043] In step S6, the fluorescence signal of the droplets is read, the number of positive droplets k and the total number of droplets n are identified, and the average copy number of the target nucleic acid in each microliter of the reaction system is calculated using a Poisson distribution model function. This further estimates the absolute content of specific nucleic acid in the pseudovirus antigen standard. Comparative Example 2: Evaluation Method for Pseudovirus Copy Numbers Extracted Using Non-Surface-Modified Magnetic Beads This comparative example illustrates the procedure for nucleic acid extraction using magnetic beads without hydroxysilane coating and surface charge neutralization modification. It differs from step S3 in this invention, but the other procedures remain consistent with the examples to construct a control group.
[0044] In step S1, pseudovirus particles containing the target gene sequence shown in SEQ ID NO:1 were constructed. HEK293T cells were co-transfected with the target sequence and helper plasmids psPAX2 and pMD2.G using the pLenti-CMV vector system. After culturing for 60 hours, the supernatant was collected, filtered through a 0.22-micron filter, and the virus was concentrated by ultracentrifugation at 70,000 g. The virus was then resuspended in sterile PBS and stored at −80°C.
[0045] In step S2, 120 μL of pseudovirus sample was taken, and 2.4 μL (2 U / μL) of DNase I solution was added. After incubation at 37°C for 25 minutes, 2.4 μL of 50 mM EDTA was added, followed by heating in a water bath at 65°C for 10 minutes to terminate the enzyme reaction.
[0046] In step S3, nucleic acid extraction was performed. After adding 360 μL of lysis buffer, 90 μL of unmodified carboxyl magnetic beads (lysis buffer to magnetic beads volume ratio 4:1) were added, and the mixture was reacted for 10 minutes, with the pH of the binding reaction controlled between 6.6 and 6.8. After binding, the sample was washed twice with 80% ethanol for 30 seconds each time, and finally, RNA was eluted with 70 μL of RNase-free water at 55°C. The obtained nucleic acid sample was analyzed by spectrophotometry and used for subsequent amplification.
[0047] In step S4, the reaction system was constructed with a total volume of 25 μL. The components included: 1× ddPCR reaction buffer, 3.5 mM MgCl2, 250 μM dNTP mixture, 500 nM each of forward primer SEQ ID NO:2 and reverse primer SEQ ID NO:3, 250 nM probe SEQ ID NO:4, 2 μL reverse transcriptase, 0.5 U high-fidelity polymerase, 5 μL RNA template, and RNase-free water to the final volume.
[0048] In step S5, the reaction solution and oil phase were mixed at a 1:2 volume ratio and emulsified at 2300 rpm for 90 seconds to generate approximately 130,000 droplets, each with a volume of approximately 1.1 nanoliters. The PCR amplification program was as follows: 50°C for 30 minutes, 95°C for 10 minutes, followed by 45 cycles (94°C for 30 seconds, 60°C for 1 minute), and finally an extension at 98°C for 10 minutes.
[0049] In step S6, fluorescence signals are collected and the number of positive droplets and the total number of droplets are counted. The nucleic acid copy number is calculated based on the Poisson distribution model to obtain the average number of target nucleic acid molecules per unit volume. The absolute copy number of the target sequence in the sample is then estimated by combining the reaction volume.
[0050] Comparative Example 3: A pseudovirus nucleic acid quantification method using mismatch-free base probes This comparative example describes the nucleic acid copy number evaluation process under conditions where fluorescent probes with mismatched structures are not used in digital PCR reactions. Most steps in this comparative example are consistent with those in Example 1, except that the probe used, SEQ ID NO:5, is a fully matched sequence and no artificial C–T mismatch sites are introduced.
[0051] In step S1, a pseudovirus standard was constructed using pLVX-IRES-Puro as the vector and psPAX2 and pMD2.G as helper plasmids. After co-transfection with HEK293T cells, the supernatant was collected after 48 hours of culture, filtered, and enriched by centrifugation at 75,000 g. The virus particles were then resuspended in PBS and stored.
[0052] In step S2, take 100 μL of sample, add 2 μL of DNase I working solution (1 U / μL), treat at 37°C for 30 minutes, then add 2 μL of 50 mM EDTA and incubate at 65°C for 10 minutes to inactivate the enzyme.
[0053] In step S3, 350 μL of lysis buffer was added and mixed with 70 μL of hydroxysilane-coated magnetic beads and reacted for 10 minutes, with the pH controlled at 6.7. After washing twice with 80% ethanol, the target RNA was eluted with 65 μL of RNase-free water at 58°C.
[0054] In step S4, a digital PCR reaction system with a total volume of 25 μL was constructed, comprising the following components: 10 μL of ddPCRSupermix, 3.5 mM of MgCl2, 250 μM of dNTP mixture, 500 nM each of forward primer SEQ ID NO:2 and reverse primer SEQ ID NO:3, probe sequence SEQ ID NO:5 with a total length of 21 bases, a complete match to the target sequence, containing no mismatched bases, labeled with FAM at the 5' end and BHQ1 at the 3' end, purified by HPLC at a concentration of 250 nM; 1.5 μL of reverse transcriptase, 0.5 U of high-fidelity DNA polymerase, and 5 μL of template RNA.
[0055] In step S5, the reaction mixture was mixed with biocompatible silicone oil and emulsified at 2300 rpm for 90 seconds, resulting in an average droplet volume of 1.0–1.2 nL and a total number of approximately 140,000 droplets. The amplification procedure was consistent with the previous examples, including reverse transcription, denaturation, 45 rounds of PCR cycles, and end extension.
[0056] In step S6, the fluorescence signal of the FAM channel is collected, the number of positive droplets and the total number of droplets are recorded, the average number λ of target molecules per unit volume is calculated using the Poisson distribution model, and the absolute copy number of the target nucleic acid is calculated by combining the reaction system volume.
[0057] Comparative experiment: To verify the effectiveness of the key technical features of this invention, quantitative experiments were conducted in Examples 1 and 2, and Comparative Examples 1, 2, and 3. Each experiment used the same batch of pseudovirus antigen standards as the detection target, with the target sequence being SEQ ID NO:1. The same digital PCR platform and detection process were employed. The number of positive droplets, the total number of droplets, and the average copy number (copies / mL) of the target nucleic acid were recorded. The experimental groups and detection results are shown in Tables 1 and 2 below. Table 1. Experimental Grouping Instructions
[0058] Table 2 Comparison of experimental results data
[0059] Data Analysis: The quantitative results from Examples 1 and 2 show that both completed the full sample preprocessing procedure, including enzyme treatment, modified magnetic bead extraction, and mismatch probe-guided digital PCR amplification. The estimated nucleic acid copy number values were within the same order of magnitude, and the λ values were similar, indicating that the present invention has good repeatability and consistency across multiple parameter ranges.
[0060] like Figure 2 and Figure 3 As shown, in Comparative Example 1, no cell-free DNA removal was performed. The residual non-encapsulated cell-free nucleic acids in the sample directly participated in the subsequent amplification reaction, resulting in an increase in the proportion of positive droplets. This led to a systematic increase in the λ value and copy number, indicating that the absence of enzyme treatment would significantly interfere with the determination of the true content of the standard.
[0061] In Comparative Example 2, unmodified ordinary carboxyl magnetic beads were used for extraction. Compared with hydroxyl silica-coated magnetic beads, they lacked the specific adsorption capacity for target nucleic acids. Although the samples were also treated with enzymes, the RNA recovery rate and purity were low, the number of positive droplets fluctuated greatly, and the λ value deviated from the example, indicating that the type of magnetic beads has a direct impact on nucleic acid extraction efficiency.
[0062] In Comparative Example 3, the probe SEQ ID NO:5, which did not employ a central mismatch structure, was replaced by a perfectly matched probe. This resulted in relatively weaker amplification-specific recognition, the appearance of some non-specific background signals, unclear droplet classification boundaries, and an overestimation of the number of positive droplets, leading to an amplified λ value. These results highlight the crucial role of the central C–T mismatch site in improving the specificity of digital PCR amplification and the accuracy of droplet identification.
[0063] In summary, the sample pretreatment mechanism, magnetic bead surface modification design, and probe mismatch strategy proposed in this invention have significant technical advantages in the absolute copy number quantification process, and can jointly ensure the accurate assessment of the target nucleic acid copy number in pseudovirus antigen standards.
[0064] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
Claims
1. A method for evaluating the copy number of specific nucleic acids in quantitative pseudovirus antigen standards, characterized in that, Includes the following steps: S1. Provision of Standards and Gene Sequence Localization: Providing pseudovirus antigen standards containing the target gene sequence to be tested, wherein the target gene sequence is shown in SEQ ID NO:1; S2. Free DNA enzyme treatment and termination: The pseudovirus antigen standard is digested with DNase enzyme to remove non-encapsulated free DNA from the standard, and the enzyme reaction is terminated by high-temperature inactivation at 65°C. S3. Extraction and purification of target nucleic acid: The processed pseudovirus nucleic acid was extracted using magnetic beads with a hydroxysilane coating. Binding, washing and temperature-controlled elution were performed at pH 6.5-7.2, with the elution temperature controlled between 55-60°C, to obtain the target nucleic acid for subsequent amplification reactions. S4. Construction of digital PCR reaction system: The reaction system contains specific primer pairs and fluorescent probes designed for the target sequence. The primer pairs are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively, with a length of 20–24 bases and a GC content controlled at 45–55%. The probe, as shown in SEQ ID NO:5, contains a central C–T mismatched base, with its 5' end labeled with a FAM or HEX fluorescent group and its 3' end labeled with a BHQ1 or MGB quenching group. S5. Microdroplet formation and amplification: The reaction system is mixed with the oil phase to form microdroplets, generating 100,000–200,000 isolated independent reaction units, and the reverse transcription and PCR amplification reactions are completed under the set temperature control curve. S6. Fluorescence Reading and Poisson Modeling Calculation: Collect droplet fluorescence signals and calculate the proportion of positive droplets based on a Poisson distribution model, where the Poisson distribution model describes the random distribution of target nucleic acids within the droplets; satisfying: ； in, This represents the number of target nucleic acid molecules in a unit droplet; k is a non-negative integer, representing the actual number of target nucleic acid molecules contained in a certain droplet; Here, is the Poisson distribution parameter, representing the average number of target nucleic acid molecules per unit droplet volume; is the base of the natural logarithm; The factorial of k; The minimum number of positive droplets was set at 500, and the relative standard deviation of the detection results was controlled within 5% to obtain the absolute copy number of specific nucleic acid in the pseudovirus antigen standard.
2. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S3, during the magnetic bead extraction operation, the volume ratio of lysis buffer to magnetic beads is 3-5:1, and the binding reaction time is 10-12 minutes. The magnetic beads are monodisperse hydroxysilicone-coated magnetic beads with a particle size of 1.2~1.5 μm, and their surfaces are modified with charge neutralization to enhance the selective adsorption capacity of the target nucleic acid. The washing buffer was 80% ethanol, and the washing was performed twice for 30 seconds each time. The elution buffer was RNase-free water, and the elution volume was 60–80 μL.
3. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S4, the design of the specific primers satisfies the following conditions: The primer binding site spacing is 90–110 bp, the primer dimer free energy ΔG is higher than −6 kcal / mol, the 3' end does not contain G bases, and during the design process, a sequence exclusion algorithm is used to remove regions that are homologous to the host genome or packaging vector sequence.
4. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S4, the fluorescent probe SEQ ID NO:4 is a TaqMan structure probe with a C–T mismatched base introduced at position 9–12 of its center. The total length of the probe is 20–22 bases. The 5' fluorescent group is selected from FAM, HEX or TET, the 3' quenching group is BHQ1, and the probe is purified by HPLC.
5. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S4, the droplet digital PCR reaction system includes the following components: The PCR reaction buffer is 1×, the MgCl2 concentration is 3.0-5.0 mM, the total concentration of the dNTP mixture is 200-400 μM, the final concentration of the primers is 500 nM, the final concentration of the probe is 200-300 nM, the reverse transcriptase is 1-2 μL, the high-fidelity DNA polymerase is 0.5 U, and the total reaction volume is 20-30 μL.
6. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S5, the emulsification conditions for forming microdroplets are a rotation speed of 2300 rpm and a time of 90 seconds. The oil phase used is biocompatible silicone oil containing 2% surfactant. The number of microdroplets formed is no less than 130,000, with an average volume of 0.9–1.2 nL.
7. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S6, the fluorescence signal is read in a dual-channel mode. Droplets corresponding to different fluorescence channels are classified and counted. The non-uniformity of droplet distribution is corrected based on the compensation calculation model of Poisson distribution to obtain the corrected nucleic acid copy number result.
8. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S3, the extracted nucleic acid samples were tested by absorbance, and the A260 / A280 ratio was controlled within the range of 1.9–2.
0. Agarose gel electrophoresis confirmed that there was no obvious degradation.
9. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, In step S6, the parameter λ in the Poisson distribution model represents the average number of target nucleic acid molecules per unit volume of the reaction system. This parameter is calculated by the ratio of the number of positive droplets to the total number of droplets, satisfying the following: ； in, is the average number of target molecules per microliter of reaction system; k is the number of positive droplets detected; n is the total number of droplets; This is the operation for the natural logarithm.
10. The method for evaluating the copy number of specific nucleic acid in the quantitative pseudovirus antigen standard according to claim 1, characterized in that, The method is applicable to the quantitative evaluation of various pseudovirus antigen standards, which include pseudovirus particles encoding at least one of the viral gene sequences of EV71 VP1, HBV preS1, HCV Core, or HIV gag.