A droplet digital PCR absolute quantification method for mixed standard substance used in high-throughput sequencer calibration

Abstract

The application belongs to the field of gene sequencing, and discloses a microdroplet digital PCR absolute calibration method for mixed standard substances for high-throughput sequencer calibration and application thereof. The method comprises the following steps: providing N sets of primer probes for N targets in the standard substance; full background specificity verification: for the i th set of primer probes, it is necessary to prove that it can specifically detect the i th target, and prove that it has no specific detection signal in the mixed DNA sample composed of the remaining N-1 targets; only using all the primer probes verified in S2, performing parallel digital PCR detection on the standard substance sample; and calculating the absolute copy number concentration of each target according to the detection result. The method ensures the quantitative "purity" of each target in a complex mixed background, provides guarantee for high-confidence calibration results from the process design, and improves the throughput and efficiency of multi-target detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a method for absolute determination of microdroplet digital PCR using mixed standard substances for high-throughput sequencer calibration and its application. Background Technology

[0002] High-throughput sequencer calibration reference materials have significant applications in sequencer monitoring and calibration, clinical diagnostics, and other fields. These reference materials typically contain multiple different target DNA sequences, and their core metrological parameters—the absolute copy number concentration and relative abundance of each DNA component—must be accurately known and traceable to be effectively used for quality control and calibration of the sequencing process, interlaboratory comparisons, and kit performance verification.

[0003] Droplet digital PCR (ddPCR) technology has become the preferred method for DNA standard reference determination due to its advantages of achieving absolute nucleic acid quantification without the need for a standard curve and its strong tolerance to PCR inhibitors. However, when the standard reference to be tested contains multiple DNA targets of equal length, existing ddPCR determination methods face a severe technical bottleneck: it is difficult to systematically ensure the absolute specificity of the primer-probe system corresponding to each target in a complex mixed background. Even the presence of weak cross-reactions can cause imperceptible systematic biases in the quantification results; these biases are further amplified when calculating relative abundance, seriously affecting the accuracy and uncertainty assessment of the determination, and failing to meet the stringent requirements for metrological reliability of high-grade standard references.

[0004] Existing techniques are based on limited negative controls for ddPCR primer and probe validation and single-target quantification. This method is currently the standard practice for ddPCR quantification of multi-component DNA samples. Specific primers and fluorescent probes (such as TaqMan probes) are designed for each DNA target to be quantified. Subsequently, PCR or ddPCR tests are typically performed using a single-target DNA template or a mixture containing a few closely related target DNAs expected to cross-react, to validate the basic specificity and amplification efficiency of the primers and probes. After successful validation, N independent ddPCR experiments are performed on a mixed sample containing N targets using N sets of primers and probes. Each experiment targets only one target and is detected under a single fluorescence channel. The absolute copy number concentration of each target is calculated based on the number of positive droplets in each independent experiment. The obtained N independent concentration values ​​are normalized to calculate the relative abundance of each target.

[0005] This method is widely documented in molecular biology lab manuals and related research papers, serving as the benchmark procedure for ddPCR in multi-target analysis. Its drawbacks are twofold: First, it cannot eliminate the risk of cross-reactions in complex backgrounds: the specificity verification of its primers and probes is performed under simplified or ideal conditions (e.g., using a single target or limited mixed template). However, in actual assays—complex mixed standard materials containing all N targets—primers and probes may exhibit weak binding or cross-reactions with non-target targets that were not exposed in simplified verification. Such latent cross-reactions under "all-background" conditions cannot be effectively detected by existing verification protocols, becoming a hidden but critical source of systematic error in the assay results. Second, the uncertainty of the assay results is underestimated: due to the failure to systematically investigate and eliminate the error source of "all-background" cross-reactions, the inherent accuracy of the target concentration values ​​obtained based on this method is questionable. The calculated relative abundance and its uncertainty evaluation are incomplete, and the actual uncertainty may be significantly higher than the value in the evaluation report, leading to a reduction in the metrological quality grade of the standard material. Third, the method lacks a systematic quality control process to ensure the reliability of the assigned values: This technical solution treats primer and probe verification as relatively independent steps from subsequent value assignment, and the verification standards are insufficient. The entire process lacks a mandatory final review step, referenced to the final value assignment object (the mixture of all components), to ensure absolute independence between each detection unit. Therefore, this method cannot guarantee high-confidence value assignment results from a process design perspective.

[0006] Another related existing technology is the direct absolute quantification method based on multicolor fluorescence multiplex ddPCR. This method is currently the most advanced solution in the field of simultaneous multi-target detection using ddPCR technology. Its multicolor probe design involves designing multiple sets of specific primers and probes for multiple DNA targets requiring simultaneous quantification. Each set of probes is labeled with a different fluorescent reporter group (such as FAM, HEX / VIC, CY5, etc.) to ensure differentiation by the instrument's optical system within the same reaction tube. Multiplex reaction system establishment and optimization: Multiple primer-probe pairs are mixed in the same ddPCR reaction system. By optimizing the concentration of each primer-probe, annealing temperature, and other conditions, efforts are made to reduce primer dimer formation and competitive inhibition between different primer pairs, ensuring that each target achieves similar amplification efficiency. Single-tube multiplex ddPCR detection and data analysis: The high-throughput sequencer calibration sample to be tested is added to the above multiplex reaction system for droplet generation and PCR amplification. The signal of each droplet in multiple fluorescence channels is detected using an ddPCR instrument. Multidimensional scatter plot analysis was used to classify droplets into: negative (no target), single positive (containing a single target), double positive, or multi-positive (containing two or more targets) based on different combinations of fluorescence signals. Absolute copy number calculation: Based on the Poisson distribution, the number of positive droplets targeting each target (including single positive and multi-positive droplets containing that target) was counted, and the absolute copy number concentration of each target in the original sample was calculated.

[0007] This method is commonly found in multiplex detection application guidelines and related literature promoted by commercial ddPCR platforms (such as Bio-Rad), aiming to improve the throughput and efficiency of multi-target detection. However, its drawbacks are twofold. First, the throughput improvement is limited by the number of fluorescence channels: the number of targets that can be detected by this method is strictly limited by the number of fluorescence channels that the instrument can distinguish. Mainstream commercial ddPCR instruments typically only support 3-4 fluorescence channels, which means that only a maximum of 3-4 targets can be absolutely quantified in a single reaction. For complex standard substances containing more components (such as 10 or more), this method cannot achieve simultaneous quantification, and it is still necessary to revert to a multiple-detection approach, which cannot fundamentally solve the efficiency problem. Second, the multiplex reaction system is complex, difficult to optimize, and prone to introducing bias: placing multiple pairs of primers and probes in the same reaction system can easily lead to inconsistent amplification efficiency and difficulties in signal interpretation due to primer dimers, competition between different primer pairs, and fluorescence spectrum overlap (crosstalk). Especially for target mixtures with highly homologous sequences and large abundance differences, optimizing a multiplex system with balanced amplification of all targets and no cross-interference is extremely difficult, and may even be impossible. The inherent instability of such a system directly introduces quantitative biases that are difficult to quantify. Thirdly, the impact of cross-reactions is unavoidable and difficult to assess: in multiplex reactions, all target DNA templates and all primers and probes are present simultaneously, making the potential risk of cross-reactions higher than in single-detection methods. However, this method lacks an independent, "all-background" validation step to assess and eliminate this risk. Once a cross-reaction occurs, it can lead to incorrect fluorescence signal assignment (e.g., misclassifying non-specific signals as weak positives), severely affecting the accuracy of the assay, and this error is difficult to detect and correct.

[0008] Therefore, even with more advanced multiplex ddPCR protocols, it is still impossible to systematically solve the core metrological challenge of ensuring the "purity" of each target quantification in complex mixed environments, which is precisely the starting point of this invention's innovation. Summary of the Invention

[0009] This invention aims to overcome the fundamental defects of existing technologies in the absolute determination of standard materials for high-throughput sequencer calibration using droplet digital PCR (ddPCR). It provides a primer and probe set for the absolute determination of standard materials for high-throughput sequencer calibration, its preparation method, and applies it to the preparation of mixed standard materials for high-throughput sequencer calibration.

[0010] This invention addresses the shortcomings of existing technologies that rely on limited negative controls for verification: it provides a method that can systematically verify whether each set of primers and probes used for quantification has absolutely no cross-reactivity in a mixed sample containing all other non-target substances, thereby fundamentally eliminating quantitative bias caused by insufficient verification.

[0011] To address the problem that existing value setting processes lack mandatory quality control steps to ensure the reliability of results and that uncertainty is underestimated, a value setting process with "full background verification" as a mandatory prerequisite is established. This ensures that only detection units that have passed the most stringent specificity verification can participate in the final value setting, thereby guaranteeing the metrological reliability of the value setting results from a methodological perspective and ensuring that the uncertainty of the final assessment truly reflects the measurement level.

[0012] To address the challenge of balancing throughput, accuracy, and reliability in existing technologies (whether single- or multi-channel), a value determination scheme is proposed that does not rely on a limited number of multiple fluorescence channels, thus theoretically applicable to the value determination of any number (N) of targets. At the same time, through independent "validation-screening" and "parallel detection" steps, it avoids the optimization difficulties and potential interference of complex multi-channel systems, achieving scalable throughput while ensuring high accuracy and reliability.

[0013] This invention provides a method for absolute determination of droplet digital PCR values ​​using mixed standard materials for high-throughput sequencer calibration. The method includes the following steps:

[0014] (S1) Provide N sets of primers and probes for N targets in the standard substance;

[0015] (S2) Full background specificity verification: For the i-th set of primers and probes, it must be proven that it can specifically detect the i-th target, and at the same time prove that it has no specific detection signal in the mixed DNA sample composed of the remaining N-1 targets;

[0016] (S3) Perform parallel digital PCR detection on the standard material samples using only all primers and probes that have passed (S2) validation;

[0017] (S4) Calculate the absolute copy number concentration of each target based on the detection results.

[0018] Specifically, step (S2) includes:

[0019] (1) Primary validation: Using the NCBI BLASTN tool, the designed primers and probes are compared with the self-built sequence library. The relevant parameters are set. Only primers and probes that completely match the target sequence and have no significant similarity to other sequences can enter the next round of validation.

[0020] (2) Cross-reaction verification: Cross-reaction verification is performed using primers and probes that have undergone primary verification. The self-built sequence library is divided into N compatible groups based on sequence similarity, ensuring that the sequence difference within the same group is greater than 30%. Dimer detection: Dimer free energy between all primers and probes is calculated. Preferably, the OligoWalk function of DNAMAN software is used. Complementarity analysis: The number of consecutive complementary bases and non-consecutive complementarity are evaluated.

[0021] Specifically, the cross-reactivity verification includes the following three stages in sequence:

[0022] Phase 1: Specificity validation and detection unit access based on "full background negative control";

[0023] Preparation of detection unit: For the N DNA targets to be determined in the mixed standard substance, N sets of pre-designed specific primers and corresponding fluorescently labeled probes are provided, preferably such as TaqMan probes;

[0024] Perform a "full background" validation experiment: For the i-th set of primers and probes, perform the following two sets of ddPCR detections independently:

[0025] a) Self-target positive control detection: Using high-purity i-th target DNA as a template, it is confirmed that the detection unit can effectively detect its target;

[0026] b) Full background negative control detection: A mixed sample containing DNA from all (N-1) targets except the i-th target is used as a template for detection;

[0027] Set admission criteria and screening: A strict judgment threshold is pre-set. The primer probe set is deemed to have passed the verification and is allowed to enter the next stage only if the i-th primer probe set produces a clear positive signal in detection (a) and does not produce a positive signal in detection (b). Units that fail the verification are excluded.

[0028] Phase 2: Standardized parallel singlet ddPCR detection:

[0029] Configure parallel reactions: Configure N sets of primers and probes that have passed the first stage of validation into N independent ddPCR reaction systems according to their respective validated optimal single reaction conditions;

[0030] Sample loading and parallel operation: Add an equal amount of appropriately diluted high-throughput sequencer calibration standard sample as a common template to each reaction system; then, place these N reaction systems in the same ddPCR instrument, use the same thermal cycling program to start and complete amplification and detection in the same batch, so as to minimize instrument fluctuations and batch-to-batch differences;

[0031] Phase 3: Data Output

[0032] After parallel testing is completed, the ddPCR instrument will output N sets of independent droplet fluorescence data. Each set of data uniquely corresponds to a specific target that has been validated and can be directly used to calculate the absolute copy number concentration of the target in the original sample.

[0033] Furthermore, in step (S2), "no specific detection signal" means that the i-th set of primers and probes has no signal in the mixed DNA sample composed of the remaining N-1 targets.

[0034] Specifically, the parallel testing in step (S3) is carried out simultaneously under uniform reaction system and thermal cycling conditions.

[0035] Furthermore, step (S4) is as follows:

[0036] Each target of the reference material was absolutely quantified using droplet digital PCR technology to accurately determine its copy number concentration. Preferably, three technical replicates were set up for each sample.

[0037] Based on the results of digital PCR testing, the required dilution volume for each sample is determined using a standard calculation formula;

[0038] Dilute each target sample using a buffer solution (e.g., TE buffer (10 mM Tris-HCl, 1 mM EDTA) at pH 8.0) and mix thoroughly.

[0039] The thoroughly mixed DNA solution is precisely aliquoted into pre-chilled sterile cryovials, specifically, each aliquot has a volume of 50 μL; optionally, the process also includes:

[0040] The dispensed standard substances are numbered according to batch, and 100 tubes are sealed in a special cryopreservation box; they are immediately transferred to an ultra-low temperature freezer at -80℃ for storage, with temperature fluctuations controlled within ±5℃.

[0041] The present invention also provides an application of a specific primer-probe set verified and screened by the method in the absolute quantification of N targets in a mixed standard substance therein.

[0042] The present invention further provides a microdroplet digital PCR quantification kit for absolute quantification of N targets in a mixed standard substance, which includes a specific primer and probe set verified and screened by the method.

[0043] This invention also provides the use of the method and the primer-probe set obtained therefrom, or the kit described herein, in the preparation of formulations for the following purposes:

[0044] (a) Assigning standard values ​​to the standard substances;

[0045] (b) To perform homogeneity testing on the said standard substance; and / or,

[0046] (c) The stability of the standard substance is monitored.

[0047] This invention, through the innovative technical solution of "full background verification-access-parallel detection," fundamentally solves the problems of cross-reactivity risks, insufficient quality control, and the difficulty in balancing throughput and accuracy in existing value determination methods, thereby bringing the following significant and verifiable beneficial technical effects:

[0048] This invention completely eliminates systematic quantitative bias caused by cross-reactions, achieving a fundamental improvement in the accuracy of quantitative determination. Traditional validation is conducted only in simplified contexts, failing to expose the weak cross-reactions that may occur in real, complex samples. These undetected non-specific signals directly contaminate the quantitative results, becoming unassessable systematic errors. This invention, through the most rigorous "full-background validation," forcibly selects detection units that maintain absolute specificity even in the most realistic and complex working environments. This means that the signal source of each primer and probe set ultimately used for quantitative determination is highly "pure," fundamentally cutting off the input path of cross-reactions, the main source of error, and ensuring that the final measured copy number of each target is as close as possible to its true value.

[0049] A proactive, high-confidence metrological quality control system was constructed, making the uncertainty assessment of the determination results more realistic and reliable. This invention shifts quality assurance from "post-assessment" to "pre-prevention" and "in-process control." The access mechanism ensures the ultra-high reliability of the measuring instruments themselves; parallel testing eliminates the time and condition variations introduced by batch experiments. Therefore, the intrinsic quality (precision and accuracy) of the raw data obtained by this method is far superior to that of traditional methods. In subsequent uncertainty assessment based on metrological standards (such as GUM), since the main systematic errors (cross-reactions) have been actively eliminated and random errors have been minimized by parallel design, the synthesized uncertainty can more realistically and completely reflect the actual measurement level, avoiding the risk of underestimation of uncertainty due to uncontrolled potential interference, thereby supporting the attainment of higher metrological quality levels for standard materials.

[0050] This invention provides a scalable and universal calibration technique that simultaneously guarantees high accuracy and high reliability. This strategy offers dual advantages. First, it does not rely on a limited number of multiple fluorescence channels, theoretically applicable to the calibration of any number (N) of targets, exhibiting strong throughput scalability. Second, it avoids the problems of competitive suppression and spectral crosstalk that are difficult to optimize in complex and unstable multiplex reaction systems. By independently configuring and validating optimal reaction systems for each target, and then operating them under strictly controlled parallel conditions, this invention enables the detection of each target to be performed in its optimal state with zero interference between them. This successfully achieves an optimal balance among the three typically mutually restrictive goals of high throughput, high accuracy (optimal single-target performance), and high reliability (independent and interference-free), providing a universal and robust solution for the accurate calibration of standard materials for complex high-throughput sequencers. Attached Figure Description

[0051] Figure 1 Optimization of the three-step dye method and probe method program.

[0052] Figure 2 Comparison of Sequence 9 dye method and probe method.

[0053] Figure 3 Comparison of the dye method and probe method for sequence 37.

[0054] Figure 4 Comparison of the dye method and probe method for sequence 41.

[0055] Figure 5 Comparison of Sequence 60 dye method and probe method.

[0056] Figure 6 Comparison of the dye method and probe method for sequence 71.

[0057] Figure 7 Comparison of the dye method and probe method for sequence 74.

[0058] Figure 8 Comparison of the dye method and probe method for sequence 83.

[0059] Figure 9 Comparison of Sequence 85 dye method and probe method.

[0060] Figure 10 Comparison of Sequence 89 dye method and probe method.

[0061] Figure 11 Comparison of the dye method and probe method for sequence 93.

[0062] Figure 12 ddPCR droplet diagrams under different primer and probe concentration systems.

[0063] Figure 13 Digital PCR amplification results at different annealing temperatures.

[0064] Figure 14 Results of crossover experiment for gene sequence 1.

[0065] Figure 15 The results of the crossover experiment for Sequence 1. Channel 1 is the test sample, and channels 2 and 3 are the mixture of all other samples (96 mix).

[0066] Figure 16 Results of cross-testing of gene sequence 3. Channel 1 is the test sample, and channels 2 and 3 are mixtures of all other samples (96 mix).

[0067] Figure 17 Results of crossover experiments for gene sequence 4. Channel 1 is the test sample, and channels 2 and 3 are mixtures of all other samples (96 mix).

[0068] Figure 18 Results of crossover assay for gene sequence 5. Channel 1 shows the test sample, and channels 2 and 3 show the mixture of all other samples (96 mix). Detailed Implementation

[0069] This invention provides an absolute determination method for droplet digital PCR (ddPCR) of standard substances used for calibration of high-throughput sequencers.

[0070] Definitions of abbreviations and key terms

[0071] Droplet Digital PCR (ddPCR): A third-generation PCR quantitative method based on water-in-oil droplet technology, which divides the reaction system into tens of thousands of independent microreaction units and calculates the absolute copy number of the target molecule through endpoint detection.

[0072] Absolute Quantification: Determines the absolute number (e.g., copies / µL) of the target molecule in a sample directly, without relying on a standard curve.

[0073] Cross-reactivity validation: Verifies whether a primer-probe pair specifically amplifies its target sequence without reacting with non-target sequences.

[0074] High-throughput sequencing (HDS): often referred to as next-generation sequencing, is a technology that can simultaneously sequence millions to billions of DNA fragments in parallel.

[0075] The core of this method lies in establishing a closed-loop quality control system of "verification-admission-parallel value determination." Its fundamental difference from existing technologies lies in the introduction of a mandatory specificity verification step using a "full background mixture" as a negative control, which serves as a prerequisite for all subsequent value determination operations. To fundamentally guarantee the specificity of primers and probes, this study first used BioEdit software to integrate 97 baseline unit sequences to construct a local reference sequence library. This database contains complete information on all test sequences, serving as the sole comparison benchmark for subsequent primer and probe design and specificity verification.

[0076] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0077] Example 1: To ensure the specificity of the primers and probes, two levels of validation were performed:

[0078] 1. Initial Validation: Using the NCBI BLASTN tool, the designed primers and probes were compared with a self-built library of 97 sequences. Primer and probe sequence information is shown in Tables 1 and 3. Relevant parameters were set. Only primers and probes that completely match the target sequence and show no significant similarity to other sequences were allowed to proceed to the next round of validation.

[0079] 2. Cross-reaction verification: Cross-reaction verification was performed using primers and probes that had undergone primary verification. Based on sequence similarity, the 97 sequences were divided into N compatible groups, ensuring that the sequence difference within each group was greater than 30%. Dimer detection: The OligoWalk function of DNAMAN software was used to calculate the dimer free energy between all primers and probes. Complementarity analysis: The number of consecutive complementary bases (≤5bp) and non-consecutive complementarity (≤8bp) were evaluated. The results are shown in Tables 2 and 4.

[0080] The actual performance of primers and probes was verified by in vitro synthesis to ensure good specificity and amplification efficiency under experimental conditions.

[0081] Table 1 Sequence Primer Information

[0082]

[0083] Table 2. Primer complementarity detection.

[0084]

[0085] ① represents the complementary comparison between the primer at the 5' end of the first sequence and the primer at the 3' end of the same sequence;

[0086] ② represents the complementary comparison between the primer at the 5' end of the first sequence and the primer at the 5' end of the second sequence;

[0087] ③ represents the complementary comparison between the primer at the 5' end of the first sequence and the primer at the 3' end of the second sequence;

[0088] ④ represents the complementary comparison between the primer at the 5' end of the next sequence and the primer at the 3' end of the second sequence;

[0089] ⑤ represents the complementary comparison between the primer at the 3' end of the first sequence and the primer at the 3' end of the second sequence.

[0090] Table 3 Primer and probe sequence information

[0091]

[0092] Table 4. Detection of complementarity between primers and probes.

[0093]

[0094] ① represents the comparison of the primer complementarity between the probe sequence and the 5' end of the first sequence;

[0095] ② represents the comparison of the primer complementarity between the probe sequence and the 3' end of the first sequence;

[0096] ③ represents the comparison of the primer complementarity at the 5' end of the probe sequence and the second sequence;

[0097] ④ represents the comparison of the primer complementarity between the probe sequence and the 3' end of the second sequence;

[0098] ⑤ represents the primer complementarity comparison between two probe sequences.

[0099] Following the above analysis process, 97 primers with good fragment specificity were obtained, as shown in Table 5.

[0100] Table 5 Specific Primer List

[0101]

[0102]

[0103]

[0104] 1. Establishment and optimization of digital PCR determination method

[0105] By comparing the three-step dye method and the probe method, a standardized scheme that ensures both accuracy and efficiency was selected.

[0106] 1) Reaction program optimization

[0107] A three-step dye method and a probe method were compared and tested on the same batch of samples. See [link to final reaction procedure and optimization]. Figure 1 .

[0108] 2) Verification using the three-step dye method and probe method

[0109] Ten RUS fragments covering different GC contents (38.7%-61.2%) were selected and parallel quantitative verification was performed using the three-step dye method and probe method, respectively.

[0110] Table 6 Comparison of quantitative results between the three-step dye method and the probe method (unit: copies / μL)

[0111]

[0112] The results are shown in Table 6 and Figures 2-11 The relative deviations of the measured values ​​for the detected fragments by both methods were less than 5%, and no systematic bias was found. There was no significant difference in the quantitative results between the three-step dye method and the probe method. Based on the comprehensive advantages of ease of operation, cost-effectiveness, and high-throughput applicability, this study selected the three-step dye method as the quantification method for all RUS sequences.

[0113] 2. Optimization of amplification conditions

[0114] Based on the established three-step dye method, key reaction parameters were optimized.

[0115] 1) Primer concentration optimization: Three primer concentration gradients were set: 300, 500, and 700 nmol / L. See the results below. Figure 12 Analysis of digital PCR scatter plots revealed that a concentration of 500 nmol / L resulted in the clearest separation of positive and negative droplet clusters and the highest signal-to-noise ratio, thus determining it as the optimal primer concentration.

[0116] 2) Annealing temperature optimization: Six temperature gradients were set within the range of 55.0℃ to 62.7℃ for testing. Results Figure 13 The results show that annealing at 55.0℃ yields the highest amplification efficiency for the vast majority of fragments and the best separation effect for droplet clusters. Therefore, 55.0℃ was chosen as the uniform annealing temperature.

[0117] Therefore, the appropriate thermal cycling conditions for ddPCR were finally obtained, as shown in Table 7.

[0118] Table 7 Thermal cycling conditions for ddPCR

[0119]

[0120] The verification process consists of the following three stages:

[0121] Phase 1: Specificity validation and detection unit access based on "full background negative control"

[0122] The goal of this stage is to screen out qualified tools that can achieve absolute specificity in complex sample contexts.

[0123] Preparation of detection unit: For the N DNA targets to be determined in the mixed standard substance, N sets of pre-designed specific primers and corresponding fluorescently labeled probes (e.g., TaqMan probes) are provided.

[0124] Perform a “full background” validation experiment: For the i-th set of primers and probes (i=1 to N), perform the following two sets of ddPCR detections independently:

[0125] a) Self-target positive control detection: Using high-purity i-th target DNA as a template, the detection unit is confirmed to be able to effectively detect its target.

[0126] b) Full-background negative control detection: A mixed sample containing the DNA of all (N-1) target DNA except for the i-th target is used as a template for detection. This mixed sample fully simulates the complex environment of non-target components in the target.

[0127] Set admission criteria and screening: A strict judgment threshold is pre-set. The primer probe set is deemed to have passed the verification and is allowed to enter the next stage only if the i-th primer probe set produces a clear positive signal in detection (a) and does not produce a positive signal in detection (b). Units that fail the verification are excluded.

[0128] To verify the specificity of the established three-step dye method and the amplification system, a crossover experiment was designed among 97 samples.

[0129] First, a certain volume of each individual sample was aspirated. Then, the remaining 96 samples (excluding the individual sample) were mixed together and simultaneously detected using the designed primers. Each sample was tested twice. The specificity of the established ddPCR method means that each specific primer can only amplify the target sequence and cannot non-specifically amplify other sequences. As shown in the figure below, when using the primer for sequence 1, only the PCR reaction result for gene sequence 1 template was positive, while the PCR reaction results for sequences 2-97 templates were all negative. This indicates that the established ddPCR detection method for gene sequence 1 is highly specific and has good specificity for detecting this DNA template.

[0130] The same crossover experimental method was used to detect 97 gene sequences, and the ddPCR detection results were the same as described above. Taking sequences 1-5 as examples, the specificity was good. For details, please refer to [link to relevant documentation]. Figures 14-18 .

[0131] The results show that the ddPCR detection method can be used for subsequent detection of standard substances and determination of characteristic values.

[0132] Phase 2: Standardized parallel singlet ddPCR detection

[0133] This stage utilizes all validated "clean" detection units to quantify the target sample under highly consistent conditions.

[0134] Configure parallel reactions: Configure N sets of primers and probes that have passed the first stage of validation into N independent ddPCR reaction systems according to their respective validated optimal single reaction conditions.

[0135] Sample loading and parallel runs: An equal volume of appropriately diluted high-throughput sequencer calibration standard material was added to each reaction system as a common template. Subsequently, these N reaction systems were placed in the same ddPCR instrument, and the same thermal cycling program was used to start and complete amplification and detection within the same batch to minimize instrument fluctuations and batch-to-batch differences.

[0136] Phase 3: Data Output

[0137] After parallel testing is completed, the ddPCR instrument will output N sets of independent droplet fluorescence data. Each set of data uniquely corresponds to a specific target that has been validated and can be directly used to calculate the absolute copy number concentration of the target in the original sample.

[0138] Example 2: Technical Variation in the "Full Background Verification" Step

[0139] Step-by-step background verification

[0140] To reduce the complexity of a single validation, the "full background" (N-1 non-target targets) validation can be broken down into multiple rounds of grouped validation. For example, all non-target targets can be first divided into several logical groups (e.g., grouped by sequence similarity), and the specificity of the i-th set of primers and probes for each group of mixtures can be validated sequentially. Full background validation is considered successful only if all grouped validations pass. This reduces the complexity of a single reaction, but the core idea (validation must cover all non-target background) remains consistent with this invention.

[0141] Competitive inhibition verification

[0142] In the "all-background negative control detection," in addition to adding the template from the non-target mixture, a trace amount of the target DNA was also added as a competitive internal standard. The validation criteria were: the primer probe must be able to specifically and quantitatively detect the trace target under this competitive background, without generating any positive signal from the non-target mixture. This protocol, while validating specificity, also examined the quantitative robustness of the detection unit under complex backgrounds.

[0143] Example 3: Technical Variation in the "Parallel Detection" Step

[0144] Multi-channel independent detection based on microfluidic chip

[0145] Instead of using traditional droplet-based ddPCR, a chip-based digital PCR (cdPCR) approach is employed. This chip pre-immobilizes different primers and probes in separate, independent reaction chambers. During detection, standard samples are introduced into the chip, distributing them evenly across all chambers, enabling physically isolated parallel detection of N targets on a single chip. This achieves the goal of "independent, parallel, and cross-contamination-free" detection.

[0146] "Primer and probe premixing - single-tube partitioning" detection

[0147] N sets of validated primers and probes are pre-mixed, but a single-tube ddPCR reaction is still used. The key is to achieve absolute signal differentiation within a single tube through extreme reaction condition optimization (such as using highly rigorous hot-start enzymes and special buffer systems) and highly specific probe signal design (such as using probes with different luminescence principles, like TaqMan probes with different quenchers and molecular beacons). This approach attempts to achieve a "parallel, interference-free" effect through chemical means without dividing the physical reaction system, representing an attempt to move towards traditional "multiplex detection" while pursuing the same level of reliability.

[0148] Example 4: Molecular absolute counting and bioinformatics correction scheme based on next-generation sequencing (NGS)

[0149] Extremely high-depth whole-genome or targeted sequencing was performed on standard material samples. The absolute number of molecules for each target was calculated by absolutely counting the sequencing reads and correcting for known sample input and molecular recovery models of the sequencing process.

[0150] This approach completely avoids the specificity crossover problem inherent in primer-probe based methods because its detection is based on the complete sequence information, rather than pre-designed short fragments. It also aims to achieve high-precision absolute quantification of components in complex mixtures through different technical approaches (sequencing and bioinformatics analysis).

[0151] Example 5: Detection of absolute copy number concentration of each target

[0152] To ensure the excellent stability and metrological consistency of the reference materials during storage and use, standardized dispensing procedures and strict storage conditions have been established. All operations are performed in a clean environment that meets the requirements of molecular biology experiments, minimizing exogenous contamination and nucleic acid degradation.

[0153] 1. Pre-packaging and concentration standardization

[0154] 1) Initial concentration determination

[0155] Absolute quantification of 97 DNA candidates was performed using droplet digital PCR technology to accurately determine their copy number concentration; three technical replicates were set up for each sample. Preliminary concentration detection results are shown in Table 8.

[0156] 2) Dilution calculation and preparation

[0157] Based on the ddPCR results, the required dilution volume for each sample was determined using a standard calculation formula:

[0158] Required dilution volume = (Initial concentration × Initial volume) / Target concentration - Initial volume

[0159] The target concentration was set at 5 nM to meet the sample introduction requirements of most high-throughput sequencing platforms.

[0160] Table 8 Preliminary Values ​​Table

[0161]

[0162] 2. Storage and Dispensing

[0163] TE buffer (10 mM Tris-HCl, 1 mM EDTA) at pH 8.0 was used as the standard dilution and storage medium. All dispensing operations were performed in a biosafety cabinet to ensure a sterile environment. The specific procedure is as follows:

[0164] 1) Sample pretreatment: Place the DNA stock solution stored at -80℃ on ice to thaw slowly; after it is completely thawed, use a vortex mixer to shake at 3000 rpm for 10 seconds to ensure that the solution is homogeneous; collect the droplets on the tube wall by short centrifugation.

[0165] 2) Dilution and mixing: Using a metered pipette, accurately transfer the calculated volume of DNA sample and TE preservation solution into a sterile container and mix. The specific volumes of each sequence are shown in Table 9. Place the mixture in a shaker at 4°C and shake at 150 rpm for 60 minutes to ensure thorough mixing.

[0166] 3) Aliquoting: Using a pipette, accurately aliquot the well-mixed DNA solution into pre-chilled 0.5 mL sterile cryovials. Aliquot volume per tube: 50 μL.

[0167] 4) Storage and management: Number the dispensed standard substances by batch and seal 100 tubes in a special cryopreservation box; immediately transfer them to an ultra-low temperature freezer at -80℃ for storage, with temperature fluctuations controlled within ±5℃.

[0168] Through the standardized dispensing and storage process described above, we ensure that this standard substance maintains good stability and quantitative consistency during storage.

[0169] Table 9 Requires a detailed volume table for each sequence.

[0170]

[0171] Example 6: Standard Reference Material Value Determination and Screening

[0172] 1) Screening and verification by joint value-setting laboratories

[0173] A multi-laboratory joint assay was used to assign values ​​to 97 genes. The main principles for selecting laboratories were: first, their relevant measurement capabilities; second, their representativeness within the industry or field; and third, consideration of different digital PCR platforms. Eight laboratories and two models of testing instruments were selected from several sectors, including the National Health Commission, agriculture, third-party testing centers, testing companies, and metrology institutions. Specific information is shown in Table 10. All eight laboratories used the Bio-Rad QX200 microdroplet digital PCR instrument from Bio-Rad Laboratories (USA), and one laboratory used the DQ-24 microdroplet digital PCR instrument from Sinopharm.

[0174] Table 10 Joint Value-Setting Laboratory Numbers and Instrument Models

[0175]

[0176] The organizing unit distributed blind samples (human (male) genomic DNA quantification standard, GBW09856) to the eight participating units. Upon receiving the blind samples, the participating units conducted value determination experiments according to the organizing unit's value determination scheme and submitted the measurement results. The organizing unit summarized the measurement results, and the data processing and result evaluation process is as follows:

[0177] (1) The original results of the blind sample test in the laboratory are shown in Table 11.

[0178] Table 11 Raw results of laboratory blind sample testing (copies / μL)

[0179]

[0180] (2) Comparison of laboratory and blinded sample results

[0181] The assigned values ​​of the participating units were compared with those of the standard reference material. Reference values ​​and uncertainty data are shown in Tables 12 and 13.

[0182] Table 12 Reference values ​​and uncertainty data for quantitative standard substances

[0183]

[0184] Table 13 Laboratory Value Set Results

[0185]

[0186] The blind sample test results showed that the measurement results from all eight participating laboratories did not exceed the range of the certified reference materials and exhibited acceptable repeatability. These laboratories possess basic and reliable detection capabilities in DNA quantification, meeting the initial requirements for participating in this multi-gene joint value determination study.

[0187] 2) Standard reference value determination scheme

[0188] Based on the initial determination results of the mother liquor, the 97 sample sequences were weighed to ensure the uniformity of each fragment throughout the mixed standard. Digital PCR was then used to determine the values ​​of the prepared mixed standard. Three tubes of the standard were randomly selected and diluted twice 100-fold using a balance. The 97 gene sequences in the diluted standard were measured twice in duplicate. The Dixon criterion was used to check for any suspicious values. The mean and standard deviation of the measured data were examined for statistical significance. If no statistical significance was found, the overall mean and standard deviation were calculated.

[0189] 3) Standard reference value determination

[0190] Nine laboratories participated in the collaborative determination. Each laboratory independently measured three sample units under repeatability conditions, and each sample unit was measured twice, resulting in a total of nine sets (six values ​​per set) of raw measurements.

[0191] During the data statistical processing stage, the following steps are performed:

[0192] Cochrane Test: First, the Cochrane test was used to test the homogeneity of variances for the nine groups of data. The test showed that the variances of each group were within the significance range. There was no significant difference at C=0.05 (C max <C 临界 This indicates that the measurement precision of the nine laboratories is consistent, and the data can be further merged for analysis.

[0193] Grubbs' test: After confirming consistent precision within the group, the results of each laboratory unit were calculated, yielding 54 independent data points. The Grubbs' test was used to test for outliers in this group. The test rule is as follows: If a measured value x... i There are residuals ,when At that time, It should be removed. It depends on the number of measurements and the given significance level. The relevant values. No outliers were found, and the means from all laboratories have been retained.

[0194] Shapiro-Wilk test: The Shapiro-Wilk method was used to test the normality of the nine laboratory data points retained after the above test. The test results show that the data follow a normal distribution.

[0195] Based on the above systematic verification, all data involved in the value determination are reliable in quality and meet the requirements of parameter statistics. Taking sequence 1 as an example, the test results from 9 laboratories are shown in Table 14. Therefore, the arithmetic mean of the results from the 9 laboratories is used as the standard value of this reference material.

[0196] Table 14 Result of Sequence 1

[0197] .

Claims

1. A method for absolute determination of droplet digital PCR values ​​using mixed standard substances for high-throughput sequencer calibration, characterized in that, The method includes the following steps: (S1) Provide N sets of primers and probes for N targets in the standard substance; (S2) Full background specificity verification: For the i-th set of primers and probes, it must be proven that it can specifically detect the i-th target, and at the same time prove that it has no specific detection signal in the mixed DNA sample composed of the remaining N-1 targets; (S3) Perform parallel digital PCR detection on the standard material samples using only all primers and probes that have passed (S2) validation; (S4) Calculate the absolute copy number concentration of each target based on the detection results.

2. The method according to claim 1, characterized in that, The step (S2) includes: (1) Primary validation: Using the NCBI BLASTN tool, the designed primers and probes are compared with the self-built sequence library. The relevant parameters are set. Only primers and probes that completely match the target sequence and have no significant similarity to other sequences can enter the next round of validation. (2) Cross-reaction verification: Cross-reaction verification is performed using primers and probes that have undergone primary verification. The self-built sequence library is divided into N compatible groups based on sequence similarity, ensuring that the sequence difference within the same group is greater than 30%. Dimer detection: Dimer free energy between all primers and probes is calculated. Preferably, the OligoWalk function of DNAMAN software is used. Complementarity analysis: The number of consecutive complementary bases and non-consecutive complementarity are evaluated.

3. The method according to claim 2, characterized in that, The cross-reactivity verification includes the following three stages in sequence: Phase 1: Specificity validation and detection unit access based on "full background negative control"; Preparation of detection unit: For the N DNA targets to be determined in the mixed standard substance, N sets of pre-designed specific primers and corresponding fluorescently labeled probes are provided, preferably such as TaqMan probes; Perform a "full background" validation experiment: For the i-th set of primers and probes, perform the following two sets of ddPCR detections independently: a) Self-target positive control detection: Using high-purity i-th target DNA as a template, it is confirmed that the detection unit can effectively detect its target; b) Full background negative control detection: A mixed sample containing DNA from all (N-1) targets except the i-th target is used as a template for detection; Set admission criteria and screening: A strict judgment threshold is pre-set. The primer probe set is deemed to have passed the verification and is allowed to proceed to the next stage only if the i-th primer probe set produces a clear positive signal in detection (a) and does not produce a positive signal in detection (b). Units that fail the verification are excluded. Phase 2: Standardized parallel singlet ddPCR detection: Configure parallel reactions: Configure N sets of primers and probes that have passed the first stage of validation into N independent ddPCR reaction systems according to their respective validated optimal single reaction conditions; Sample loading and parallel runs: Add an equal amount of appropriately diluted high-throughput sequencer calibration standard sample of the unknown value to each reaction system as a common template; Subsequently, these N reaction systems were placed in the same ddPCR instrument, and the same thermal cycling program was used to start and complete amplification and detection within the same batch to minimize instrument fluctuations and batch-to-batch differences. Phase Three: Data Output After parallel testing is completed, the ddPCR instrument will output N sets of independent droplet fluorescence data. Each set of data uniquely corresponds to a specific target that has been validated and can be directly used to calculate the absolute copy number concentration of the target in the original sample.

4. The method according to claim 2, characterized in that, In step (S2), "no specific detection signal" means that the i-th set of primers and probes has no signal in the mixed DNA sample composed of the remaining N-1 targets.

5. The method according to claim 1, characterized in that, The parallel testing in step (S3) is carried out simultaneously under uniform reaction system and thermal cycling conditions.

6. The method according to claim 2, characterized in that, Step (S4) is as follows: 1) For each target of the target, the candidate is absolutely quantified using droplet digital PCR technology to accurately determine its copy number concentration. Preferably, three technical replicates are set up for each sample. 2) Based on the results of digital PCR testing, determine the required dilution volume for each sample using a standard calculation formula; 3) Dilute each target sample using a buffer solution (e.g., TE buffer (10 mM Tris-HCl, 1 mM EDTA) at pH 8.0) and mix them thoroughly. 4) Accurately aliquot the well-mixed DNA solution into pre-chilled sterile cryovials, specifically, each aliquot should be 50 μL; optionally, this also includes: 5) After the standard substances have been dispensed, they are numbered according to batch number, and 100 tubes are sealed in a special cryopreservation box; Immediately transfer to an ultra-low temperature freezer at -80℃ for storage, with temperature fluctuations controlled within ±5℃.

7. The application of a specific primer-probe set verified and screened by the method of any one of claims 1 to 6 in the absolute quantification of N targets in a mixed standard substance therein.

8. A microdroplet digital PCR quantification kit for absolute quantification of N targets in a mixed standard substance, characterized in that, It comprises a set of specific primers and probes that have been verified and screened by the method described in any one of claims 1 to 6.

9. The use of the method of any one of claims 1 to 6 and the primer-probe set obtained therefrom, or the kit of claim 8, in the preparation of formulations for the following purposes: (a) Assigning standard values ​​to the standard substances; (b) To perform homogeneity testing on the said standard substance; and / or, (c) The stability of the standard substance is monitored.

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