A detection process for identifying genes associated with spontaneous abortion

Abstract

The application relates to the technical field of nucleic acid detection, and discloses a detection process for identifying spontaneous abortion associated genes, which comprises the following steps: establishing a reaction system containing to-be-detected nucleic acid, a blocking probe, betaine and dimethyl sulfoxide, using the betaine and the dimethyl sulfoxide to construct a solvent environment with consistent base pair melting temperature; placing the reaction system at an annealing temperature, so that the blocking probe is combined with a wild type background sequence and induces degradation fragments to form an intramolecular hairpin structure; heating to a target temperature at a preset control rate to dissociate the intramolecular hairpin structure; and using a polymerase or releasing the polymerase in situ in the reaction system and implementing an extension reaction with the target mutant sequence as a template, wherein the double-strand stability is converted from being dependent on base composition to being dependent on fragment length through the cooperation of chemical components and temperature gradient, non-specific interference generated by degraded nucleic acid is eliminated, and high-specificity enrichment of low-abundance pathogenic variations is ensured.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, and in particular relates to a detection process for identifying genes associated with spontaneous abortion. Background Technology

[0002] With the current development of clinical reproductive medicine, molecular diagnosis targeting cell-free nucleic acids in chorionic villus tissue from spontaneous abortion or peripheral blood of pregnant women is a way to clarify the cause of miscarriage and guide subsequent pregnancy. In molecular diagnostic practice, multiplex polymerase chain reaction is usually used to target and enrich the target nucleic acid sequence, and pathogenic mutation sites are identified by fluorescence detection or high-throughput sequencing. Spontaneous abortion samples have significant characteristics. The target nucleic acids in embryonic arrest tissue or maternal peripheral blood are highly fragmented, and the samples contain a large amount of normal maternal gene sequence background. Since the pathogenic mutations associated with spontaneous abortion and wild-type sequences usually only differ by a single nucleotide, and their hybridization thermodynamic properties are highly similar, extracting trace mutation signals from background noise under this physiological background has long been a technical challenge in the field of nucleic acid detection.

[0003] In existing reaction systems, amplification primers, wild-type templates, and mutant templates are all in the same thermodynamic environment. Conventional techniques attempt to expand the hybridization energy difference by optimizing the annealing temperature or introducing locked nucleic acid modifications. However, in practical applications, the melting temperature of nucleic acid double strands is not only limited by fragment length but also strongly depends on the internal base composition of the sequence. Highly fragmented degraded single strands of nucleic acid are prone to forming intramolecular hairpin structures or random mismatched dimers rich in guanine and cytosine base pairs when the ambient temperature decreases. Since guanine and cytosine base pairs have higher hydrogen bond dissociation energies, the thermodynamic stability of these non-specific hybrids composed of short fragments is even higher than that of specific double strands with longer sequences but rich in adenine and thymine base pairs.

[0004] Therefore, the technical problem to be solved by this invention is to construct a specific recognition mechanism that can smooth out differences in base composition and generate physical occupancy solely based on fragment length during the biochemical reaction stage without adding hardware assistance. Summary of the Invention

[0005] This invention provides a detection process for identifying genes associated with spontaneous abortion, comprising the following steps: Step S101: Establish a reaction system containing the nucleic acid to be tested, a blocking probe, betaine, and dimethyl sulfoxide; the nucleic acid to be tested contains the target mutant sequence and the wild-type background sequence; wherein, betaine and dimethyl sulfoxide constitute a solvent environment that makes the melting temperature of different base pairs uniform; Step S102: The reaction system is placed under annealing temperature and kept under preset annealing time for annealing hybridization to block the binding of the probe with the wild-type background sequence to form complementary double strands, and the degradation fragments in the nucleic acid to be tested form intramolecular hairpin structures. In step S103, the reaction system is heated from the endpoint temperature of step S102 to the target temperature at a preset control rate. During the heating process, intramolecular hairpin structures with fragment lengths lower than complementary double strands dissociate, while complementary double strands remain bound. In step S104, polymerase is used in the reaction system to perform an extension reaction using the target mutant sequence in the unbound state of the nucleic acid to be tested as a template. The complementary double strand blocks the polymerase from reading the wild-type background sequence during the extension process.

[0006] Preferably, the specific temperature path of step S103 is as follows: when the reaction system is in the first heating range of 45°C to 55°C, the temperature is increased at a first preset rate; when the reaction system is in the second heating range of 55°C to 65°C, the temperature is increased at a second preset rate; wherein, the second preset rate is lower than the first preset rate, and the second preset rate is in the range of 0.1°C / s to 0.15°C / s.

[0007] Preferably, the 3' end of the blocking probe is modified with a blocking group, which is at least one of a phosphate group, an amino group, an inverted deoxythymidine, or a C3 spacer.

[0008] Preferably, the molar ratio of the blocking probe to the wild-type background sequence in the nucleic acid to be tested is 50:1 to 200:1.

[0009] Preferably, the polymerase is a hot-start high-fidelity DNA polymerase; step S104 specifically involves: after the reaction system is heated to the target temperature and kept at a constant temperature, the hot-start high-fidelity DNA polymerase is released in situ or the hot-start high-fidelity DNA polymerase is added to the reaction system to carry out the extension reaction.

[0010] Preferably, the nucleic acid to be tested is extracted from chorionic villus tissue of spontaneous abortion or cell-free DNA from peripheral blood of pregnant women, and the target mutation sequence contains single nucleotide variant sites or copy number variant sites; the detection process also includes: sequencing the products generated by the extension reaction, and determining the genotype of the target mutation sequence based on the sequencing results.

[0011] Preferably, a nonionic surfactant is also added in step S101; the nonionic surfactant is polyoxyethylene sorbitan monolaurate, and its volume fraction in the reaction system is 0.05% to 0.1%.

[0012] Preferably, the cooling rate in step S102 is 2°C / s to 5°C / s.

[0013] Preferably, the sequence length of the blocking probe is 25 bp to 45 bp, and the denaturation temperature of the blocking probe is 3°C to 8°C higher than the original denaturation temperature of the wild-type background sequence.

[0014] Preferably, the concentration of betaine in the reaction system is 1.5 mol / L to 2.5 mol / L, and the volume fraction of dimethyl sulfoxide in the reaction system is 5% to 10%; the annealing temperature in step S102 is 35°C to 45°C, and the preset annealing time is 30 s to 60 s; the preset control rate in step S103 is 0.1°C / s to 0.2°C / s, and the target temperature is 62°C to 65°C.

[0015] Compared with existing technologies, the detection process for identifying genes associated with spontaneous abortion in this invention has the following advantages: 1. In the detection process for identifying genes associated with spontaneous abortion, a specific concentration of chemical modifiers is introduced into the reaction system to construct an equipotential buffer environment. This transforms the stability of double-stranded hybridization between nucleic acid sequences from a traditional base composition-dependent to a fragment length-dependent process. Based on this, precise temperature control using a reverse temperature gradient forms a molecular-scale thermodynamic filter at the surface level. This deep coupling of chemical environment reshaping and physical temperature control timing enables the blocking probe with extension blocking modification to form a stable long-chain physical occupancy structure with the wild-type sequence. Simultaneously, it induces ordered thermodynamic dissociation of the random mismatch structure formed by highly fragmented degradation background sequences. This mechanism eliminates the interference of sequence composition heterogeneity in degradation samples on detection specificity and enhances the detection system's ability to identify target sequences in complex genetic backgrounds.

[0016] 2. By utilizing the kinetic impact of the rapid drop in the reaction environment temperature field from the denaturation temperature zone to the nucleation temperature zone, the intramolecular folds and ineffective inter-fragment aggregation that spontaneously form in highly fragmented samples during normal cooling are forcibly broken. This rapid cooling physical intervention increases the effective kinetic collision frequency between the blocking probe and the background sequence template, overcoming the implicit shielding of the target recognition site caused by the steric hindrance effect of the degradation fragments. This mechanism ensures that before the specific extension reaction is initiated, a large number of wild-type background sequences have achieved physical locking with the modified probe, thereby cutting off the extension path of polymerase to non-specific background.

[0017] 3. By logically reshaping the temperature control timeline in the detection process, the original single annealing stage is broken down into a synergistic chain involving supercooled nucleation and reverse melting locking. This physical action sequence accurately captures the critical energy difference between perfectly complementary double strands and randomly mismatched double strands in the phase transition dissociation process of degraded nucleic acid samples. This allows the target mutant sequence to be clearly distinguished from the massive background sequence in the early stage of the biochemical reaction. This multi-mechanism linkage scheme solves the resource competition problem caused by the high fragmentation of samples in traditional processes, enabling substrate resources and enzyme activity in the detection system to accurately converge on the pathogenic mutation sites with lower abundance, thereby achieving structural optimization of the detection signal-to-noise ratio. Attached Figure Description

[0018] Figure 1 This is a flowchart of the pathogenic mutation enrichment process of the present invention, which utilizes the synergistic effect of chemical components and temperature gradient. Figure 2 This is a diagram showing the interaction between the hardware and software functions of the molecular diagnostic system of this invention in the detection of degraded samples. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] It should be noted that all directional and positional terms used in this invention, such as: up, down, left, right, front, back, vertical, horizontal, inner, outer, top, bottom, transverse, longitudinal, center, etc., are only used to explain the relative positional relationship and connection between components in a specific state (as shown in the accompanying drawings). They are only for the convenience of describing this invention and do not require that this invention be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention. In addition, the descriptions of "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms installation, connection, and linking should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to the internal connection of two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0022] In the description of this specification, references to the terms "an embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0023] A detection process for identifying genes associated with spontaneous abortion includes the following steps: Step S101: Establish a reaction system containing the nucleic acid to be tested, a blocking probe, betaine, and dimethyl sulfoxide; the nucleic acid to be tested contains the target mutant sequence and the wild-type background sequence; wherein, betaine and dimethyl sulfoxide constitute a solvent environment that makes the melting temperature of different base pairs uniform; Step S102: The reaction system is placed under annealing temperature and kept under preset annealing time for annealing hybridization to block the binding of the probe with the wild-type background sequence to form complementary double strands, and the degradation fragments in the nucleic acid to be tested form intramolecular hairpin structures. In step S103, the reaction system is heated from the endpoint temperature of step S102 to the target temperature at a preset control rate. During the heating process, intramolecular hairpin structures with fragment lengths lower than complementary double strands dissociate, while complementary double strands remain bound. In step S104, polymerase is used in the reaction system to perform an extension reaction using the target mutant sequence in the unbound state of the nucleic acid to be tested as a template. The complementary double strand blocks the polymerase from reading the wild-type background sequence during the extension process.

[0024] Preferably, the specific temperature path of step S103 is as follows: when the reaction system is in the first heating range of 45°C to 55°C, the temperature is increased at a first preset rate; when the reaction system is in the second heating range of 55°C to 65°C, the temperature is increased at a second preset rate; wherein, the second preset rate is lower than the first preset rate, and the second preset rate is in the range of 0.1°C / s to 0.15°C / s.

[0025] Preferably, the 3' end of the blocking probe is modified with a blocking group, which is at least one of a phosphate group, an amino group, an inverted deoxythymidine, or a C3 spacer.

[0026] Preferably, the molar ratio of the blocking probe to the wild-type background sequence in the nucleic acid to be tested is 50:1 to 200:1.

[0027] Preferably, the polymerase is a hot-start high-fidelity DNA polymerase; step S104 specifically involves: after the reaction system is heated to the target temperature and kept at a constant temperature, the hot-start high-fidelity DNA polymerase is released in situ or the hot-start high-fidelity DNA polymerase is added to the reaction system to carry out the extension reaction.

[0028] Preferably, the nucleic acid to be tested is extracted from chorionic villus tissue of spontaneous abortion or cell-free DNA from peripheral blood of pregnant women, and the target mutation sequence contains single nucleotide variant sites or copy number variant sites; the detection process also includes: sequencing the products generated by the extension reaction, and determining the genotype of the target mutation sequence based on the sequencing results.

[0029] Preferably, a nonionic surfactant is also added in step S101; the nonionic surfactant is polyoxyethylene sorbitan monolaurate, and its volume fraction in the reaction system is 0.05% to 0.1%.

[0030] Preferably, the cooling rate in step S102 is 2°C / s to 5°C / s.

[0031] Preferably, the sequence length of the blocking probe is 25 bp to 45 bp, and the denaturation temperature of the blocking probe is 3°C to 8°C higher than the original denaturation temperature of the wild-type background sequence.

[0032] Preferably, the concentration of betaine in the reaction system is 1.5 mol / L to 2.5 mol / L, and the volume fraction of dimethyl sulfoxide in the reaction system is 5% to 10%; the annealing temperature in step S102 is 35°C to 45°C, and the preset annealing time is 30 s to 60 s; the preset control rate in step S103 is 0.1°C / s to 0.2°C / s, and the target temperature is 62°C to 65°C.

[0033] Example 1: In the clinical molecular diagnostic scenario targeting chorionic villus tissue samples from spontaneous abortions, technicians face the dual physical challenges of highly fragmented target nucleic acids accompanied by a massive amount of maternal wild-type background sequences. Under this physiological background, the short nucleic acid fragments rich in guanine and cytosine generated during degradation can rapidly form intramolecular hairpin structures or random mismatched dimers with extremely high thermodynamic stability during conventional cooling annealing processes due to their high hydrogen bond dissociation energies. This surface phase transition state, dominated by heterogeneity in the underlying base composition, creates physical steric hindrance in space, shielding the target recognition sites on the wild-type template. This renders the conventional specific hybridization mechanism, which relies on static temperature steps, ineffective. Ultimately, the wild-type background, which has a concentration advantage, rapidly consumes the substrate and polymerase resources in the system during the initial stage of the biochemical reaction, and the trace signals of spontaneous abortion-associated mutant genes are masked by background noise.

[0034] A detection reaction system was established comprising the target nucleic acid, a blocking probe, betaine, and dimethyl sulfoxide (DMSO). An oligonucleotide with an inverted deoxythymidine modified at its 3' end was selected as the blocking probe. The molar ratio of the blocking probe to the wild-type background sequence in the target nucleic acid was set to 100:1. Betaine at a concentration of 2.0 mol / L and DMSO at a volume fraction of 6.6% were injected into the system. The reaction was then carried out according to the formula... The stability equilibrium coefficient R was calculated and calibrated to be 0.30. This specific solvent environment forcibly smooths the difference in hydrogen bond dissociation energy between guanine / cytosine base pairs and adenine / thymine base pairs at the underlying chemical interface, completely eliminating the dependence of the hybridization stability of the nucleic acid double strand on base composition and transforming it into a pure fragment length dependence. Addressing the steric hindrance and disordered entropy changes in the loop regions of residual short-sequence hairpin structures in highly degraded nucleic acids, the large-volume nonpolar solvent dimethyl sulfoxide molecules in the system can competitively intercalate into the single-strand microenvironment of the short-sequence hairpin loop regions due to their hydrophobic properties. Through physical steric hindrance, they forcibly disrupt the residual base stacking effect of free nucleotides within the loop regions, thereby thermodynamically offsetting the phase transition entropy difference caused by different folding morphologies. This forces the physical phase transition critical point of the micro-folding structure in this ion buffer system to converge into a single and quantifiable complementary stem length variable. The reaction system is controlled to rapidly decrease to 40°C at a cooling rate of 3°C / s and maintain this temperature for 45s. Annealing hybridization, the physical and kinetic impact of this supercooled environment forcibly breaks down the physical barrier of the slow self-assembly of degraded nucleic acids, driving the blocking probe with sequence length advantage to fully collide and combine with the wild-type background sequence to form complementary double strands. At the same time, the massive number of short degraded fragments in the nucleic acid to be tested also form mismatched intramolecular hairpin structures at this temperature. In this rapid cooling stage, the extremely rapid drop in temperature field is essentially using the surface time difference effect of Brownian motion to artificially cut off the thermodynamic path of the slow evolution of short chain degraded fragments to the lowest energy complementary state, instantly freezing them in a shallow mismatch kinetic trap with extremely high structural tension and extreme instability. Meanwhile, the free blocking probe, with its absolute concentration suppression of hundreds of times and the conformational flexibility of long chains, forcibly completes the physical integration with the wild-type background sequence through extremely high frequency molecular collisions within an extremely short cooling window. This rapid cooling mechanism, guided by the situation, achieves the overall suppression of effective assembly of short chains, maintains them in a high-energy mismatch state, and thus ensures the dynamic phase separation of the stable occupation of specific long chains.

[0035] The control reaction system slowly raises the ambient temperature from 40°C to 64°C through a first heating zone and a second heating zone. Specifically, in this embodiment, the temperature actually rises to 64°C when entering the second heating zone (55°C to 64°C), at a slow heating rate of 0.15°C / s. In this physical environment constructed by a chemically equipotential buffer solution and a slow reverse heating action, the chemical polarity regulation provides a base-free phase change medium for the dynamic evolution of the temperature field, transforming the heating process into a thermodynamic filter based on molecular size. With the continuous injection of thermodynamic kinetic energy, the short-chain intramolecular hairpin structure with a fragment length lower than the complementary double strand of the blocking probe, due to insufficient physical size, accelerates... Thermodynamic dissociation occurs in stages, and the blocking probe with a sequence length set at 35 bp and a melting temperature 5°C higher than the original melting temperature of the wild-type background sequence, maintains a stable complementary double-stranded binding state with the wild-type background sequence at a high temperature of 64°C due to its completely complementary length advantage. Through this mechanism of synergy between chemical composition and temperature gradient, the originally intertwined background interference structure is stripped away, and the wild-type sequence is formed into a long-chain physical occupant structure by the complementary double strand. In the specific implementation process, the system adopts thermally melted phase change microcapsule pre-encapsulation technology, pre-sealing the thermally started high-fidelity DNA polymerase inside micro / nano-scale inert paraffin capsules with a melting point precisely calibrated to 62°C, and then allowing it to settle. Suspended at the bottom of the liquid surface in the reaction tube; when the reaction system passes the critical phase transition point of 62℃ through a programmed temperature-controlled rise and stabilizes at an isothermal state of 64℃, the outer paraffin shell undergoes a phase transition and melts, then floats to the surface due to density differences. Utilizing the surface thermal convection effect, the internal polymerase is spontaneously and passively released in situ under isothermal and isotonic conditions. The entire process requires no opening of the tube cap or external mechanical sample addition, avoiding abrupt changes in the surface temperature field and disruption of the precise hybridization thermodynamic equilibrium caused by physical pipetting operations. After the reaction system is heated to 64℃ and maintained at an isothermal state, a thermally initiated high-fidelity DNA polymerase is released into the reaction system. At this point, the free target mutant sequence is fully exposed, and the polymerase... The target mutant sequence in the unbound state of the nucleic acid to be tested serves as a template to trigger the extension reaction. The wild-type background sequence, which is in greater numbers, is tightly occupied by complementary double strands that are closed at the 3' end, thus interrupting the polymerase's reading path for the wild-type background sequence during the extension process. The product generated by this extension reaction is extracted and sent to a high-throughput sequencing platform for genotyping. This detection process transcends the dependence on deep modification of complex primers and relies solely on the physical reconstruction of chemical potential energy boundaries and thermodynamic time axis. At the source, it transforms the thermodynamic blind zone unique to highly degraded samples into a physical barrier to screen out background noise, allowing the specific enrichment of extremely low abundance pathogenic variant signals to break free from the constraints of sequence heterogeneity.

[0036] Example 2: For highly fragmented spontaneous abortion villus tissue samples, a simulated physiological noise environment containing excessive wild-type DNA fragments was constructed. Standard nucleic acid samples containing the target mutant sequence were prepared. Wild-type genome sonication fragmentation products at a mass concentration 1000 times that of the target mutant sequence were incorporated into the standard nucleic acid samples. The fragment lengths of the fragmentation products were concentrated in the 30bp to 50bp range. A high background interference experimental sample was constructed, using a temperature control precision of 0.01... A thermal cycler with programmed temperature control was used as the reaction equipment, and a single-molecule fluorescence sequencing platform was used as the result detection equipment to determine the concentration of chemical components in the reaction system. Betaine and dimethyl sulfoxide were selected to eliminate the difference in hydrogen bond dissociation energy of base pairs. The concentration was set to balance the isostatic effects and polymerase activity. When the concentration was below the lower limit, the short fragments rich in guanine and cytosine maintained thermodynamic stability above the target value. When the concentration exceeded the upper limit, the high osmotic pressure changed the protein spatial conformation of the polymerase, causing loss of enzyme activity. Based on this technical balance, the betaine concentration in the experimental group was set at 2.0 mol / L and the volume fraction of dimethyl sulfoxide was 7.5%. At the same time, a first control group was set with a betaine concentration of 0 mol / L and a volume fraction of dimethyl sulfoxide of 0%. A second out-of-range control group was set with a betaine concentration of 3.0 mol / L and a volume fraction of dimethyl sulfoxide of 12%. A third deletion control group was also set. The heating rate was set at 0.5℃ / s.

[0037] The thermal cycler was started, and the reaction systems of each group were cooled to 40℃ and held for 45 seconds. The experimental group, the first control group, and the second out-of-range control group were heated to 64℃ at a rate of 0.15℃ / s. During this phase transition range, real-time fluorescence melting data of the system were collected. The measurement data showed that the melting temperature distribution range of short fragments with different base compositions in the experimental group was concentrated between 58.2℃ and 58.6℃, with a range of 0.4℃. In the first control group, the melting temperature distribution range of the corresponding short fragments was dispersed to 54.1℃ to 62.3℃, with a range of 8.2℃. This physical characterization objectively confirmed that the chemical buffer solution of a specific concentration smoothed out the phase transition differences caused by the number of hydrogen bonds inside the sequence. The melting temperature was controlled by the physical length of the fragment, so that the short fragment interferences completed thermodynamic dissociation before reaching 64℃.

[0038] After heating the reaction system to 64℃, the polymerase was released in situ or used in the reaction system to trigger the extension reaction. The extracted products were input into a single-molecule fluorescence sequencing platform to determine the mutation detection rate. The detection rate of the target mutation sequence in the experimental group reached 99.2%, and no non-specific amplified chimeras were detected. The mutation detection rate of the first control group was only 14.5%, and the sequencing data contained a large number of extension interference products of wild-type background short fragments. The mutation detection rate of the second out-of-range control group was 0.1%, confirming that the high concentration of chemical components caused polymerase inactivation. The mutation detection rate of the third partial deletion control group decreased to 41.3%, confirming that the excessively rapid heating rate caused the hairpin structure in the short chain molecule to not be fully dissociated, and the residual physical steric hindrance blocked the target occupancy of the probe. The mass concentration of the incorporated wild-type genome sonication fragmentation product was adjusted to construct a 100-fold... The performance response of the aforementioned chemical component and temperature gradient synergy mechanism under different noise pressures was tested using progressive noise gradients of 1000x, 5000x, and 10000x. The measurement data showed that the mutation detection rate of the experimental group remained stable at 99.1% and 99.2% at noise gradients of 100x and 1000x, respectively. When the background noise concentration increased to 5000x, the mutation detection rate showed a nonlinear inflection point and dropped to 62.4%. When the background noise concentration reached 10000x, the mutation detection rate dropped to 8.1%. This nonlinear degradation trend confirmed that the spatial physical collision frequency of total nucleic acid molecules exceeded the kinetic occupancy limit of the blocking probe, causing system overload. The measurement results objectively defined the optimal working window of the detection process in the 1000x background noise range and quantitatively verified the objective effect of the technical solution in filtering out specific physical interferences.

[0039] Example 3: This example combines Figures 1 to 2 A description of a detection process for identifying genes associated with spontaneous abortion, such as... Figure 1 As shown, the process, from top to bottom, includes the following steps: Nucleic acid input step, in which free deoxyribonucleic acid containing the target mutant sequence and wild-type background sequence is extracted; reaction system setup step, in which blocking probes, betaine, and dimethyl sulfoxide are added. This reaction system setup step is linked to an equipotential buffer environment module to construct a solvent environment with a consistent base pair melting temperature; annealing hybridization step is then performed, allowing the blocking probe to bind to the wild-type sequence and degrade the fragment to form an intramolecular hairpin structure; followed by a temperature-increasing dissociation step, causing the intramolecular hairpin structure of the low-length fragment to dissociate while the complementary double strand remains bound. This temperature-increasing dissociation step is linked to a reverse temperature gradient control module to raise the temperature to the target temperature at a preset control rate to eliminate non-specific fragments; the extension reaction step continues downwards, using polymerase or releasing polymerase in situ to extend the unbound target mutant sequence as a template; finally, the specific extension product output step is performed, achieving complementary double-strand blocking background reading and ensuring specific enrichment of low-abundance pathogenic variants.

[0040] like Figure 2 As shown, multiple device entities and their corresponding operation actions are defined within the boundary. Among them, the technician is connected to the action of establishing the reaction system, the capillary electrophoresis instrument is connected to the action of measuring the fragment length distribution matrix, the measurement and control unit is independently connected to the action of acquiring the inherent temperature control error data of the hardware and the action of outputting the target temperature and calibrating the heating rate, the thermal cycler is connected to the action of applying the temperature field and the action of triggering the extension reaction, and the single-molecule fluorescence sequencing platform is connected to the action of measuring the mutation detection rate and the action of determining the genotype.

[0041] Example 4: When the molecular diagnostic system processes spontaneous abortion chorionic villus tissue samples, the degree of nucleic acid degradation varies physically among different samples. When processing samples with a constant heating rate and a fixed target temperature, the hairpin structure formed by short nucleic acid fragments does not dissociate within the preset time window. This residual physical steric hindrance blocks the targeted binding path of the probe. The system receives an ex vivo sample tube containing nucleic acid extracts from spontaneous abortion chorionic villus tissue, reads the RFID tag on the outside of the sample tube, and uses a one-way hash algorithm to convert the plaintext information of the subject contained in the RFID tag into a unique, featureless hash code consisting of 64 characters. This severs the reverse tracing path between the physical sample and specific individual privacy data. In all subsequent processes, the data bus only allocates and transmits the featureless hash code as an information flow index. The initial conductivity of the solution in the ex vivo sample tube is measured by immersion in a microelectrode array, and the initial conductivity is converted into an initial ionic strength parameter. A 2μL sample of the nucleic acid to be tested is extracted and input into a capillary electrophoresis instrument to determine the fragment length distribution matrix. The median fragment length and the fragment length distribution range are extracted from the matrix and calculated according to the formula... Calculate the reference temperature; among which The reference temperature is k, and the linear mapping coefficient is k. is the median segment length, and C is the temperature constant.

[0042] Calculate the temperature compensation value based on the range of fragment length distribution, add the temperature compensation value to the reference temperature, output the target temperature, extract the range of the theoretical unwinding temperature range, and then apply the formula... Calculate the calibrated heating rate; where To calibrate the heating rate, As the reference heating rate, To determine the theoretical melting temperature range, based on the Debye-Hückel electrolyte solution theory, the environmental ionic strength of the nucleic acid system directly alters the hydrogen bond dissociation energy of base pairs. The initial ionic strength parameters are extracted from the system and then analyzed according to the formula... Calculate the betaine concentration for compensation; among which This indicates the betaine concentration compensation, which physically means the calibrated target concentration after offsetting the interference of impurity ions, and the value is greater than 0. The value represents the standard betaine concentration under standard conditions, and γ represents the ionic strength compensation coefficient, with a value range of 0.15 to 0.25. The formula represents the initial ionic strength parameter. Essentially, it is based on an empirical decay model from the whole to the surface, constructed from the competitive hydration effect between ions. Free inorganic impurity salt ions in the system preferentially and massively capture dipole water molecules, thereby weakening the electrostatic shielding envelope formed by betaine zwitterions on the nucleic acid backbone. The ionic strength compensation coefficient γ in the formula is a dimensional conversion constant derived by using multiple linear regression to continuously fit the thermodynamic shift of double-strand dissociation curves under multiple sets of standard salt concentration gradients. It is defined within the range of 0.15 to 0.25 to ensure that betaine molecules can be added precisely in small increments without exceeding the overall osmotic pressure collapse threshold of the system, thereby filling the key hydration sites captured by inorganic salts. The fluid control unit injects the corresponding volume of betaine reserve solution into the established reaction system according to the compensated betaine concentration to offset the physical disturbance of the residual salt ions in the sample extraction process on the stability of the solvent and other components.

[0043] The capillary electrophoresis system output fragments with a median length of 42 bp and a fragment length distribution range of 18 bp. A linear mapping coefficient of 0.5℃ / bp and a temperature constant of 41.5℃ were set. The calculated baseline temperature was 62.5℃, and the calculated temperature compensation value was 1.8℃. Adding these two values, the target temperature was 64.3℃. The calculated theoretical melting temperature range range was 6.2℃. A baseline heating rate of 1℃ / s was set, and the calculated calibration heating rate was 0.16℃ / s. The calibration heating rate and target temperature were input into the thermal cycler's control program. A temperature field was applied to the reaction system containing the target nucleic acid, blocking probe, betaine, and dimethyl sulfoxide. The reaction system reached the target temperature of 64.3℃ at the calibration heating rate of 0.16℃ / s. The gradient thermodynamic kinetic energy injection process matched the hairpin structure inside the sample. The dissociation kinetics curve shows that the hairpin structure within the short-chain molecule undergoes thermodynamic dissociation. In the reaction system, polymerase is used or polymerase is released in situ to trigger the extension reaction. Thermodynamic reconstruction eliminates the physical steric hindrance of fragment length distribution. The target mutation sequence in the nucleic acid to be tested undergoes specific amplification. The specifically amplified nucleic acid fragment is extracted and delivered to a single-molecule fluorescence sequencing platform to obtain the original light signal trajectory. The sequencing control terminal decodes the original light signal trajectory into a discrete base sequence file, compares the discrete base sequence file with the reference genome, calculates the ratio of the number of characteristic fragments covering the target mutation to the total read depth covering the target physical site, and generates a mutation abundance quantification index. The mutation abundance quantification index is stored in non-volatile memory as a neutral technical parameter characterizing the molecular composition ratio of the isolated nucleic acid sample for use by downstream professional equipment. The system does not output medical diagnostic conclusions.

[0044] Example 5: When a molecular diagnostic system faces the challenge of changing chemical reagents or sequencing platform hardware from batch to batch, the static values ​​of the temperature constant and linear mapping coefficient cause temperature drift in the thermodynamic screening network. A calibration reaction system is constructed by injecting target concentrations of betaine and dimethyl sulfoxide into a set of synthetic deoxyribonucleic acid (DNA) standards containing multiple known fragment lengths. A thermal cycler is used to apply temperature cycling to the calibration reaction system, and real-time fluorescence intensity changes of each DNA standard are collected during the heating process. The extreme points of the first derivative of the fluorescence signal are extracted to determine the standard melting temperature for the corresponding fragment length. A discrete data coordinate set is established based on the fragment length and the standard melting temperature. The least squares method is used to solve the discrete data coordinate set, and the slope of the fitted line is extracted as the linear mapping coefficient k. The intercept of the fitted line is also extracted as the temperature constant C.

[0045] The linear mapping coefficient k and the temperature constant C are input into the register of the measurement and control unit. The measurement and control unit obtains the inherent temperature control error data of the specific instrument as the hardware error compensation value. Based on the median length of the nucleic acid sample fragment and the range of the fragment length distribution, as well as the linear mapping coefficient k and the temperature constant C, the target temperature and the calibrated heating rate are calculated and output in real time in combination with the hardware error compensation value. The phase transition deviation between batches of chemical reagents and the hardware temperature control error cancel each other out in the physical measurement and calculation feedback. The static parameter setting is transformed into a closed-loop control process based on the physical feedback of the instrument on site. The molecular diagnostic system maintains the specific enrichment state of pathogenic mutation signals in a cross-platform deployment environment.

[0046] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A detection process for identifying genes associated with spontaneous abortion, characterized in that, Includes the following steps: Step S101: Establish a reaction system containing the nucleic acid to be tested, a blocking probe, betaine, and dimethyl sulfoxide; the nucleic acid to be tested contains the target mutant sequence and the wild-type background sequence; wherein, betaine and dimethyl sulfoxide constitute a solvent environment that makes the melting temperature of different base pairs uniform; Step S102: The reaction system is placed under annealing temperature and kept under preset annealing time for annealing hybridization to block the binding of the probe with the wild-type background sequence to form complementary double strands, and the degradation fragments in the nucleic acid to be tested form intramolecular hairpin structures. In step S103, the reaction system is heated from the endpoint temperature of step S102 to the target temperature at a preset control rate. During the heating process, intramolecular hairpin structures with fragment lengths lower than complementary double strands dissociate, while complementary double strands remain bound. In step S104, polymerase is used in the reaction system to perform an extension reaction using the target mutant sequence in the unbound state of the nucleic acid to be tested as a template. The complementary double strand blocks the polymerase from reading the wild-type background sequence during the extension process.

2. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The specific temperature path of step S103 is as follows: when the reaction system is in the first heating range of 45℃ to 55℃, the temperature is increased at the first preset rate; when the reaction system is in the second heating range of 55℃ to 65℃, the temperature is increased at the second preset rate; wherein, the second preset rate is lower than the first preset rate, and the second preset rate is in the range of 0.1℃ / s to 0.15℃ / s.

3. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The 3' end of the blocking probe is modified with a blocking group, which is at least one of a phosphate group, an amino group, an inverted deoxythymidine, or a C3 spacer.

4. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The molar ratio of the blocking probe to the wild-type background sequence in the nucleic acid to be tested is 50:1 to 200:

1.

5. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The polymerase is a hot-start high-fidelity DNA polymerase; step S104 specifically involves: after the reaction system is heated to the target temperature and kept at a constant temperature, the hot-start high-fidelity DNA polymerase is released in situ or the hot-start high-fidelity DNA polymerase is added to the reaction system to carry out the extension reaction.

6. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The nucleic acid to be tested is extracted from cell-free DNA in chorionic villus tissue from spontaneous abortion or peripheral blood of pregnant women, and the target mutation sequence contains single nucleotide variant sites or copy number variant sites; the detection process also includes: sequencing the products generated by the extension reaction, and determining the genotype of the target mutation sequence based on the sequencing results.

7. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, In step S101, a nonionic surfactant is also added; the nonionic surfactant is polyoxyethylene sorbitan monolaurate, and its volume fraction in the reaction system is 0.05% to 0.1%.

8. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The cooling rate in step S102 is 2℃ / s to 5℃ / s.

9. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The blocking probe has a sequence length of 25 bp to 45 bp, and the denaturation temperature of the blocking probe is 3°C to 8°C higher than the original denaturation temperature of the wild-type background sequence.

10. The detection process for identifying genes associated with spontaneous abortion according to claim 1, characterized in that, The concentration of betaine in the reaction system is 1.5 mol / L to 2.5 mol / L, and the volume fraction of dimethyl sulfoxide in the reaction system is 5% to 10%; the annealing temperature in step S102 is 35℃ to 45℃, and the preset annealing time is 30s to 60s; the preset control rate in step S103 is 0.1℃ / s to 0.2℃ / s, and the target temperature is 62℃ to 65℃.

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