A nucleic acid detection composition for screening of cisplatin resistance of non-small cell lung cancer

Abstract

The application relates to the technical field of biochemical detection, and discloses a nucleic acid detection composition for screening of cisplatin drug resistance of non-small cell lung cancer, which comprises a specific amplification primer pair for targeting an NFAT2 gene, a TaqMan detection probe, a hot-start Taq DNA polymerase and a reaction buffer solution; the reaction buffer solution comprises tetrahydro pyrimidine and tetramethylammonium chloride, and the molar ratio of the two is controlled to be between 3.2:1 and 3.8:1 to construct a micro function phase structure. The application blocks cisplatin through a dense hydration shell layer formed by tetrahydro pyrimidine on the surface of an enzyme, and eliminates the base pair hydrogen bond energy difference of a nucleic acid major groove by using tetramethylammonium chloride, so that efficient protection of polymerase activity and regulation of primer hybridization specificity are simultaneously realized in a single reaction system, and the sensitivity and accuracy of drug resistance gene detection in complex clinical samples are improved.

Description

Technical Field

[0001] This invention relates to a nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer, belonging to the field of biochemical detection technology. Background Technology

[0002] Current clinical screening for carboplatin resistance in non-small cell lung cancer relies on quantitative detection of the expression level of nuclear factor 2 gene in activated T cells in tumor tissue. Conventional reverse transcription polymerase chain reaction requires the reaction system to maintain DNA polymerase activity and maintain high specificity of primer-template hybridization to ensure that the cycle threshold and target concentration are linearly related.

[0003] Clinical biopsy tissue or pleural effusion samples often contain cell necrosis, releasing high concentrations of non-targeted genomic DNA fragments and residual chemotherapy drugs. Carboplatin, a DNA binding agent and protein cross-linking agent, attacks the active site of DNA polymerase, causing amplification failure and maintaining the conformational stability of the enzyme in the drug residue environment. Current techniques often add high concentrations of sequencing agents or macromolecular crowding agents to the buffer to enhance the hydrogen bond network of water molecules. High ionic strength or highly ordered solvent environments utilize the salt bridge effect to stabilize the enzyme protein. However, according to solution thermodynamics, the environment compresses the double layer around the DNA backbone, lowering the electrostatic repulsion barrier between non-specific sequences and primers, leading to non-specific amplification of high GC-content background DNA. Introducing conventional disordering agents or isoenergetic reagents eliminates thermodynamic deviations due to differences in base composition, directly destroying the protective enzyme solvation shell, causing the polymerase to be rapidly inactivated under carboplatin attack. Existing single-solvent systems cannot construct a protective enzyme-ordered aqueous environment and an isoenergetic ring that enhances hybridization specificity in the same reaction tube. The current environment necessitates complex sample pretreatment for clinical testing, increasing the risk of trace target loss and operational costs. Existing technologies for addressing drug resistance detection focus on finding new biomarkers to avoid the risk of enzyme activity inhibition, neglecting the improvement of the tolerance of the reaction medium itself. This leads to the detection process relying on sample pretreatment. For example, Chinese invention patent CN114350812A discloses NSCLC-free RNA-3 and its application related to osimertinib resistance. Although it determines drug resistance by detecting the expression level of specific small molecule RNA in serum, the technical path relies on nucleic acid extraction and reverse transcription purification steps to isolate sample matrix interference. This strategy avoids the problem of complex sample environment reactions and cannot solve the problem of DNA-binding chemotherapy drugs such as carboplatin directly attacking the polymerase active site. It does not provide a solution for simultaneously achieving anti-drug chemical corrosion and eliminating background DNA non-specific interference in a single liquid phase system. Clinical testing requires nucleic acid purification operations, increasing the risk of trace target loss and operational costs.

[0004] Therefore, how to construct a reaction system that shields the enzyme from the toxicity of residual drugs and eliminates non-specific interference from background DNA has become the technical problem to be solved by this invention. Summary of the Invention

[0005] To address the problems mentioned in the background art, the technical solution of the present invention is as follows: A nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer, the composition comprising a specific amplification primer pair targeting the coding region of the NFAT2 gene, a TaqMan fluorescent detection probe, a hot-start Taq DNA polymerase, and a reaction buffer:

[0006] The reaction buffer contains tetrahydropyrimidine and tetramethylammonium chloride; wherein the molar concentration of tetrahydropyrimidine in the reaction system is limited to 320 mM to 380 mM, the molar concentration of tetramethylammonium chloride in the reaction system is limited to 95 mM to 105 mM, and the molar ratio of tetrahydropyrimidine to tetramethylammonium chloride is controlled between 3.2:1 and 3.8:1.

[0007] This molar ratio is used to construct a differentiated solvent environment in the reaction buffer, allowing excess tetrahydropyrimidine to maintain a hydrated shell on the surface of the hot-start Taq DNA polymerase through preferential repulsion to block carboplatin molecule contact. At the same time, tetramethylammonium chloride binds to the major groove of the nucleic acid to eliminate the thermal stability difference between GC and AT base pairs. Thus, activity protection for polymerase and specific regulation of primer hybridization are achieved simultaneously in a single liquid phase system.

[0008] Preferably, the pH of the reaction buffer is limited to 8.3 to 8.7 at 25°C. This pH range is used to maintain the zwitterionic state of the tetrahydropyrimidine molecule and the complete dissociation state of tetramethylammonium chloride, ensuring that tetrahydropyrimidine and tetramethylammonium chloride establish a stable electrostatic interaction equilibrium in the solution.

[0009] Preferably, the reaction buffer does not contain dimethyl sulfoxide, glycerol or betaine, and tetramethylammonium chloride is the only small molecule quaternary ammonium salt added to the reaction buffer, in order to avoid exogenous disordering agents interfering with the hydration network maintained by tetrahydropyrimidine or competing for the binding site of tetramethylammonium chloride in the major groove of DNA.

[0010] Preferably, the specific amplification primer pairs are designed for high GC content fragments in the exon regions of the NFAT2 gene, and the melting curve peak area of ​​the non-specific products generated in the amplification reaction is less than 1% of the total product peak area; the differential solvent environment makes the binding stability of the primer pairs to the template DNA mainly depend on the sequence length, reducing the risk of non-specific binding caused by differences in template GC content.

[0011] Preferably, the concentration relationship between tetrahydropyrimidine and tetramethylammonium chloride in the reaction system satisfies the following molar ratio characteristic value. Limitations: ,in, This represents the molar concentration of tetrahydropyrimidine. This represents the molar concentration of tetramethylammonium chloride, and It is a dimensionless ratio; when Under defined conditions, the reaction system is in the solvation equilibrium range, which extends the half-life of the hot-start Taq DNA polymerase to more than five times that of the uncomposite environment in the presence of carboplatin residues.

[0012] Preferably, the composition is used to directly detect non-small cell lung cancer tissue thermal lysis fluid or pleural effusion samples that have not undergone nucleic acid purification; the reaction buffer is tolerant to heme and immunoglobulin inhibitors carried in the sample, and maintains the NFAT2 gene amplification efficiency in the range of 95% to 105% under the condition that the crude lysis buffer accounts for 10% of the final volume of the reaction system.

[0013] Preferably, the reaction buffer is prepared by the following steps: completely dissolving solid tetrahydropyrimidine in deionized water to form a basic hydrated solution, adding a stock solution of tetramethylammonium chloride dropwise to it at a rate not exceeding 10 mmol / min while continuously stirring until the molar ratio is reached, so as to ensure that tetramethylammonium chloride is uniformly dispersed in the solution system without destroying the hydrated environment established by tetrahydropyrimidine.

[0014] Preferably, the purity of tetrahydropyrimidine is greater than 99%, and tetramethylammonium chloride is a molecular biology grade reagent; the reaction buffer also includes potassium chloride for maintaining ionic strength and magnesium chloride for providing cofactors, wherein the concentration of magnesium chloride is 1.5 mM to 2.5 mM, and the concentration of potassium chloride is 10 mM to 50 mM, thereby enhancing the specificity of primer hybridization in conjunction with tetramethylammonium chloride under low ionic strength background.

[0015] Preferably, the composition is provided in kit form and is packaged in a premixed solution tube, or separately packaged in a first container and a second container; the first container contains a pre-prepared mixed solution of tetrahydropyrimidine and tetramethylammonium chloride, and the second container contains a hot-start Taq DNA polymerase, primer pairs, and a TaqMan detection probe; in use, the contents of the first and second containers are mixed to form a reaction system.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. In carboplatin resistance screening for non-small cell lung cancer, the molar ratio of tetrahydropyrimidine to tetramethylammonium chloride is limited to a specific range. A differential solvent environment targeting the interface between macromolecules and small molecules is established in the reaction buffer. High concentrations of tetrahydropyrimidine utilize preferential repulsion to form a dense hydrated shell on the surface of DNA polymerase, maintaining the conformational stability and catalytic activity of the enzyme protein in the residual environment of carboplatin chemotherapy drugs. Tetramethylammonium chloride, embedded in the interstitial hydration network, is restricted to functioning at the nucleic acid hybridization interface. The quaternary ammonium cation eliminates the difference in hydrogen bond energy between GC and AT base pairs. The dual solvation mechanism ensures that the reaction system protects the polymerase from chemical inhibition, and that the primer-template binding strength depends only on the sequence length and is no longer affected by the local base composition. This achieves decoupling of anti-drug interference and high-specificity recognition function within a single reaction tube.

[0018] 2. Based on the physical shielding effect of the hydration shell in the buffer system on the enzyme active site, the composition enhances the tolerance threshold of the amplification reaction to endogenous inhibitors and exogenous drug residues in biological samples. When processing pleural effusion or tissue homogenate from non-small cell lung cancer patients, the nucleic acid column purification or magnetic bead extraction steps can be omitted, and the thermal lysis products can be used directly as amplification templates. This avoids the degradation and loss of trace drug resistance gene transcripts during multi-step purification, ensures the detection rate of low-abundance targets, simplifies clinical operation procedures, reduces the risk of sample cross-contamination, and establishes advantages for engineering applications in rapid point-of-care testing scenarios.

[0019] 3. By utilizing the saturated binding properties of tetramethylammonium chloride to the major groove of DNA double helix at a specific ratio, an orthogonal thermodynamic hybridization environment with genomic background noise is constructed. This suppresses non-specific mismatches between high-GC-content non-target DNA fragments and primers, eliminates cycle threshold drift caused by background noise amplification, and ensures a kinetic linear relationship between the fluorescence signal accumulation rate and the initial copy number of the template in the detection of key NFAT2 resistance genes. This improves the resolution of identifying subtle changes in gene expression levels and provides a reliable data basis for the accurate clinical determination of carboplatin resistance levels. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the single-phase detection process for screening carboplatin resistance in non-small cell lung cancer according to the present invention.

[0021] Figure 2 This is a graph showing the relationship between the molar ratio characteristic value Ω of this invention and the amplification Ct value and efficiency.

[0022] Figure 3 This is a schematic diagram illustrating the microscopic mechanism by which the binary solvent system of this invention simultaneously achieves enzyme protection and hybridization-specific regulation. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0024] This invention provides a nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer, comprising a specific amplification primer pair targeting the coding region of the NFAT2 gene, a TaqMan fluorescent detection probe, a hot-start Taq DNA polymerase, and a reaction buffer. The reaction buffer is constructed with specific components and proportions to create a solvation-level pinned liquid environment, used to simultaneously maintain DNA polymerase activity and regulate nucleic acid hybridization specificity in a single reaction system. The preparation of the reaction buffer must follow thermodynamic ordering procedures to ensure that each component forms the expected microscopic solvent structure. The preparation process includes completely dissolving solid tetrahydropyrimidine with a purity greater than 99% in deionized water, utilizing the zwitterionic properties of tetrahydropyrimidine to induce water molecules to form an ordered tetrahedral hydrogen bond network, i.e., a basic hydration solution, and adding water to the basic hydration solution at a rate not exceeding [per minute] under continuous stirring. Tetramethylammonium chloride stock solution was added dropwise at a rate that allowed the quaternary ammonium cations dissociated from tetramethylammonium chloride to slowly embed into the interstitial spaces of the hydration network constructed by tetrahydropyrimidine, forming a ternary associated structure. Finally, the pH of the system was adjusted using a Tris-HCl buffer. The pH value is limited to to Within a certain range, to lock the ionization state of the tetrahydropyrimidine molecule and maintain its electrostatic interaction equilibrium with tetramethylammonium chloride; in the reaction buffer, the molar concentration of tetrahydropyrimidine in the reaction system. Limited to to molar concentration of tetramethylammonium chloride in the reaction system Limited to to The molar ratio of tetrahydropyrimidine to tetramethylammonium chloride satisfies the characteristic value. Limitations: ,in, This represents the molar concentration of tetrahydropyrimidine. This represents the molar concentration of tetramethylammonium chloride, and It is a dimensionless ratio.

[0025] The above molar ratio window to Within a confined system, the solvation competition equilibrium occurs when the ratio Below When the ratio is relatively excessive, tetramethylammonium chloride can destroy the protective hydration shell formed by tetrahydropyrimidine on the enzyme surface, causing the DNA polymerase to be directly exposed to an environment containing carboplatin residues and thus inactivated. Higher than At this time, the over-ordering effect dominated by tetrahydropyrimidine compresses the electrical double layer around the DNA double helix, lowering the electrostatic repulsion barrier between the non-target sequence and the primer, leading to non-specific amplification of background DNA with high GC content, only amplifying the target sequence. In to Within the specified range, excess tetrahydropyrimidine maintains a dense hydrated shell on the surface of the hot-start Taq DNA polymerase through preferential repulsion, thus blocking contact with carboplatin molecules. Simultaneously, tetramethylammonium chloride embedded in the hydrated network is released and binds to the major groove of the nucleic acid at high temperatures, eliminating the thermal stability difference between GC and AT base pairs. This achieves dual regulation of enzyme activity protection and primer hybridization specificity. The solvation-level pinned state was defined by a combination of dynamic light scattering and differential scanning calorimetry. It showed that the average hydrodynamic radius of the tetrahydropyrimidine hydrated clusters in the solution was stable in the range of 2.8 nm to 3.2 nm at 25 °C. Compared with the pure water system, the rate of change of specific heat capacity was reduced by more than 15% in the range of 0 °C to 100 °C. This physical indicator confirms that the tetramethylammonium chloride quaternary ammonium cation is embedded in the interstitial space of the tetrahedral hydrogen bond network of tetrahydropyrimidine and restricts the translational degree of freedom of water molecules, enabling the solution system to have a hysteretic response to temperature fluctuations and external chemical interferences.

[0026] This nucleic acid detection composition is suitable for direct detection of non-small cell lung cancer tissue thermal lysis fluid or pleural effusion samples that have not undergone nucleic acid purification. The reaction buffer is tolerant to inhibitors such as heme and immunoglobulins present in the sample. With the crude lysis buffer accounting for 10% of the final reaction volume, this buffer environment maintains the NFAT2 gene amplification efficiency within the range of 95% to 105%, and ensures a linear relationship between the cycle threshold and the target concentration. Furthermore, the reaction buffer is free of dimethyl sulfoxide, glycerol, or betaine, and tetramethylammonium chloride is the only small-molecule quaternary ammonium salt added to the reaction buffer to prevent external contamination. The source of the disordered sequence interferes with the hydration network maintained by tetrahydropyrimidine or competes for the binding site of tetramethylammonium chloride in the major groove of DNA; the specific amplification primer pairs are designed for high GC content fragments in the exon region of the NFAT2 gene. In the isoenergetic environment constructed in the above buffer, the binding stability of the primer pairs to the template DNA depends mainly on the sequence length, not the GC content of the sequence. The melting curve peak area of ​​the non-specific products generated in the amplification reaction is less than 1% of the total product peak area. The reaction system also contains potassium chloride to maintain ionic strength and magnesium chloride to provide cofactors, wherein the concentration of magnesium chloride is [missing information]. to The concentration of potassium chloride is to This low ionic strength background synergistically enhances the specificity of primer hybridization with tetramethylammonium chloride; in terms of product form, the composition is packaged in a premixed liquid tube, or separately packaged in a first container and a second container. In the split package, the first container contains a pre-prepared mixed solution of tetrahydropyrimidine and tetramethylammonium chloride, and the second container contains hot-start Taq DNA polymerase, primer pairs, and TaqMan detection probes. When in use, the contents of the first and second containers are mixed to form a reaction system.

[0027] Example 1: In the application scenario of screening for carboplatin resistance in non-small cell lung cancer patients who have received chemotherapy, the detection system directly processes pleural effusion samples containing high concentrations of necrotic genomic DNA fragments and residual carboplatin molecules. This condition requires the reaction system to simultaneously overcome the attack of chemical inhibitors on the enzyme active site and the competitive interference of background noise on trace targets at the physicochemical level. In this example, the aforementioned nucleic acid detection composition is used, and the molar concentration of tetrahydropyrimidine in its reaction buffer is set to... The molar concentration of tetramethylammonium chloride is set to Thus, the molar ratio characteristic value of the two is... Locked to This method aims to create a solvated, pinned liquid environment within the reaction tube. Under this specific ratio, a high concentration of tetrahydropyrimidine components preferentially repels and forms a dense, ordered hydration shell on the surface of the hot-start Taq DNA polymerase. This steric hindrance prevents residual carboplatin molecules from penetrating the hydrophobic core of the enzyme protein, maintaining the polymerase's conformational stability in the presence of inhibitors. Simultaneously, the addition of tetramethylammonium chloride in this ratio does not disrupt the integrity of the protective hydration shell; instead, it utilizes the specificity of its quaternary ammonium cation during high-temperature denaturation and annealing. By binding to the major groove region of the DNA double helix, the difference in hydrogen bond energy between the high GC content background sequence and the target NFAT2 gene sequence is eliminated through this binding interaction. This makes the thermodynamic stability of the hybridization between the primer and the template mainly depend on the sequence length rather than the local base composition. This binary solvation regulation mechanism enables the detection system to achieve specific amplification of low-abundance NFAT2 transcripts without nucleic acid column purification. The amplification efficiency is maintained in the range of 98% to 102%, and the melting curve signal of non-specific amplification products is suppressed to less than 1% of the total signal.

[0028] Example 2: To verify the effectiveness of the nucleic acid detection composition of the present invention in achieving both anti-chemical inhibition and high-specificity recognition in complex clinical samples, this example designed a set of comparative experiments containing gradient variables. The experimental platform used an ABI 7500 real-time fluorescence quantitative PCR instrument to simulate a real clinical testing environment. The experimental samples used standards containing known concentrations of NFAT2 plasmid (… The simulated pleural effusion lysis fluid (copy / reaction) contained not only normal human genomic DNA background ( / reaction), and also artificially added a final concentration of Carboplatin and The hemoglobin was used to construct an engineered stress test scenario with high background noise and strong chemical inhibition. The amplification reaction program was set to pre-denature at 95°C for 5 minutes to activate the hot-start enzyme, followed by 45 cycles. Each cycle included 15 seconds of denaturation at 95°C and 45 seconds of annealing extension at 60°C, and fluorescence signals were collected. The annealing temperature was set based on the primer melting temperature correction value in the presence of tetramethylammonium chloride. The heating and cooling rates were uniformly set to 2.5°C per second to match the thermal relaxation time of the tetrahydropyrimidine hydration shell, to prevent the dynamic reconstruction of the enzyme surface protective layer from lagging behind temperature changes during rapid temperature changes.

[0029] This experiment consisted of five parallel experimental groups, with the concentration of tetrahydropyrimidine in the reaction buffer of each group fixed at [value missing]. The only variable is the concentration of tetramethylammonium chloride, which results in different molar ratio characteristic values. The specific grouping is as follows: Comparative sample group A ( Tetramethylammonium chloride Sample B of this invention Tetramethylammonium chloride ), Sample C of this invention ( Tetramethylammonium chloride ), Sample D of the present invention ( Tetramethylammonium chloride ) and the control group E ( Tetramethylammonium chloride In addition, an ideal control group without carboplatin and background DNA was set up. Each group underwent three technical replicates, and the cycle threshold (Ct value) and amplification efficiency of the amplification curve were recorded. The experimental results showed characteristic values. The nonlinear regulation of amplification performance is shown in Table 1. The Ct value of control group A was delayed to 29.8, and the amplification efficiency was only 78%, indicating that excessive tetramethylammonium chloride damaged the protective hydration shell on the enzyme surface, leading to significant inactivation of Taq enzyme under carboplatin challenge. When the Ct value entered the 3.2 to 3.8 range defined in this invention (sample groups B, C, and D), the Ct value rapidly recovered to around 24.5, and the amplification efficiency stabilized between 98% and 101%, showing no difference from the ideal control group (Ct = 24.3, efficiency 99%). This confirms that enzyme activity is effectively protected and hybridization specificity is maintained at an optimal level within this range. However, when... When the value was further increased to 4.5 (comparative sample group E), although the Ct value was still 24.4, obvious non-specific peaks appeared in the melting curve analysis, indicating that the concentration of tetramethylammonium chloride was insufficient to offset the double layer compression effect caused by high concentration of tetrahydropyrimidine, resulting in an increased risk of non-specific amplification.

[0030] Table 1: Characteristic values ​​of different molar ratios The following table compares the amplification performance of NFAT2.

[0031]

[0032] The above data reveals the performance inflection point of the binary solvation system: when In to At a specific window, the reaction system achieves a dynamic equilibrium of solvation hierarchical pinning at the microscopic level. Under this equilibrium, the enzyme protective shell constructed by the preferential repulsion of tetrahydropyrimidine and the DNA major groove binding dominated by tetramethylammonium chloride do not interfere with each other and work synergistically, resolving the thermodynamic contradiction between enzyme stability and hybridization specificity.

[0033] Example 3: This example combines Figures 1 to 3 This describes a nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer, as follows: Figure 1 As shown, the process begins with clinical sample input, specifically pleural effusion or tissue samples containing carboplatin and necrotic DNA. After thermal lysis pretreatment to obtain a crude lysate that does not require nucleic acid purification, the sample enters a single-phase liquid-phase amplification reaction system. This system utilizes the preferential repulsion of tetrahydropyrimidine to construct a dense hydration shell to block carboplatin contact, and uses tetramethylammonium chloride to bind the major groove of nucleic acids to eliminate the thermal difference between GC and AT and to construct an isoenergetic environment. Under the low ionic strength environment composed of potassium chloride and magnesium chloride, the hot-start Taq DNA polymerase maintains a highly active and protected state, while the primer and template hybridization is in a specifically regulated state, jointly promoting the amplification of the high GC content exon region of the NFAT2 gene. Finally, real-time signal acquisition and Ct value calculation are performed by TaqMan fluorescence detection, and normalized differential analysis is conducted through standardized data interpretation. It outputs the drug resistance classification results for drug-resistant and sensitive types with high expression.

[0034] like Figure 2As shown, the horizontal axis represents the molar ratio characteristic value Ω, i.e., the concentration ratio of tetrahydropyrimidine to tetramethylammonium chloride, ranging from 2.5 to 4.5. The left vertical axis represents the Ct value, represented by a solid line, and the right vertical axis represents the amplification efficiency (%), represented by a dashed line. The data shows that as the Ω value increases from 2.5, the Ct value decreases and stabilizes at a low level of around 24.5 when Ω is in the 3.2 to 3.8 range. Simultaneously, the amplification efficiency rapidly increases from 78% and remains stable around 100% within the same range. Figure 3 As shown, in the enzyme protection zone on the left, tetrahydropyrimidine molecules are arranged in an orderly manner around Taq polymerase to form the hydration shell shown by the dashed line. Through steric hindrance, the carboplatin molecule labeled X is blocked to the outside, preventing it from attacking the enzyme's active site. In the DNA hybridization zone on the right, tetramethylammonium chloride molecules are specifically embedded in the major groove region of the DNA double strand, acting between the GC and AT base pairs to eliminate the thermal stability differences between different base pairs.

[0035] Example 4: To define the engineering tolerance boundaries of the nucleic acid detection composition of the present invention in actual clinical operation and to establish standardized data interpretation logic, this example constructs a system stability verification model that includes physical boundary calibration and algorithmic decision-making process. It also addresses the two most critical interference variables in the reaction system: the volume ratio of the crude lysis buffer. molar concentration of magnesium chloride A two-way gradient pressure test was performed using simulated high-viscosity pleural effusion lysis samples containing fixed concentrations of NFAT2 and GAPDH internal control targets. In the first dimension, the sample load limit test, the basic formulation of the reaction buffer remained constant. The proportion of crude pyrolysis liquid added to the total volume of the reaction system is... Set to five gradient levels: , , , and The test results show that when In to When the range is within a certain range, the amplification efficiency of NFAT2 remains stable at a certain level. And the cycle threshold The change range is less than This study confirmed that within this range, high concentrations of tetrahydropyrimidine can effectively maintain the hydrated shell on the enzyme surface, resisting the penetration of heme and protein impurities. achieve At that time, the amplification efficiency dropped sharply to ,and Value lag exceeds One cycle; when Further increase to At that time, the amplification reaction was completely suppressed. This data clearly indicates that the physical tolerance threshold of the reaction system to the crude lysis buffer is [missing information - likely a percentage of the total volume]. Verify the recommendations in the instruction manual. The sample volume has sufficient engineering safety margin.

[0036] In the second dimension of ion environment sensitivity testing, the fixed sample proportion is: Adjusting the reaction system The concentration from Increment to Data shows that when Below At this time, due to insufficient cofactors, the polymerase catalytic rate decreases, resulting in insufficient fluorescence plateau signal intensity; when In to When the system reaches the range defined by this invention, it achieves an optimal balance between sensitivity and specificity; while when Exceed At this time, excess divalent magnesium ions neutralize the negative charge of the DNA phosphate backbone through a strong electrostatic shielding effect, weakening the destabilizing effect of tetramethylammonium chloride (monovalent cation) on mismatched bases. This results in a significant nonspecific amplification signal in the negative control wells, confirming the effect of low concentrations of magnesium ions. It is a necessary condition for maintaining the thermodynamic control of tetramethylammonium chloride.

[0037] Based on the establishment of the above physical boundaries, this embodiment further defines a standardized data interpretation procedure for carboplatin resistance, which is solidified into the following three ordered steps: Step 1, Data Acquisition and Baseline Correction: The raw fluorescence intensity signals of the NFAT2 probe (FAM channel) and GAPDH internal reference probe (VIC channel) in the reaction tube are acquired by a quantitative real-time PCR instrument, and then... to The baseline is calculated from the background signal of each cycle, and the cycle threshold for each channel is determined, denoted as follows: and The second step is to calculate the normalized difference. Computational logic, namely: This step aims to eliminate systematic errors caused by fluctuations in sample addition or differences in lysis efficiency. The third step, state classification, involves determining the calculated... Values ​​compared to the preset clinical cutoff value In comparison, under the calibration system of this embodiment, The value is set to The threshold K=6.5 was determined based on retrospective clinical sample data from 120 cases confirmed by immunohistochemical gold standard. The threshold was determined by selecting the point of maximum Youden's index through receiver operating characteristic (ROC) curve analysis. The median ΔCt distribution for carboplatin-resistant samples was 4.2, and for sensitive samples, it was 8.9. This statistical limit ensures that the sum of sensitivity and specificity is maximized within the 95% confidence interval. Furthermore, the system has pre-set correction coefficients for K values ​​for different batches of reagents, automatically generated from the ΔCt offset of the factory quality control standard. The sample was determined to be carboplatin-resistant (highly expressed); if The sample was determined to be carboplatin-sensitive (low expression). This interpretation logic is based on... In clinical double-blind sample validation, the concordance rate with the immunohistochemistry (IHC) gold standard reached [percentage missing]. .

[0038] Example 5: To ensure the adaptability of nucleic acid detection compositions to different batches of raw materials and varying environmental temperatures before actual clinical deployment, this example constructs a standardized offline calibration and data filling procedure. For each new batch of synthesized tetrahydropyrimidine and tetramethylammonium chloride raw materials, thermodynamic fingerprinting based on differential scanning calorimetry (DSC) is performed. This is achieved by recording the raw materials' temperature and humidity conditions. to The endothermic peak shift and peak area within the temperature range were used to establish a baseline database of raw material purity and hydration capacity. Buffer systems with different ratios were placed within a preset temperature gradient. , , The solution was incubated at a temperature of 100°C, and the hydrodynamic radius distribution of micro-clusters in the solution was monitored using dynamic light scattering (DLS) technology.

[0039] Furthermore, considering the potential differences in PCR instrument temperature control among different medical institution laboratories, this embodiment establishes a set of on-site pre-calibration procedures, requiring that before using this composition for the first time, the specific melting temperature (…) be used. The standard reference for the calibrator was used to perform a melting curve correction procedure on the target PCR instrument, and the results were compared with actual measurements. The deviation between the measured value and the standard value was used to calculate the instrument's temperature compensation coefficient. Based on this, the annealing temperature parameters in the reaction program were fine-tuned. Simultaneously, a sensitivity confirmation experiment was performed using a set of pre-calibrated low-concentration positive controls to ensure the amplification curve's starting cycle number was within the specified optical path system of the instrument. It falls within the preset quality control range.

[0040] Example 6: This example constructs an optimization and boundary verification model for the core parameters, and focuses on the molar concentration of tetrahydropyrimidine in the reaction buffer. Perform gradient stress testing, setting a series of concentration gradients, covering a range of... to , interval is and at a constant tetramethylammonium chloride concentration and molar ratio characteristic value The study was conducted at a baseline level, monitoring the thermostability half-life of Taq DNA polymerase at different concentrations. and the number of start cycles in the NFAT2 amplification curve The test results show that when Below At this time, the enzyme's thermostability decreases, leading to insufficient amplification efficiency; while when Exceed At this time, excessively high viscosity hinders effective collision between the primer and the template, leading to Value lag, based on this nonlinear response curve, determine to To balance enzyme protection and reaction kinetics, a working window was established.

[0041] Regarding the activation time of hot-start Taq DNA polymerase in the reaction system, this embodiment designs a set of control parameters based on amplification efficiency. With nonspecific product formation rate The time optimization procedure with dual indicators, in At the activation temperature, set the activation time. from to The gradient, experimental data shows, when Less than At this time, the enzyme activity is not fully released, leading to reduced amplification efficiency. Below ;when Exceed Although the enzyme activity is fully released, prolonged exposure to high temperatures reduces the formation rate of primer dimers. Ascend, through the and Perform weighted scoring to determine to To achieve an optimal activation time range that balances high sensitivity and high specificity, and to address potential signal drift issues in the detection system, this embodiment introduces a dynamic threshold calibration logic based on the internal reference gene GAPDH. This logic no longer relies on a single fixed fluorescence threshold, but instead adjusts the calibration based on the baseline noise level of the GAPDH channel in each reaction. Dynamically calculate signal judgment threshold ,set up ,in The baseline mean. The baseline standard deviation, The confidence coefficient is usually taken as 1. .

[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer, characterized in that, The composition includes a specific amplification primer pair targeting the coding region of the NFAT2 gene, a TaqMan fluorescent detection probe, a hot-start Taq DNA polymerase, and a reaction buffer: The reaction buffer contains tetrahydropyrimidine and tetramethylammonium chloride; wherein the molar concentration of tetrahydropyrimidine in the reaction system is limited to 320 mM to 380 mM, the molar concentration of tetramethylammonium chloride in the reaction system is limited to 95 mM to 105 mM, and the molar ratio of tetrahydropyrimidine to tetramethylammonium chloride is controlled between 3.2:1 and 3.8:

1. This molar ratio is used to construct a differentiated solvent environment in the reaction buffer, so that excess tetrahydropyrimidine maintains a hydrated shell on the surface of the hot-start Taq DNA polymerase through preferential repulsion to block carboplatin molecule contact. At the same time, tetramethylammonium chloride binds to the major groove of the nucleic acid to eliminate the thermal stability difference between GC and AT base pairs, thereby simultaneously achieving activity protection for polymerase and specific regulation of primer hybridization in a single liquid phase system. Furthermore, the reaction buffer is free of dimethyl sulfoxide, glycerol, or betaine, and tetramethylammonium chloride is the only small-molecule quaternary ammonium salt added to the reaction buffer to prevent exogenous disordering agents from interfering with the hydration network maintained by tetrahydropyrimidine or competing for the binding site of tetramethylammonium chloride in the major groove of DNA. The concentration relationship between tetrahydropyrimidine and tetramethylammonium chloride in the reaction system satisfies the following molar ratio characteristics. Limitations: ,in, This represents the molar concentration of tetrahydropyrimidine. This represents the molar concentration of tetramethylammonium chloride, and It is a dimensionless ratio; when Under defined conditions, the reaction system is in the solvation equilibrium range, which extends the half-life of hot-start Taq DNA polymerase to more than five times that of the composition-free environment in the presence of carboplatin residues. The composition is used to directly detect non-small cell lung cancer tissue thermal lysis buffer or pleural effusion samples that have not undergone nucleic acid purification; the reaction buffer is tolerant to heme and immunoglobulin inhibitors carried in the sample, and maintains the NFAT2 gene amplification efficiency in the range of 95% to 105% when the crude lysis buffer accounts for 10% of the final volume of the reaction system.

2. The nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer according to claim 1, characterized in that, The pH of the reaction buffer is limited to 8.3 to 8.7 at 25°C. This pH range is used to maintain the zwitterionic state of the tetrahydropyrimidine molecule and the complete dissociation state of tetramethylammonium chloride, ensuring that tetrahydropyrimidine and tetramethylammonium chloride establish a stable electrostatic interaction equilibrium in the solution.

3. The nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer according to claim 1, characterized in that, The specific amplification primer pairs are designed for high GC content fragments in the exon regions of the NFAT2 gene, and the melting curve peak area of ​​the non-specific products generated in the amplification reaction is less than 1% of the total product peak area; the differential solvent environment makes the binding stability of the primer pairs to the template DNA mainly dependent on the sequence length, reducing the risk of non-specific binding caused by differences in template GC content.

4. The nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer according to claim 1, characterized in that, The reaction buffer is prepared by the following steps: solid tetrahydropyrimidine is completely dissolved in deionized water to form a basic hydrated solution, and a stock solution of tetramethylammonium chloride is added dropwise to it at a rate not exceeding 10 mmol / min while continuously stirring until the molar ratio is reached to ensure that tetramethylammonium chloride is uniformly dispersed in the solution system without destroying the hydrated environment established by tetrahydropyrimidine.

5. The nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer according to claim 1, characterized in that, The purity of tetrahydropyrimidine is greater than 99%, and tetramethylammonium chloride is a molecular biology grade reagent. The reaction buffer also includes potassium chloride to maintain ionic strength and magnesium chloride to provide cofactors, wherein the concentration of magnesium chloride is 1.5 mM to 2.5 mM and the concentration of potassium chloride is 10 mM to 50 mM. The specificity of primer hybridization is enhanced by tetramethylammonium chloride in conjunction with the low ionic strength background.

6. The nucleic acid detection composition for screening carboplatin resistance in non-small cell lung cancer according to claim 1, characterized in that, The composition is provided as a kit and is packaged in a premixed solution tube, or separately in a first container and a second container; the first container contains a pre-prepared mixed solution of tetrahydropyrimidine and tetramethylammonium chloride, and the second container contains a hot-start Taq DNA polymerase, primer pairs, and a TaqMan detection probe; in use, the contents of the first and second containers are mixed to form a reaction system.

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