Fluorescent PCR (Polymerase Chain Reaction) method and system for synchronously detecting KRAS mutation and tumor immune microenvironment

Abstract

The invention relates to a fluorescent PCR (Polymerase Chain Reaction) method and system for synchronously detecting KRAS mutation and a tumor immune microenvironment. According to the method, a staged temperature control amplification strategy of RNA reverse transcription, KRAS DNA preferential amplification and low-abundance cDNA amplification is sequentially executed, and a probe dynamic quenching technology for selectively activating / inhibiting a fluorescence signal by utilizing an illumination or temperature response mechanism of a photosensitive quenching probe is adopted; kRAS gene mutation detection and tumor-related macrophage marker expression analysis are synchronously achieved in a single tube, the problems of DNA / RNA co-amplification interference, signal crosstalk and tedious operation in a traditional method are solved, the detection sensitivity, specificity and timeliness are remarkably improved, and an integrated solution is provided for colorectal cancer targeted therapy and immune microenvironment evaluation.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnostics, and particularly relates to a fluorescence PCR method and system for synchronous detection of KRAS mutations and tumor immune microenvironment. Background Art

[0002] KRAS gene mutations in colorectal cancer are closely related to the immunosuppressive microenvironment mediated by tumor-associated macrophages (TAMs). Existing fluorescence PCR technologies have the following limitations:

[0003] Firstly, traditional fluorescence PCR needs to separately detect KRAS mutations (DNA) and TAM markers (such as CKIP-1 mRNA, CD206 mRNA), resulting in increased sample consumption and operation complexity.

[0004] Secondly, when high-abundance KRAS DNA coexists with low-abundance CKIP-1 mRNA, DNA amplification inhibits reverse transcription efficiency.

[0005] Therefore, there is an urgent need for a fluorescence PCR technology that can achieve synchronous detection of KRAS mutations and TAM-related RNA markers. Summary of the Invention

[0006] Based on this, it is necessary to provide a fluorescence PCR method and system for synchronous detection of KRAS mutations and tumor immune microenvironment in view of the above technical problems.

[0007] In a first aspect, the present application provides a fluorescence PCR method for synchronous detection of KRAS mutations and tumor immune microenvironment, including:

[0008] S1: Pretreat the sample to obtain a pretreated sample; mix the pretreated sample with a premixed system to obtain a new mixed system;

[0009] Wherein, the sample is a colorectal cancer puncture biopsy fluid or a blood ctDNA sample, the pretreatment includes lysis treatment and centrifugation treatment, the premixed system includes phosphorothioate-modified primers and photosensitive quenching probes, the phosphorothioate-modified primers are synthetic oligonucleotides with a phosphorothioate bond introduced at the 3' end of the primer, and the photosensitive quenching probes include FAM channel probes and Cy5 channel probes;

[0010] S2: Perform amplification treatment on the new mixed system through a staged amplification control strategy to obtain an amplification product;

[0011] Wherein, the amplification treatment is to sequentially perform RNA reverse transcription, preferential amplification of KRAS DNA, and amplification of low-abundance cDNA, and the staged amplification control strategy is to regulate the order and efficiency of reverse transcription and amplification reactions through temperature gradients; the phosphorothioate-modified primers are used to: inhibit the formation of primer dimers and improve amplification specificity during amplification treatment;

[0012] S3: Use the probe dynamic quenching technology to perform signal separation processing on the amplification product, obtain FAM and Cy5 dual-channel fluorescence signal data, and synchronously output the KRAS mutation typing analysis result and the tumor-associated macrophage activity score based on the FAM and Cy5 dual-channel fluorescence signal data;

[0013] Among them, the probe dynamic quenching technology dynamically controls the release and quenching of fluorescence signals through the light or temperature response mechanism of the photosensitive quenching probe.

[0014] In a second aspect, the present application also provides a fluorescence PCR system for synchronous detection of KRAS mutations and the tumor immune microenvironment, including:

[0015] A sample processing module for preprocessing the sample to obtain a preprocessed sample; mixing the preprocessed sample with a premixed system to obtain a new mixed system;

[0016] Among them, the sample is a colorectal cancer puncture biopsy fluid or a blood ctDNA sample, the preprocessing includes lysis treatment and centrifugation treatment, the premixed system includes a phosphorothioate-modified primer and a photosensitive quenching probe, the phosphorothioate-modified primer is a synthetic oligonucleotide with a phosphorothioate bond introduced at the 3' end of the primer, and the photosensitive quenching probe includes a FAM channel probe and a Cy5 channel probe;

[0017] A staged temperature control amplification module for performing amplification processing on the new mixed system through a staged amplification control strategy to obtain an amplification product;

[0018] Among them, the amplification process is to sequentially perform RNA reverse transcription, preferential amplification of KRAS DNA, and amplification of low-abundance cDNA, and the staged amplification control strategy is to control the order and efficiency of the reverse transcription and amplification reactions through temperature gradient regulation; the phosphorothioate-modified primer is used for: inhibiting the formation of primer dimers and improving the amplification specificity during the amplification process;

[0019] A probe dynamic quenching dual-channel analysis module for performing signal separation processing on the amplification product using the probe dynamic quenching technology to obtain FAM and Cy5 dual-channel fluorescence signal data, and synchronously outputting the KRAS mutation typing analysis result and the tumor-associated macrophage activity score based on the FAM and Cy5 dual-channel fluorescence signal data;

[0020] Among them, the probe dynamic quenching technology dynamically controls the release and quenching of fluorescence signals through the light or temperature response mechanism of the photosensitive quenching probe.

[0021] In a third aspect, the present application also provides a computer device, including a memory and a processor. The memory stores a computer program, and when the processor executes the computer program, it implements a fluorescence PCR method for synchronous detection of KRAS mutations and tumor immune microenvironment as described in the first aspect.

[0022] In a fourth aspect, the present application also provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, it implements a fluorescence PCR method for synchronous detection of KRAS mutations and tumor immune microenvironment as described in the first aspect.

[0023] The above fluorescence PCR method and system for synchronous detection of KRAS mutations and tumor immune microenvironment, through a staged temperature control amplification strategy that sequentially performs RNA reverse transcription, preferential amplification of KRAS DNA, and amplification of low-abundance cDNA, and a probe dynamic quenching technique that selectively activates / inhibits fluorescence signals using the light or temperature response mechanism of photosensitive quenching probes, synchronously realizes KRAS gene mutation detection and analysis of the expression of tumor-related macrophage markers in a single tube, solves the problems of DNA / RNA co-amplification interference, signal crosstalk, and cumbersome operation in traditional methods, significantly improves detection sensitivity, specificity, and timeliness, and provides an integrated solution for targeted therapy of colorectal cancer and evaluation of the immune microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or related technologies, the following will briefly introduce the drawings required for use in the description of the embodiments or related technologies. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is a flowchart showing a fluorescence PCR method for synchronous detection of KRAS mutations and tumor immune microenvironment provided by the present invention;

[0026] Figure 2 It is a flowchart showing the amplification process of a new mixed system in an optional embodiment of the present invention;

[0027] Figure 3 It is a schematic structural diagram of a fluorescence PCR system for synchronous detection of KRAS mutations and tumor immune microenvironment provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In order to make the objectives, technical solutions and advantages of this application more clearly understood, the following further details this application in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for explaining this application and are not used to limit this application.

[0029] Reference Figure 1 , which shows a schematic flow chart of a fluorescence PCR method for synchronous detection of KRAS mutations and tumor immune microenvironment provided by this application. This method includes the following steps:

[0030] S1: Pretreat the sample to obtain a pretreated sample; mix the pretreated sample with a premixed system to obtain a new mixed system. Among them, the sample is a colorectal cancer puncture biopsy fluid or a blood ctDNA sample. The pretreatment includes lysis treatment and centrifugation treatment. The premixed system includes phosphorothioate-modified primers and photosensitive quenching probes. The phosphorothioate-modified primers are synthetic oligonucleotides with a phosphorothioate bond introduced at the 3' end of the primer. The photosensitive quenching probes include FAM channel probes and Cy5 channel probes.

[0031] Specifically, the colorectal cancer puncture biopsy fluid is a sample directly obtained from the tumor tissue site, which contains abundant tumor cells and related genetic material information. This sample can more directly reflect the KRAS mutation situation in the tumor local area. The blood ctDNA (circulating tumor DNA) sample is a non-invasive detection source. ctDNA is the DNA fragment released into the blood circulation during the growth, apoptosis or necrosis of tumor cells, which carries the gene characteristics of the tumor. The advantage of using blood samples is that they can be easily collected multiple times for dynamic monitoring of tumor development and treatment effects. For example, during the treatment of advanced colorectal cancer patients, regularly drawing blood to detect the KRAS mutation changes in ctDNA can timely detect whether the tumor has drug-resistant mutations and other situations.

[0032] The lysis treatment can be carried out by using a specific lysis reagent, which can destroy the cell membrane and nuclear membrane structures of cells, so that the DNA inside the cells is fully released into the solution. During this process, strictly control the dosage and action time of the lysis reagent to ensure that it can effectively lyse cells without over-degrading DNA. For example, if some chemical lysis reagents are used in excess or the action time is too long, it may cause DNA strand breakage and affect the subsequent PCR amplification effect.

[0033] The purpose of the centrifugation treatment is to separate the cell debris, proteins and other impurities after lysis from the released DNA. By setting appropriate centrifugation speed and time, the DNA can be located in the supernatant, while the impurities precipitate at the bottom of the centrifuge tube. For example, under general experimental conditions, after centrifuging at a medium speed for several minutes, a relatively pure supernatant containing DNA can be obtained.

[0034] A phosphorothioate - modified primer is a primer with special chemical modification. A phosphorothioate bond is introduced at the 3' end of the primer. The change in this structure makes the primer have higher stability during the PCR reaction. It can resist the degradation of certain nucleases and can more effectively guide DNA polymerase to extend the chain when binding to template DNA. For example, in some complex biological sample environments, ordinary primers may be rapidly degraded by nucleases, while phosphorothioate - modified primers can maintain activity for a longer time, improving the success rate of the PCR reaction.

[0035] The FAM - channel probe and Cy5 - channel probe in the light - sensitive quenching probe are respectively for different fluorescence signal detection channels. The FAM - channel probe is used to detect relatively common gene target sequences, and the fluorescence signal it emits can be accurately captured by fluorescence detection equipment within a specific wavelength range. The Cy5 - channel probe can be used to detect other specific gene sequences or, in complex gene detection scenarios, work in cooperation with other probes to distinguish different signal sources. When the above two probes are not bound to specific DNA sequences, they are in a quenched state; if they specifically bind to the target sequence, during the PCR amplification process, with the action of DNA polymerase, the structure of the probe changes, and the fluorescence signal will be released, thus realizing real - time quantitative detection of the target gene.

[0036] S2: The new mixed system is amplified by a staged amplification control strategy to obtain amplification products. Among them, the amplification process is to sequentially perform RNA reverse transcription, preferential amplification of KRAS DNA, and amplification of low - abundance cDNA. The staged amplification control strategy is to regulate the order and efficiency of reverse transcription and amplification reactions through temperature gradients; the phosphorothioate - modified primer is used to: inhibit the formation of primer dimers and enhance amplification specificity during the amplification process.

[0037] Specifically, the RNA reverse transcription stage is to convert RNA in the sample into cDNA. Although the PCR technique mainly amplifies DNA, the expression of many tumor - related genes also has important diagnostic and research value at the RNA level. By using reverse transcriptase, under specific temperature and buffer system conditions, cDNA corresponding to the RNA template is synthesized. For example, in some tumor types, the mRNA expression level of certain genes is positively correlated with the malignancy of the tumor. After reverse transcription, the gene expression can be quantitatively analyzed during the subsequent amplification process.

[0038] The purpose of the KRAS DNA preferential amplification stage is to ensure the accurate detection of KRAS gene mutations. The KRAS gene is an important oncogene, and its mutations are relatively common in various tumors such as colorectal cancer. Moreover, different mutation sites and types have a crucial impact on the selection of treatment regimens. In this stage, by optimizing PCR reaction conditions, such as adjusting parameters like primer concentration, magnesium ion concentration, and annealing temperature, KRAS DNA can be preferentially amplified. For example, by increasing the binding efficiency of primers to the KRAS DNA template while inhibiting the amplification of other non-target DNA sequences, the sensitivity and specificity of KRAS gene mutation detection can be significantly improved.

[0039] The low-abundance cDNA amplification stage mainly aims at amplifying cDNA with relatively low content in the sample. Low-abundance cDNA may represent certain gene transcripts with special functions or playing key roles in the occurrence and development of tumors. By adopting special amplification techniques, such as adding appropriate enhancers to the PCR reaction system or using long-fragment amplification strategies, etc., low-abundance cDNA can be effectively amplified, thus providing more information for a comprehensive understanding of the gene expression profile and immune microenvironment of tumors.

[0040] Temperature gradient regulation is achieved by setting a series of different temperature conditions during the PCR reaction to precisely control the sequence and efficiency of reverse transcription and amplification reactions.

[0041] In the reverse transcription stage, a relatively low but appropriate temperature can be set to ensure the activity of reverse transcriptase while avoiding the degradation of RNA templates. For example, at a temperature of about 42 °C, reverse transcriptase can efficiently convert RNA into cDNA.

[0042] In the amplification stage, by gradually increasing the temperature, the DNA double strands are gradually dissociated, primers can specifically bind to the template DNA, and strand extension occurs under the action of DNA polymerase. The setting of the temperature gradient can be optimized according to different gene sequences and primer characteristics to ensure that the reactions in each stage can proceed under the optimal temperature conditions. For example, for some DNA sequences with a high GC content, a higher annealing temperature can be set to ensure specific binding of primers; while for some sequences with a high AT content, amplification can be carried out at a relatively low annealing temperature.

[0043] Inhibiting primer dimer formation is an important advantage of phosphorothioate-modified primers. In a normal PCR reaction, primers may form dimers due to their complementary sequences, consuming a large amount of primers and interfering with the normal PCR amplification process, resulting in reduced amplification efficiency and increased non-specific products. Phosphorothioate-modified primers, due to their special chemical structure, weaken the intermolecular interaction between primer molecules, thus greatly reducing the formation of primer dimers.

[0044] In terms of improving amplification specificity, phosphorothioate-modified primers can bind more precisely to the target DNA sequence. Their modified structure makes the binding of primers to template DNA more stable during the annealing process, reducing the possibility of non-specific binding. For example, when detecting some highly homologous gene sequences, phosphorothioate-modified primers can effectively distinguish the target gene from non-target genes, ensuring that the amplified products are mainly the target gene sequences and improving the reliability of gene detection.

[0045] S3: The amplified products are subjected to signal separation using the probe dynamic quenching technique to obtain FAM and Cy5 dual-channel fluorescence signal data, and the KRAS mutation typing analysis results and tumor-associated macrophage activity scores are synchronously output based on the FAM and Cy5 dual-channel fluorescence signal data. Among them, the probe dynamic quenching technique dynamically controls the release and quenching of fluorescence signals through the light or temperature response mechanism of photosensitive quenching probes.

[0046] Specifically, the light response mechanism of the photosensitive quenching probe is based on the fact that the probe molecule undergoes a structural change under light of a specific wavelength, resulting in the release or quenching of its fluorescence signal. For example, when there is no light, the molecular structure of some photosensitive quenching probes is in a compact state, causing the energy of the fluorescent group to be absorbed by the internal quenching group and unable to emit a fluorescence signal. When stimulated by light of a specific wavelength, the structure of the probe molecule changes, and the energy transfer between the fluorescent group and the quenching group is blocked, allowing the fluorescence signal to be released. This light response mechanism can achieve dynamic regulation of the fluorescence signal by precisely controlling parameters such as the intensity, time, and wavelength of the light.

[0047] The temperature response mechanism uses the changes in the physical and chemical properties of the probe molecule at different temperatures to control the fluorescence signal. At a lower temperature, the structure of the probe molecule is relatively stable, and the fluorescence signal is in a quenched state. As the temperature increases, the structure of the probe molecule undergoes a certain degree of relaxation, and the fluorescence signal is gradually released. For example, during the extension stage of the PCR reaction, as the temperature increases, the fluorescence signal of the photosensitive quenching probe gradually increases, and quantitative analysis of the amplified products can be carried out by real-time monitoring of the fluorescence signal changes.

[0048] The fluorescence signal of the FAM channel is used to detect KRAS mutation typing. Different KRAS mutation sites and types will result in different binding efficiencies with specific FAM channel probes, thus generating fluorescence signals of different intensities. By analyzing parameters such as the intensity and appearance time of the FAM channel fluorescence signal, the mutation type of the KRAS gene can be accurately determined. For example, the G12D mutation and G13R mutation of the KRAS gene will have different characteristics in the FAM channel fluorescence signal. By establishing a corresponding fluorescence signal analysis model, the above two mutation types can be quickly distinguished.

[0049] The fluorescence signal of the Cy5 channel is mainly used for the activity scoring of tumor-associated macrophages. Tumor-associated macrophages play an important role in the tumor immune microenvironment, and the level of their activity is closely related to processes such as tumor growth, invasion, and immune escape. By designing specific Cy5 channel probes, the gene expression situation related to macrophages can be detected. For example, detecting the expression level of certain marker genes on the surface of macrophages, or the expression situation of cytokine genes related to macrophage function. According to the intensity and pattern of the Cy5 channel fluorescence signal, the activity of tumor-associated macrophages can be quantitatively scored, thereby evaluating the state of the tumor immune microenvironment.

[0050] The advantage of synchronously outputting the KRAS mutation typing analysis result and the tumor-associated macrophage activity score is that it can provide comprehensive tumor-related information. In clinical diagnosis, this synchronous detection method can help doctors quickly understand the gene mutation situation and immune microenvironment state of tumors, so as to formulate more precise treatment plans. For example, for colorectal cancer patients with both KRAS mutations and high tumor-associated macrophage activity, a comprehensive treatment strategy combining targeted therapy and immunotherapy can be adopted.

[0051] In the field of tumor research, the above synchronous detection method can provide important data support for in-depth research on the occurrence and development mechanism of tumors. By analyzing the relationship between the KRAS mutation situation and the activity of tumor-associated macrophages in a large number of samples, the interaction mechanism between tumor gene mutations and the immune microenvironment can be explored, providing a theoretical basis for the development of new tumor treatment targets and drugs.

[0052] The above fluorescence PCR method for synchronous detection of KRAS mutations and tumor immune microenvironment realizes the synchronous detection of KRAS gene mutations and the expression analysis of tumor-associated macrophage markers in a single tube by successively implementing a staged temperature control amplification strategy of RNA reverse transcription, preferential amplification of KRAS DNA, and amplification of low-abundance cDNA, and a probe dynamic quenching technique that selectively activates / inhibits fluorescence signals by using the light or temperature response mechanism of photosensitive quenching probes, solving the problems of DNA / RNA co-amplification interference, signal crosstalk, and cumbersome operation in traditional methods, significantly improving the detection sensitivity, specificity, and timeliness, and providing an integrated solution for targeted therapy of colorectal cancer and evaluation of immune microenvironment.

[0053] Reference Figure 2 , in an optional embodiment, the amplification products include amplified KRAS DNA, amplified cDNA, FAM fluorescence signal, and Cy5 fluorescence signal, and S2 includes the following steps:

[0054] S21: Perform RNA reverse transcription treatment and pre-denaturation treatment of KRAS DNA on the new mixed system to obtain cDNA and pre-denatured KRAS DNA; wherein, the RNA reverse transcription treatment is: by activating a temperature-sensitive reverse transcriptase, reverse transcribing CKIP-1 mRNA and CD206 mRNA into cDNA; the pre-denaturation treatment of KRAS DNA is: under specific temperature and time conditions, making the double-stranded KRAS DNA dissociate into single strands by incubation treatment to obtain pre-denatured KRAS DNA.

[0055] Specifically, the temperature-sensitive reverse transcriptase has high activity within a specific temperature range. By precisely controlling the reaction temperature, it can efficiently convert RNA into cDNA. For example, within the temperature range of 37°C - 45°C, this type of reverse transcriptase can tightly bind to the RNA template and use RNA as a template to synthesize a complementary cDNA strand using dNTPs (deoxynucleotide triphosphates).

[0056] The protein encoded by the CKIP-1 (cAMP-dependent kinase anchor protein 1) gene plays a key regulatory role in the cell signal transduction pathway, and changes in its expression level may be closely related to processes such as the proliferation, migration, and invasion of tumor cells. CD206 is a gene related to macrophage function, and the expression of its mRNA can reflect the activation state of tumor-associated macrophages. By reverse transcribing the above two mRNAs into cDNA, it can provide a template for subsequent low-abundance amplification treatment, so as to deeply understand the biological behavior of tumor cells and the activity of macrophages in the tumor immune microenvironment.

[0057] Incubation under specific temperature and time conditions is crucial for the dissociation of KRAS DNA double strands into single strands. The temperature of the pre-denaturation treatment can be set high enough to break the hydrogen bonds between the DNA double strands, separating the double-stranded DNA into single strands. For example, under high-temperature conditions of 94°C - 98°C and after 1 - 5 minutes of incubation, the KRAS DNA double strands can be fully dissociated. The purpose of this process is to prepare for subsequent preferential amplification, as single-stranded DNA is more likely to bind to primers, thereby improving the amplification efficiency and specificity.

[0058] S22: Perform preferential amplification on pre-denatured KRAS DNA to obtain FAM fluorescence signals, including:

[0059] Specifically amplify the pre-denatured KRAS DNA using phosphorothioate-modified primers to obtain amplified KRAS DNA;

[0060] The amplified KRAS DNA specifically binds to the FAM channel probe, activating the photolytic quenching of the FAM channel probe and releasing FAM fluorescence signals.

[0061] Specifically, when the phosphorothioate-modified primers specifically amplify the pre-denatured KRAS DNA, the modified phosphorothioate bond structure makes the binding between the primers and the KRAS DNA template more stable. During the PCR reaction, DNA polymerase can extend along the primers with higher fidelity, synthesizing new DNA strands, thereby obtaining amplified KRAS DNA. Specific amplification can effectively avoid the generation of non-specific products and improve the accuracy and reliability of amplification.

[0062] For example, in some complex gene detection samples, there may be multiple non-target DNA sequences. Ordinary primers may bind to non-target sequences to a certain extent, resulting in non-specific fragments in the amplification products. Due to its special chemical structure, the phosphorothioate-modified primers can more accurately identify the specific sequences of KRAS DNA, making the amplification products mainly KRAS DNA, greatly improving the sensitivity and specificity of detection.

[0063] When the FAM channel probe is not bound to the amplified KRAS DNA, the light emitted by its fluorophore is absorbed by the quencher group of the probe itself, thus being in a quenched state. When the probe specifically binds to the amplified KRAS DNA, under the action of DNA polymerase, the structure of the probe changes, increasing the distance between the fluorophore and the quencher group, and activating the photolytic quenching mechanism. At this time, the FAM fluorescence signal emitted by the fluorophore can be smoothly released and captured by the fluorescence detection system. The generation of this fluorescence signal is closely related to the amplification degree of KRAS DNA. By analyzing the intensity and variation of the FAM fluorescence signal, quantitative and typing analysis of KRAS mutations can be carried out.

[0064] S23: Perform low-abundance amplification on cDNA to obtain Cy5 fluorescence signals, including:

[0065] Use phosphorothioate-modified primers to specifically amplify the cDNA of CKIP-1 mRNA and the cDNA of CD206 mRNA at a specific annealing temperature to generate amplified cDNA;

[0066] By regulating the temperature, the Cy5 channel probe specifically binds to the amplified cDNA, dissociating the Cy5 fluorophore and the quencher group of the Cy5 channel probe, and releasing the Cy5 fluorescence signal.

[0067] Specifically, when specifically amplifying the cDNA of CKIP-1 mRNA and the cDNA of CD206 mRNA, the phosphorothioate-modified primers can improve the amplification efficiency and specificity. Since the above two kinds of mRNA are usually present in low abundance in the sample, ordinary primers may lead to low amplification efficiency or the production of non-specific products due to binding to non-target sequences. The phosphorothioate-modified primers can bind more precisely to the target cDNA sequence and can effectively amplify even in the case of low abundance to generate amplified cDNA.

[0068] For example, in some tumor samples, the mRNA expression levels of CKIP-1 and CD206 may be very low. When using ordinary primers for amplification, weak amplification signals or no amplification may occur. Due to its special chemical modification, the phosphorothioate-modified primers can enhance the affinity with the target cDNA, enabling better amplification effects even under low-abundance conditions and providing a reliable template for subsequent fluorescence signal detection.

[0069] The key to releasing the Cy5 fluorescent signal is to dissociate the Cy5 fluorescent group and the quenching group of the Cy5 channel probe by regulating the temperature. At lower temperatures, the structure of the probe is more compact, the distance between the fluorescent group and the quenching group is closer, and the fluorescent signal is quenched. As the temperature rises, the structure of the probe molecule changes to a certain extent, and the distance between the fluorescent group and the quenching group increases, thereby releasing the quenching state and releasing the Cy5 fluorescent signal. This temperature regulation mechanism can be precisely controlled according to different stages of the PCR reaction process to ensure that accurate Cy5 fluorescent signals are released under appropriate temperature conditions, which is used for quantitative analysis of amplified cDNA and then to evaluate the activity of tumor-associated macrophages.

[0070] In an optional embodiment, the condition for determining that the RNA reverse transcription process is completed in S21 is: the reverse transcription efficiency of the RNA reverse transcription process is higher than the first preset threshold. The calculation formula of the reverse transcription efficiency is:

[0071]

[0072] Among them, E RT is the reverse transcription efficiency, k RT is the activity constant of the temperature-sensitive reverse transcriptase, t1 is the reaction time, T1 is the actual reaction temperature of the RNA reverse transcription process, T opt is the optimal temperature for reverse transcription reaction.

[0073] Specifically, during the RNA reverse transcription process, a reasonable reverse transcription efficiency threshold can be set to determine whether the process is complete. The threshold can be determined based on a large amount of experimental data and experience, and it represents the minimum reverse transcription efficiency standard that can meet the requirements of subsequent experiments. For example, through preliminary experimental exploration and comparative analysis, it is found that when the reverse transcription efficiency reaches more than 80%, the subsequent amplification reaction can obtain more accurate and reliable results, so 80% is used as the first preset threshold. Only when the reverse transcription efficiency actually measured is higher than the threshold can the RNA reverse transcription process be considered to have been completed, thereby ensuring the smooth progress of subsequent experiments.

[0074] The calculation formula used in this embodiment comprehensively considers the influence of various factors on the reverse transcription efficiency and can more accurately reflect the actual effect of the reverse transcription reaction. The following is an explanation of the parameters in the formula:

[0075] k RT(The activity constant of a temperature-sensitive reverse transcriptase) is a key parameter reflecting the activity of reverse transcriptase and is closely related to the catalytic efficiency of the enzyme. Different reverse transcriptases have different activity constants. Under the same reaction conditions, the higher the activity constant of an enzyme, the stronger its ability to catalyze the reverse transcription of RNA into cDNA. For example, for some novel temperature-sensitive reverse transcriptases, after genetic engineering modification, their k RT value is significantly increased, enabling efficient reverse transcription reactions to be completed in a shorter time.

[0076] t1 (reaction time) is one of the important factors affecting reverse transcription efficiency. Under other unchanged conditions, as the reaction time prolongs, the reverse transcription efficiency usually gradually increases. However, when the reaction reaches a certain extent, the improvement in efficiency tends to level off. Therefore, reasonably controlling the reaction time is crucial for obtaining the optimal reverse transcription efficiency. For example, in some rapid reverse transcription kits, by optimizing the reaction system and conditions, efficient reverse transcription reactions can be achieved within 15 - 30 minutes, greatly shortening the experimental time.

[0077] T1 (the actual reaction temperature for RNA reverse transcription treatment) has important effects on both the activity of reverse transcriptase and the stability of RNA. Too high a temperature may cause the inactivation of reverse transcriptase or the degradation of RNA by denaturation, while too low a temperature will reduce the enzyme's activity and affect reverse transcription efficiency. By comparing the actual reaction temperature with T opt (the optimal temperature for reverse transcription reaction) and incorporating it into the calculation formula, the influence of temperature on reverse transcription efficiency can be more accurately evaluated. For example, for a certain temperature-sensitive reverse transcriptase, its optimal reaction temperature is 42°C. When the actual reaction temperature deviates from this temperature, the term in the formula will change, thus affecting the finally calculated reverse transcription efficiency.

[0078] T opt (the optimal temperature for reverse transcription reaction) is the temperature at which reverse transcriptase exhibits the best catalytic activity. At this temperature, a better balance can be achieved between the enzyme's activity and the stability of RNA, thus maximizing the reverse transcription efficiency. Different types of reverse transcriptases may have different optimal temperatures. Therefore, when selecting reverse transcriptases and designing reaction conditions, this factor can be fully considered. For example, for some heat-resistant reverse transcriptases, their optimal temperature may be above 50°C, suitable for reverse transcription reactions that need to be carried out at relatively high temperatures.

[0079] By using the above calculation formula, the efficiency of RNA reverse transcription can be monitored in real time during the experiment, and the reaction conditions can be adjusted in a timely manner or the experimental protocol can be optimized according to the calculation results. For example, during large-scale RNA reverse transcription experiments, samples can be taken regularly to measure the reverse transcription efficiency. If the efficiency is found to be lower than the preset threshold, measures such as extending the reaction time, adjusting the reaction temperature, or replacing the reverse transcriptase can be taken to improve the efficiency and ensure the accuracy of subsequent experiments.

[0080] In addition, this formula can also be used to compare the reverse transcription efficiencies under different reverse transcriptases, different reaction systems, or different experimental conditions, providing a scientific basis for optimizing experimental methods and selecting the best experimental conditions. For example, when screening new reverse transcriptases, this formula can be used to quantitatively compare the reverse transcription efficiencies of different enzymes, so as to quickly determine excellent enzyme varieties.

[0081] In an alternative embodiment, the condition for determining that the photolytic quenching of the FAM channel probe is effective in S22 is: the quenching efficiency of the photolytic quenching of the FAM channel probe is higher than the second preset threshold; wherein, the calculation formula for the quenching efficiency is:

[0082]

[0083] where α is the photolytic coefficient, I is the light intensity at a wavelength of 365 nm, and t2 is the illumination duration.

[0084] Specifically, during the photolytic quenching of the FAM channel probe, a reasonable quenching efficiency threshold can be set. This threshold is called the second preset threshold, which can be determined comprehensively according to factors such as experimental requirements, probe characteristics, and instrument detection sensitivity. Only when the actually measured quenching efficiency is higher than this threshold can the photolytic quenching be considered effective. For example, through a large number of experimental verifications, it is found that when the quenching efficiency reaches more than 90%, the accuracy and reliability of subsequent fluorescence signal detection can be ensured. Therefore, 90% is set as the second preset threshold.

[0085] This embodiment concisely and precisely describes the relationship between the photolytic quenching efficiency and related parameters with this calculation formula. The following is an explanation of the related parameters:

[0086] α (photolytic coefficient) is a parameter reflecting the ability of the FAM channel probe to undergo photolysis under specific illumination conditions.

[0087] I (light intensity at a wavelength of 365 nm) is one of the key factors affecting the photolytic quenching efficiency. Light with a wavelength of 365 nm is the appropriate wavelength for the photolysis of the FAM fluorophore. Within a certain range, the greater the light intensity, the more photons per unit time, and the more opportunities to interact with the probe molecules, resulting in a higher probability of photolysis and quenching efficiency.

[0088] t2 (light illumination duration) determines the total energy received by the probe molecule under light illumination. Under the conditions of a certain light intensity and photolysis coefficient, appropriately extending the light illumination duration can increase the occurrence probability of the photolysis reaction, thereby improving the quenching efficiency. However, an overly long light illumination time may lead to other adverse reactions, such as probe degradation or sample damage. Therefore, the light illumination duration can be reasonably selected according to specific circumstances.

[0089] By using this calculation formula, the photolysis quenching efficiency of the FAM channel probe can be monitored in real time during the experiment, and experimental parameters such as light intensity or light illumination time can be adjusted in a timely manner according to the calculation results to ensure the effectiveness of the quenching process. For example, during a fluorescence PCR experiment, if it is found that the quenching efficiency is lower than the second preset threshold, the quenching efficiency can be improved by increasing the light intensity or extending the light illumination time, thereby ensuring the accuracy of subsequent fluorescence signal detection.

[0090] In addition, this formula can also be used to optimize experimental conditions and probe design. By analyzing the influence of each parameter in the formula on the quenching efficiency, the experimental scheme can be improved targeted, such as selecting a probe material with a higher photolysis coefficient, or using a more suitable light illumination device and parameter settings to improve the performance and reliability of the entire fluorescence detection system.

[0091] In an alternative embodiment, the intensity of the Cy5 fluorescence signal released in S23 is regulated by the following temperature-responsive Sigmoid function:

[0092]

[0093] where F is the intensity of the Cy5 fluorescence signal, F max is the maximum fluorescence intensity of the Cy5 channel probe, T m is the melting temperature of the Cy5 channel probe, T2 is the actual reaction temperature of the low-abundance amplification treatment, and δ is the temperature response slope.

[0094] Specifically, during the fluorescence PCR detection process, the intensity of the Cy5 fluorescence signal needs to be precisely regulated to ensure that it can accurately reflect the amplification of low-abundance cDNA. The temperature-responsive Sigmoid function is a mathematical model used to describe the non-linear relationship between the fluorescence signal intensity and temperature. By using this function, the intensity of the Cy5 fluorescence signal can be dynamically regulated according to the actual reaction temperature, thereby improving the sensitivity and specificity of the detection.

[0095] The following is an explanation of this Sigmoid function:

[0096] F (intensity of Cy5 fluorescence signal) is the output value of the function, representing the strength of the fluorescence signal emitted by the Cy5 channel probe under specific temperature conditions. The magnitude of the fluorescence signal intensity is directly related to the detection result of the amplification product. Therefore, precise function regulation is required to ensure its accuracy.

[0097] F max (maximum fluorescence intensity of Cy5 channel probe) is the maximum fluorescence intensity that the Cy5 channel probe can emit under optimal conditions, which is a theoretical limit value. This limit value depends on factors such as the structure of the probe, the properties of the fluorescent group, and the sensitivity of the detection instrument. In practical applications, F max can be determined experimentally or estimated based on instrument parameters.

[0098] T m (melting temperature of Cy5 channel probe) is an important indicator of the binding stability between the probe and the target sequence. For the Cy5 channel probe, T m refers to the temperature at which the double-stranded structure between the probe and the target sequence begins to dissociate. When the reaction temperature is lower than T m , the probe binds tightly to the target sequence and the fluorescence signal is weak; when the reaction temperature is higher than T m , the probe gradually dissociates from the target sequence and the fluorescence signal gradually increases.

[0099] T2 (actual reaction temperature for low-abundance amplification treatment) is the actual temperature of the PCR reaction system during the low-abundance amplification treatment. By substituting T2 into the Sigmoid function, the corresponding Cy5 fluorescence signal intensity can be calculated, thereby achieving real-time regulation of the fluorescence signal.

[0100] δ (temperature response slope) describes the steepness of the change in fluorescence signal intensity with temperature. A larger δ value indicates that the fluorescence signal intensity is more sensitive to temperature changes, and within the temperature range near T m , the fluorescence signal intensity will rise rapidly; a smaller δ value indicates that the fluorescence signal intensity changes more gently with temperature and the rising speed is slower. By adjusting the δ value, the regulation effect of the fluorescence signal can be optimized to better adapt to different experimental conditions and detection requirements.

[0101] This Sigmoid function dynamically regulates the Cy5 fluorescence signal intensity by comparing the actual reaction temperature T2 with the melting temperature T m and combining the temperature response slope δ. During the low-abundance amplification treatment process, as the reaction temperature gradually increases, when T2 approaches T mWhen the temperature reaches a certain value, the fluorescence signal intensity begins to increase rapidly, showing a sigmoid-shaped change curve. This regulation mechanism can ensure that within the optimal temperature range, the Cy5 fluorescence signal intensity reaches the maximum value, thereby improving the detection sensitivity for low-abundance cDNA.

[0102] For example, when detecting the cDNA of CKIP-1 mRNA and CD206 mRNA, since they are usually present in low abundance in the sample, by using this temperature-responsive sigmoid function to regulate the Cy5 fluorescence signal intensity, the amplification signals of low-abundance cDNA can be more accurately captured during the amplification process, avoiding detection errors caused by weak signals. At the same time, this regulation mechanism can also effectively reduce the interference of non-specific signals and improve the specificity of detection.

[0103] In an alternative embodiment, S3 includes the following steps:

[0104] S31: Dynamically separate the FAM fluorescence signal and the Cy5 fluorescence signal in the amplification product through the light or temperature response mechanism of the photosensitive quenching probe to obtain FAM and Cy5 dual-channel fluorescence signal data.

[0105] Specifically, the photosensitive quenching probe realizes the dynamic separation of the FAM and Cy5 fluorescence signals during the fluorescence PCR reaction through its unique light or temperature response mechanism. In the light response mechanism, when light of a specific wavelength irradiates the probe, the structure of the probe changes, resulting in the energy transfer between the fluorescent group and the quenching group connected to it being blocked, thereby releasing the fluorescence signal. For example, when ultraviolet light with a wavelength of 365 nm irradiates the FAM channel probe, the light emitted by the FAM fluorescent group is activated, and the quenching group connected to it undergoes a structural change under the action of light and can no longer absorb the energy of the FAM fluorescent group, thus releasing the FAM fluorescence signal.

[0106] In the temperature response mechanism, as the temperature of the PCR reaction changes, the structure of the probe also changes accordingly, thereby affecting the release of the fluorescence signal. When the temperature rises to a certain extent, the binding stability between the probe and the target DNA sequence decreases, the conformation of the probe molecule changes, resulting in an increase in the distance between the fluorescent group and the quenching group, thereby releasing the quenching state and releasing the fluorescence signal. For example, in the Cy5 channel probe, when the reaction temperature approaches its melting temperature, the Cy5 fluorescence signal gradually increases.

[0107] Through the light or temperature response mechanism of the photosensitive quenching probe, the two fluorescence signals of FAM and Cy5 in the amplification product can be dynamically separated to obtain the FAM and Cy5 dual-channel fluorescence signals. This process is crucial for subsequent signal analysis because it can ensure the accurate detection and differentiation of the two fluorescence signals, thus providing a reliable signal basis for KRAS mutation typing analysis and tumor-associated macrophage activity scoring.

[0108] S32: According to the FAM channel fluorescence signal intensity data in the FAM and Cy5 dual-channel fluorescence signal data, perform melting curve analysis to obtain the melting temperature of the KRAS mutation subtype; distinguish the G12D, G12V, and G12C mutation subtypes of KRAS based on the melting temperature to obtain the KRAS mutation typing analysis result.

[0109] Specifically, melting curve analysis is a technique for determining the melting temperature (Tm) of double-stranded DNA by monitoring the change in fluorescence signal with temperature. After the PCR reaction ends, as the temperature gradually increases, the double-stranded DNA gradually dissociates into single strands, and the fluorescent probe bound to it will also release fluorescence signals accordingly. By real-time monitoring the change in fluorescence signal, a melting curve can be plotted to determine the melting temperature of the DNA.

[0110] In this method, melting curve analysis is performed based on the intensity data of the FAM channel fluorescence signal. The specific operation is that after the PCR reaction ends, the temperature is gradually increased at a certain temperature gradient while the intensity change of the FAM fluorescence signal is monitored in real time. As the temperature increases, the FAM channel probe bound to the KRAS DNA will gradually release fluorescence signals. When the temperature reaches the melting temperature of the KRAS DNA, the fluorescence signal will change significantly, resulting in a peak on the melting curve, and the temperature corresponding to this peak is the melting temperature of the KRAS mutation subtype.

[0111] Due to the differences in their base sequences, different KRAS mutation subtypes (such as G12D, G12V, G12C) will have different melting temperatures. By comparing the differences in melting temperatures, the mutation subtypes can be accurately distinguished. For example, the melting temperature of the G12D mutation subtype may be slightly higher than that of the G12V mutation subtype, and the melting temperature of the G12C mutation subtype is different from the previous two. Accurately distinguishing mutation subtypes is of great significance for clinical diagnosis and treatment plan selection because different mutation subtypes may have differences in drug sensitivity and prognosis.

[0112] S33: According to the Cy5 channel fluorescence signal intensity data in the FAM and Cy5 dual-channel fluorescence signal data, perform baseline correction and relative quantification to obtain the relative expression levels of CKIP-1 mRNA and CD206 mRNA as the relative expression levels of mRNA.

[0113] Specifically, the purpose of baseline correction is to eliminate background noise and non-specific signal interference during the detection of fluorescence signals, thereby improving the accuracy and reliability of fluorescence signals. During the fluorescence PCR reaction process, due to factors such as the noise of the instrument itself and the non-specific reaction of the reagent, certain background fluorescence signals may be generated. Through baseline correction, the background signal can be deducted, making the detection of fluorescence signals more accurate.

[0114] The method of baseline correction is usually to collect fluorescence signal data for a period of time at the early stage of fluorescence signal detection, that is, before or just at the beginning of the PCR reaction, and use it as the baseline signal. Then, in the subsequent fluorescence signal analysis, subtract the baseline signal corresponding to each time point from the fluorescence signal intensity to obtain the corrected fluorescence signal intensity.

[0115] Relative quantification is to evaluate the changes of target genes (CKIP-1 and CD206) during tumorigenesis and development by comparing their relative expression levels in different samples. Its principle is based on the linear relationship between fluorescence signal intensity and gene expression level. By comparing the relative changes in fluorescence signal intensity in different samples, the relative changes in gene expression level are inferred.

[0116] The specific calculation method is to first normalize the corrected fluorescence signal intensity data, usually using a reference gene for normalization to eliminate differences between samples. Then, by comparing the fluorescence signal intensities of the target gene in different samples, calculate its relative expression level. For example, the 2^(-ΔΔCt) method can be used for calculation, where ΔCt is the difference between the Ct value of the target gene and the Ct value of the reference gene, and ΔΔCt is the difference in ΔCt values between different samples.

[0117] S34: Input the KRAS mutation typing analysis results and the relative mRNA expression level into a preset scoring model to obtain the tumor-associated macrophage activity score.

[0118] Specifically, the preset scoring model is established based on a large amount of experimental data and clinical research results for comprehensively evaluating the activity of tumor-associated macrophages. This model usually uses multiple linear regression, logistic regression or other machine learning algorithms, takes the KRAS mutation typing analysis results and the relative mRNA expression level as input parameters, and calculates the tumor-associated macrophage activity score through certain mathematical formulas or algorithms.

[0119] For example, the preset scoring model can be expressed as: Score = a × KRAS mutation type coefficient + b × CKIP-1 mRNA relative expression level + c × CD206 mRNA relative expression level, where a, b, and c are model coefficients, and the KRAS mutation type coefficient can be assigned different values according to different mutation subtypes.

[0120] The tumor-associated macrophage activity score can comprehensively reflect the activation status and functional changes of macrophages in the tumor immune microenvironment, and is of great significance for evaluating the malignancy, prognosis, and treatment effect of tumors. A higher score may indicate that tumor-associated macrophages are in a highly activated state, which may promote tumor growth, invasion, and immune escape, and the prognosis is poor; while a lower score may indicate that the activity of tumor-associated macrophages is low, the inhibitory effect on tumors is strong, and the prognosis is relatively good.

[0121] In an alternative embodiment, S1 includes the following steps:

[0122] S11: Perform lysis treatment on the sample, mix the colorectal cancer puncture biopsy fluid or blood ctDNA sample with the lysis buffer to obtain a lysis product.

[0123] Specifically, the lysis buffer is a solution specifically designed to disrupt cell structure and release intracellular nucleic acids (DNA and RNA). It can contain various components such as detergents, salts, reducing agents, etc. Detergents can disrupt the lipid bilayer structure of cell membranes and nuclear membranes, releasing cell contents into the solution; salts can adjust the ionic strength of the solution, helping to maintain the stability of nucleic acids; reducing agents can break some disulfide bonds in cells, further promoting the disintegration of cell structure.

[0124] When performing lysis treatment, first mix the colorectal cancer puncture biopsy fluid or blood ctDNA sample with the lysis buffer in a certain proportion. The determination of the mixing ratio can be comprehensively considered according to factors such as the type, concentration of the sample, and the performance of the lysis buffer. For example, for a puncture biopsy fluid with a higher cell density, a higher proportion of lysis buffer can be used to ensure sufficient lysis; while for a blood ctDNA sample, since its cell components are relatively few, the proportion of lysis buffer can be appropriately reduced.

[0125] The lysis reaction is usually carried out under certain temperature and time conditions. Generally speaking, a higher temperature can accelerate the lysis process, but too high a temperature may cause nucleic acid degradation. Therefore, a suitable temperature range can be selected, such as 37°C - 55°C, and the lysis time can be strictly controlled, such as 10 - 30 minutes. During the lysis process, the sample can be gently shaken to promote the full contact between the lysis buffer and cells, improving the lysis efficiency.

[0126] S12: Centrifuge the lysate to remove cell debris and protein precipitates, and collect the nucleic acid mixture in the supernatant as the pre-treated sample.

[0127] Specifically, the purpose of centrifugation is to separate impurities such as cell debris and protein precipitates in the lysate from nucleic acids, so as to obtain a relatively pure nucleic acid mixture. The principle of centrifugation is to use centrifugal force to make substances with different densities receive different centrifugal forces in the centrifugal field, thereby achieving separation. During centrifugation, cell debris and protein precipitates will settle to the bottom of the centrifuge tube due to their higher density, while nucleic acids will be distributed in the supernatant due to their lower density.

[0128] Generally speaking, the higher the centrifugation speed, the better the separation effect, but too high a centrifugation speed may cause damage to nucleic acids. Therefore, an appropriate centrifugation speed can be selected according to the characteristics of the sample and the performance of the centrifuge equipment, which can be set to 10000 - 15000 r / min. The centrifugation time can be set to 5 - 10 min, but it can also be adjusted according to the actual situation.

[0129] To improve the centrifugation effect, the sample can be pre-cooled at 4°C before centrifugation to reduce nucleic acid degradation. In addition, using appropriate centrifuge tubes and centrifuge adapters to ensure the stability and uniformity of the sample during centrifugation is also an important factor in obtaining good centrifugation results.

[0130] S13: Mix the nucleic acid mixture with the premixed system to obtain a new mixed system.

[0131] Specifically, the premixed system contains key components such as phosphorothioate-modified primers and photosensitive quenching probes. Phosphorothioate-modified primers can improve the specificity and amplification efficiency of the PCR reaction, and photosensitive quenching probes are used to achieve dynamic regulation and detection of fluorescence signals in subsequent amplification processes.

[0132] In addition, the premixed system can also contain other necessary components such as DNA polymerase, dNTPs (deoxynucleotide triphosphates), buffer, etc. to ensure the smooth progress of the PCR reaction.

[0133] When mixing the nucleic acid mixture with the premixed system, ensure that the two are fully and evenly mixed to ensure the uniformity and consistency of the PCR reaction. Mixing can be carried out by gently shaking or pipetting, but avoid excessive operation that may cause nucleic acid degradation or the generation of bubbles.

[0134] The mixed system is the new mixed system, which will be used for subsequent staged amplification processing. The quality and uniformity of the new mixed system directly affect the effect of the subsequent PCR reaction. Therefore, strictly control the conditions and quality of the mixing operation to ensure the stability and reliability of the new mixed system.

[0135] The above-mentioned fluorescence PCR method for synchronous detection of KRAS mutation and tumor immune microenvironment, through a staged temperature control amplification strategy and a probe dynamic quenching technique, combined with phosphorothioate-modified primers to inhibit non-specific amplification and a primer pool design optimized by Monte Carlo simulation, synchronously completes the efficient detection of KRAS mutation and tumor immune microenvironment markers in a single tube, effectively overcoming problems such as DNA / RNA co-amplification interference, primer dimer formation, and signal crosstalk, and providing a rapid and accurate integrated detection solution for targeted therapy and immune microenvironment evaluation of colorectal cancer.

[0136] It should be understood that although the steps in the flowcharts involved in the above-described embodiments are sequentially shown according to the indications of the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless there is a clear indication in this article, the execution of these steps has no strict order limit, and these steps can be executed in other orders. Moreover, at least a part of the steps in the flowcharts involved in the above-described embodiments may include multiple steps or multiple stages, and these steps or stages are not necessarily executed at the same moment, but can be executed at different moments, and the execution order of these steps or stages is not necessarily sequential, but can be executed alternately or alternately with at least a part of other steps or steps or stages in other steps.

[0137] Based on the same inventive concept, the embodiments of the present application also provide a system for implementing the above-mentioned fluorescence PCR method for synchronous detection of KRAS mutation and tumor immune microenvironment. The solution provided by this system to solve the problem is similar to the solution described in the above method. Therefore, the specific limitations in one or more embodiments of the fluorescence PCR system for synchronous detection of KRAS mutation and tumor immune microenvironment provided below can refer to the limitations for a fluorescence PCR method for synchronous detection of KRAS mutation and tumor immune microenvironment in the above text, and will not be repeated here.

[0138] In an exemplary embodiment, as Figure 3 shown, a fluorescence PCR system 30 for synchronous detection of KRAS mutation and tumor immune microenvironment is provided, including:

[0139] A sample processing module 31, configured to preprocess a sample to obtain a preprocessed sample; mix the preprocessed sample with a premixed system to obtain a new mixed system.

[0140] Wherein, the sample is a colorectal cancer puncture biopsy fluid or a blood ctDNA sample, the preprocessing includes lysis treatment and centrifugation treatment, the premixed system includes phosphorothioate-modified primers and photosensitive quenching probes, the phosphorothioate-modified primers are synthetic oligonucleotides with a phosphorothioate bond introduced at the 3' end of the primer, and the photosensitive quenching probes include FAM channel probes and Cy5 channel probes.

[0141] The staged temperature control amplification module 32 is used to perform amplification processing on the new mixed system through a staged amplification control strategy to obtain an amplification product.

[0142] Among them, the amplification process is to sequentially perform RNA reverse transcription, preferential amplification of KRAS DNA, and amplification of low-abundance cDNA. The staged amplification control strategy is to regulate the sequence and efficiency of reverse transcription and amplification reactions through temperature gradients; the phosphorothioate-modified primer is used to: inhibit the formation of primer dimers and improve amplification specificity during the amplification process.

[0143] The probe dynamic quenching dual-channel analysis module 33 is used to perform signal separation processing on the amplification product using the probe dynamic quenching technique to obtain FAM and Cy5 dual-channel fluorescence signal data, and synchronously output the KRAS mutation typing analysis result and the tumor-associated macrophage activity score based on the FAM and Cy5 dual-channel fluorescence signal data.

[0144] Among them, the probe dynamic quenching technique is to dynamically control the release and quenching of fluorescence signals through the light or temperature response mechanism of the photosensitive quenching probe.

[0145] Optionally, the amplification product includes amplified KRAS DNA, amplified cDNA, FAM fluorescence signal, and Cy5 fluorescence signal. The staged temperature control amplification module 32 includes:

[0146] The preliminary processing unit 321 is used to perform RNA reverse transcription processing and KRAS DNA pre-denaturation processing on the new mixed system to obtain cDNA and pre-denatured KRAS DNA; among them, the RNA reverse transcription process is to reverse transcribe CKIP-1 mRNA and CD206 mRNA into cDNA by activating a temperature-sensitive reverse transcriptase; the KRAS DNA pre-denaturation process is to dissociate the double-stranded KRAS DNA into single strands by incubation under specific temperature and time conditions to obtain pre-denatured KRAS DNA.

[0147] The preferential amplification unit 322 is used to perform preferential amplification processing on the pre-denatured KRAS DNA to obtain a FAM fluorescence signal, including:

[0148] Specifically amplify the pre-denatured KRAS DNA with a phosphorothioate-modified primer to obtain amplified KRAS DNA;

[0149] The amplified KRAS DNA specifically binds to the FAM channel probe, activating the photolytic quenching of the FAM channel probe and releasing the FAM fluorescence signal.

[0150] The low-abundance amplification unit 323 is used to perform low-abundance amplification processing on the cDNA to obtain a Cy5 fluorescence signal, including:

[0151] Using phosphorothioate-modified primers, specifically amplify the cDNA of CKIP-1 mRNA and the cDNA of CD206 mRNA at a specific annealing temperature to generate amplified cDNA;

[0152] By regulating the temperature, the Cy5 channel probe specifically binds to the amplified cDNA, dissociating the Cy5 fluorophore of the Cy5 channel probe from the quenching group and releasing the Cy5 fluorescence signal.

[0153] Optionally, the preliminary processing unit 321 includes a first determination subunit 3211 for determining whether the RNA reverse transcription process has been completed, and the determination condition is that the reverse transcription efficiency of the RNA reverse transcription process is higher than a first preset threshold.

[0154] Among them, the calculation formula for the reverse transcription efficiency is:

[0155]

[0156] Among them, E RT is the reverse transcription efficiency, k RT is the activity constant of the temperature-sensitive reverse transcriptase, t1 is the reaction time, T1 is the actual reaction temperature of the RNA reverse transcription process, and T opt is the optimal temperature for the reverse transcription reaction.

[0157] Optionally, the priority amplification unit 322 includes a second determination subunit 3221 for determining whether the photolytic quenching of the FAM channel probe is effective, and the determination condition is that the quenching efficiency of the photolytic quenching of the FAM channel probe is higher than a second preset threshold.

[0158] Among them, the calculation formula for the quenching efficiency is:

[0159]

[0160] Among them, α is the photolytic coefficient, I is the light intensity at a wavelength of 365 nm, and t2 is the illumination duration.

[0161] Optionally, the low-abundance amplification unit 323 includes a Cy5 fluorescence signal intensity regulation subunit 3231 for regulating the intensity of the Cy5 fluorescence signal obtained by the low-abundance amplification unit 323 through a temperature-responsive sigmoid function. Among them, the temperature-responsive sigmoid function is:

[0162]

[0163] Among them, F is the intensity of the Cy5 fluorescence signal, and F max is the maximum fluorescence intensity of the Cy5 channel probe, and T mT1 is the melting temperature of the Cy5 channel probe, T2 is the actual reaction temperature of the low-abundance amplification process, and δ is the temperature response slope.

[0164] Optionally, the probe dynamic quenching dual-channel analysis module 33 includes:

[0165] A fluorescence signal separation unit 331, configured to dynamically separate the FAM fluorescence signal and the Cy5 fluorescence signal in the amplification product through the light illumination or temperature response mechanism of the photosensitive quenching probe, and obtain FAM and Cy5 dual-channel fluorescence signal data.

[0166] The FAM channel fluorescence signal analysis unit 332 is configured to perform melting curve analysis processing according to the FAM channel fluorescence signal intensity data in the FAM and Cy5 dual-channel fluorescence signal data to obtain the melting temperature of the KRAS mutation subtype; distinguish the G12D, G12V, and G12C mutation subtypes of KRAS according to the melting temperature, and obtain the KRAS mutation typing analysis result.

[0167] The Cy5 channel fluorescence signal analysis unit 333 is configured to perform baseline correction and relative quantification processing according to the Cy5 channel fluorescence signal intensity data in the FAM and Cy5 dual-channel fluorescence signal data, and obtain the relative expression levels of CKIP-1 mRNA and CD206 mRNA as the relative expression level of mRNA.

[0168] The model scoring unit 334 is configured to input the KRAS mutation typing analysis result and the relative expression level of mRNA into a preset scoring model to obtain the tumor-associated macrophage activity score.

[0169] Optionally, the sample processing module 31 includes:

[0170] A lysis processing unit 311, configured to perform lysis processing on the sample, mix the colorectal cancer puncture biopsy fluid or blood ctDNA sample with a lysis buffer to obtain a lysis product.

[0171] A centrifugation processing unit 312, configured to perform centrifugation processing on the lysis product, remove cell debris and protein precipitation, and collect the nucleic acid mixture in the supernatant as a pretreatment sample.

[0172] A mixing processing unit 313, configured to mix the nucleic acid mixture with a premixed system to obtain a new mixed system.

[0173] An embodiment of the present application further provides a computer device, including a memory and a processor, where the memory stores a computer program, and when the processor executes the computer program, the steps in the foregoing method embodiments are implemented.

[0174] Embodiments of the present application also provide a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, the steps in the above method embodiments are implemented.

[0175] For the device embodiments, since they basically correspond to the method embodiments, the relevant parts can be referred to the descriptions of the method embodiments. The device embodiments described above are only illustrative. The components described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed to multiple network units. Some or all of the modules can be selected according to actual needs to achieve the purpose of the present disclosure solution. Those of ordinary skill in the art can understand and implement it without creative efforts.

[0176] The above embodiments only represent several implementation manners of the embodiments of the present application. The descriptions are relatively specific and detailed, but should not be construed as a limitation on the patent scope of the embodiments of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the embodiments of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the embodiments of the present application.

Claims

1. A fluorescent PCR method for synchronous detection of KRAS mutation and tumor immune microenvironment, characterized in that: The method comprises: S1: pretreating a sample to obtain a pretreated sample; mixing the pretreated sample with a premixed system to obtain a new mixed system; Wherein, the sample is a colorectal cancer puncture biopsy fluid or a blood ctDNA sample, the pretreatment includes a lysis treatment and a centrifugation treatment, the premix system includes a thiophosphate-modified primer and a photosensitive quenching probe, the thiophosphate-modified primer is a synthetic oligonucleotide with a thiophosphate bond introduced at the 3' end of the primer, and the photosensitive quenching probe includes a FAM channel probe and a Cy5 channel probe; S2: amplifying the new mixed system through a staged amplification control strategy to obtain an amplified product; The amplification process is to sequentially perform RNA reverse transcription, KRAS DNA preferential amplification, and low-abundance cDNA amplification, and the staged amplification control strategy is to regulate the order and efficiency of reverse transcription and amplification reactions through a temperature gradient; the thiophosphorylated modified primers are used to: inhibit primer dimer formation and improve amplification specificity during the amplification process; S3: performing signal separation processing on the amplified product using probe dynamic quenching technology to obtain FAM and Cy5 dual-channel fluorescence signal data, and synchronously outputting KRAS mutation typing analysis results and tumor-associated macrophage activity scores based on the FAM and Cy5 dual-channel fluorescence signal data; The dynamic quenching technology of the probe is to dynamically control the release and quenching of the fluorescence signal through the light or temperature response mechanism of the photosensitive quenching probe.

2. The method according to claim 1, characterized in that: The S2 includes: S21: performing RNA reverse transcription treatment and KRAS DNA pre-denaturation treatment on the new mixed system to obtain cDNA and pre-denatured KRAS DNA; wherein the RNA reverse transcription treatment is: activating a temperature-sensitive reverse transcriptase to reverse transcribe CKIP-1 mRNA and CD206 mRNA into the cDNA; the KRAS DNA pre-denaturation treatment is: under specific temperature and time conditions, incubating to dissociate the double-stranded KRAS DNA into single-stranded KRAS DNA to obtain the pre-denatured KRAS DNA; S22: performing preferential amplification treatment on the pre-denatured KRAS DNA to obtain a FAM fluorescent signal, including: The pre-denatured KRAS DNA is specifically amplified by the phosphorothioate-modified primers to obtain amplified KRAS DNA; The amplified KRAS DNA specifically binds to the FAM channel probe, activates the photolysis quenching of the FAM channel probe, and releases the FAM fluorescence signal; S23: performing low-abundance amplification processing on the cDNA to obtain a Cy5 fluorescent signal, including: Using the phosphorothioate modified primers, specifically amplifying the cDNA of CKIP-1 mRNA and the cDNA of CD206 mRNA at a specific annealing temperature to generate amplified cDNA; By regulating the temperature, the Cy5 channel probe specifically binds to the amplified cDNA, so that the Cy5 fluorescent group of the Cy5 channel probe dissociates from the quenching group, releasing the Cy5 fluorescent signal; Wherein, the amplified product includes the amplified KRAS DNA, the amplified cDNA, the FAM fluorescent signal and the Cy5 fluorescent signal.

3. The method according to claim 2, characterized in that The condition for determining that the RNA reverse transcription process has been completed in S21 is: The reverse transcription efficiency of the RNA reverse transcription process is higher than a first preset threshold value; Wherein, the calculation formula of the reverse transcription efficiency is: Among them, E RT is the reverse transcription efficiency, k RT is the activity constant of the temperature-sensitive reverse transcriptase, t1 is the reaction time, T1 is the actual reaction temperature of the RNA reverse transcription process, T opt is the optimal temperature for reverse transcription reaction.

4. The method according to claim 2, characterized in that The condition for determining that the photolysis quenching of the FAM channel probe is effective in S22 is: The quenching efficiency of the photolysis quenching of the FAM channel probe is higher than a second preset threshold; Wherein, the calculation formula of the quenching efficiency is: Wherein, α is the photolysis coefficient, I is the light intensity at a wavelength of 365 nanometers, and t2 is the duration of illumination.

5. The method according to claim 2, characterized in that: The intensity of the Cy5 fluorescent signal released in the S23 is regulated by the following temperature-responsive Sigmoid function: Wherein, F is the intensity of the Cy5 fluorescence signal, F max is the maximum fluorescence intensity of the Cy5 channel probe, T m is the melting temperature of the Cy5 channel probe, T2 is the actual reaction temperature of the low-abundance amplification process, and δ is the temperature response slope.

6. The method according to any one of claims 2 to 5, characterized in that: The S3 includes: S31: dynamically separating the FAM fluorescence signal and the Cy5 fluorescence signal in the amplification product through the light or temperature response mechanism of the photosensitive quenching probe to obtain the FAM and Cy5 dual-channel fluorescence signal data; S32: performing melting curve analysis and processing according to the FAM channel fluorescence signal intensity data in the FAM and Cy5 dual-channel fluorescence signal data to obtain the melting temperature of the KRAS mutant subtype; distinguishing the G12D, G12V, and G12C mutant subtypes of KRAS according to the melting temperature to obtain the KRAS mutation typing analysis result; S33: performing baseline correction and relative quantitative processing according to the Cy5 channel fluorescence signal intensity data in the FAM and Cy5 dual-channel fluorescence signal data to obtain the relative expression amount of CKIP-1 mRNA and the relative expression amount of CD206 mRNA as the mRNA relative expression amount; S34: Inputting the KRAS mutation typing analysis results and the relative expression level of the mRNA into a preset scoring model to obtain the tumor-associated macrophage activity score.

7. The method according to claim 6, characterized in that The S1 includes: S11: performing a lysis treatment on the sample, mixing the colorectal cancer puncture biopsy fluid or blood ctDNA sample with a lysis buffer to obtain a lysis product; S12: centrifuging the lysate to remove cell debris and protein precipitate, and collecting the nucleic acid mixture in the supernatant as the pre-treated sample; S13: Mixing the nucleic acid mixture with the premixed system to obtain the new mixed system.

8. A fluorescent PCR system for simultaneous detection of KRAS mutation and tumor immune microenvironment, characterized in that: The system comprises: The sample processing module is used to pre-process the sample to obtain a pre-processed sample; and mix the pre-processed sample with a pre-mixed system to obtain a new mixed system; Wherein, the sample is a colorectal cancer puncture biopsy fluid or a blood ctDNA sample, the pretreatment includes a lysis treatment and a centrifugation treatment, the premix system includes a thiophosphate-modified primer and a photosensitive quenching probe, the thiophosphate-modified primer is a synthetic oligonucleotide with a thiophosphate bond introduced at the 3' end of the primer, and the photosensitive quenching probe includes a FAM channel probe and a Cy5 channel probe; A staged temperature-controlled amplification module, used to amplify the new mixed system through a staged amplification control strategy to obtain an amplification product; The amplification process is to sequentially perform RNA reverse transcription, KRAS DNA preferential amplification, and low-abundance cDNA amplification, and the staged amplification control strategy is to regulate the order and efficiency of reverse transcription and amplification reactions through a temperature gradient; the thiophosphorylated modified primers are used to: inhibit primer dimer formation and improve amplification specificity during the amplification process; A probe dynamic quenching dual-channel analysis module, which is used to perform signal separation processing on the amplification product using probe dynamic quenching technology to obtain FAM and Cy5 dual-channel fluorescence signal data, and synchronously output KRAS mutation typing analysis results and tumor-associated macrophage activity scores based on the FAM and Cy5 dual-channel fluorescence signal data; The dynamic quenching technology of the probe is to dynamically control the release and quenching of the fluorescence signal through the light or temperature response mechanism of the photosensitive quenching probe.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, wherein: When the processor executes the computer program, the method according to any one of claims 1 to 7 is implemented.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the method according to any one of claims 1 to 7 is implemented.

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