Multiplex fluorescence-based pcr method for detecting thyroid cancer driver gene mutations

Abstract

The application discloses a method for detecting thyroid cancer driving gene mutations based on multiplex fluorescence PCR, and belongs to the technical field of molecular biology detection. The method simultaneously detects nine mutation sites, namely BRAF V600E, NRAS Q61R, NRAS Q61K, HRAS Q61R, HRAS Q61K, KRAS G12V, KRAS G12D, TERT C228T and TERT C250T in a single tube reaction. The method enhances the distinguishing ability of wild type and mutant types by using a locked nucleic acid modified allele-specific primer, suppresses wild type amplification by using a peptide nucleic acid clamp, introduces a competitive internal reference probe to correct amplification efficiency difference, and reduces the sample usage to one fifth of that of a traditional multi-tube scheme. The mutation detection lower limit reaches 0.5% allele frequency, and the total time consumption is not more than 2.5 h.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a method for detecting thyroid cancer driver gene mutations based on multiplex fluorescent PCR. Background Technology

[0002] Thyroid cancer is the most common malignant tumor of the endocrine system, and its global incidence has been rising steadily in recent years. In the molecular pathogenesis of thyroid cancer, driver gene mutations in the mitogen-activated protein kinase pathway and the phosphatidylinositol 3-kinase pathway play a central role. Among them, the BRAF V600E mutation has a detection rate of 40% to 80% in papillary thyroid carcinoma and has been established as a highly specific molecular marker for papillary thyroid carcinoma. RAS family gene mutations, including point mutations in codons 61 of NRAS, 61 of HRAS, and codons 12 / 13 of KRAS, have an overall detection rate of 10% to 50% in follicular thyroid carcinoma and follicular papillary thyroid carcinoma. C228T and C250T mutations in the promoter region of the telomerase reverse transcriptase gene have a detection rate of 10% to 20% in differentiated thyroid cancer, and can reach as high as 40% to 45% in poorly differentiated and undifferentiated carcinomas, and are closely associated with high tumor invasiveness and poor prognosis. When BRAF V600E mutations coexist with TERT promoter mutations, patients have a significantly increased risk of recurrence and disease-specific mortality. Therefore, multi-gene combined detection is of great value for accurately assessing the biological behavior and prognostic stratification of thyroid cancer.

[0003] Currently, the main methods for detecting driver gene mutations in thyroid cancer include Sanger sequencing, ARMS-PCR, digital PCR, and next-generation sequencing. Sanger sequencing, as the gold standard for mutation detection, has a sensitivity of only 15% to 20% allele frequency, making it difficult to detect low-abundance mutations. ARMS-PCR, through allele-specific primer design, can improve sensitivity to 1% to 5% allele frequency; however, existing products mostly use a single-gene, single-tube detection mode. Detecting nine mutation sites across five categories—BRAF, NRAS, HRAS, KRAS, and TERT promoters—usually requires establishing separate reaction systems, which is not only cumbersome but also consumes a large amount of samples. While digital PCR has extremely high sensitivity, the equipment is expensive and has limited throughput, making it unsuitable for routine clinical screening. Next-generation sequencing can achieve comprehensive genomic analysis; however, its long detection cycle, complex data analysis, and high cost pose a risk of over-detection for applications requiring only the detection of known high-frequency mutation sites. Therefore, there is an urgent need to establish a multiplex quantitative PCR detection method that can simultaneously detect multiple core driver gene mutation sites in thyroid cancer in a single-tube reaction, and that combines high sensitivity with ease of operation. Summary of the Invention

[0004] To address the technical problems of low efficiency, large sample consumption, and insufficient sensitivity in existing thyroid cancer driver gene mutation detection technologies, this invention provides a method for detecting thyroid cancer driver gene mutations based on multiplex fluorescent PCR. This method enhances the distinguishing ability between mutant and wild-type templates by designing allele-specific primers modified with locked nucleic acids, selectively inhibits wild-type template amplification using peptide-nucleic acid clamping technology to improve detection sensitivity, and introduces a competitive internal control probe to correct for differences in amplification efficiency of each target in multiplex reactions to ensure quantitative accuracy. This enables simultaneous detection of nine core thyroid cancer driver gene mutation sites—BRAF V600E, NRAS Q61R, NRAS Q61K, HRAS Q61R, HRAS Q61K, KRASG12V, KRAS G12D, TERT C228T, and TERT C250T—in a single-tube reaction system.

[0005] The technical solution of this invention has the following technical advantages. In terms of sensitivity, through the synergistic effect of locked nucleic acid modification and peptide nucleic acid clamping, the lower limit of mutation detection reaches 0.5% allele frequency, which is 2 to 10 times higher than the conventional ARMS-PCR method. In terms of detection efficiency, nine mutation sites can be detected in the same reaction tube, requiring only 20 ng to 100 ng of sample DNA, reducing sample consumption to less than one-fifth compared to traditional multi-tube sorting methods. In terms of quantitative accuracy, the introduction of a competitive internal control probe effectively corrects for amplification efficiency deviations caused by template quality differences and PCR inhibitors, achieving a reliable semi-quantitative assessment of mutation abundance. In terms of ease of operation, the entire detection process can be completed on a conventional real-time PCR instrument without special equipment, and the total time from sample addition to result interpretation does not exceed 2.5 hours. Attached Figure Description

[0006] Figure 1 This is a schematic diagram of the overall process of the thyroid cancer driver gene mutation detection method based on multiplex fluorescent PCR of the present invention.

[0007] Figure 2 A schematic diagram comparing the molecular mechanisms by which nucleic acid modification allele-specific primers bind to mutant and wild-type templates, respectively.

[0008] Figure 3 This is a schematic diagram illustrating the molecular mechanism by which peptide-nucleic acid clamp formulations selectively inhibit the amplification of wild-type templates.

[0009] Figure 4 This is a schematic diagram illustrating the temporal separation principle of peptide nucleic acid clamping and primer annealing under a two-stage temperature gradient annealing mode.

[0010] Figure 5A schematic diagram of the configuration of five-channel fluorescent probe labeling and the allocation of nine target fluorescent channels.

[0011] Figure 6 This is a schematic diagram of the melting curve analysis results of the amplification products of ROX channel positive samples, where the horizontal axis represents temperature and the vertical axis represents the negative first derivative of the fluorescence signal.

[0012] Figure 7 A schematic diagram illustrating the mechanism by which stabilizers such as betaine synergistically eliminate differences in the amplification efficiency of various targets in a multiplex reaction system with dimethyl sulfoxide.

[0013] Figure 8 This is a schematic diagram showing a quantitative comparison of the contributions of each level of technology in the five-fold synergistic mechanism to the sensitivity of mutation detection. Detailed Implementation

[0014] The core of the method for detecting driver gene mutations in thyroid cancer based on multiplex fluorescent PCR provided by this invention lies in the organic integration of locked nucleic acid modified allele-specific primer technology, peptide nucleic acid clamp wild-type inhibition technology, and competitive internal reference correction technology into a single multiplex fluorescent quantitative PCR reaction system, achieving simultaneous detection of nine core driver gene mutation sites in thyroid cancer in a single tube. Figure 1 As shown, the overall process of this method includes four core steps: primer and probe system construction, multiplex reaction system optimization, sample nucleic acid amplification, and result determination. The following provides a detailed description of each technical aspect of this invention.

[0015] Regarding the construction of the primer-probe system, this invention first establishes a detection target panel based on the high-frequency driver gene mutation sites identified in molecular pathology studies of thyroid cancer. This panel covers the V600E mutation (c.1799T>A) at codon 600 of exon 15 of the BRAF gene, the Q61R mutation (c.182A>G) and Q61K mutation (c.181C>A) at codon 61 of exon 3 of the NRAS gene, the Q61R mutation (c.182A>G) and Q61K mutation (c.181C>A) at codon 61 of exon 3 of the HRAS gene, the G12V mutation (c.35G>T) and G12D mutation (c.35G>A) at codon 12 of exon 2 of the KRAS gene, and the C228T mutation (c.1-124C>T) and C250T mutation (c.1-146C>T) in the promoter region of the TERT gene. The selection of the above nine sites is based on their high mutation frequency in various pathological subtypes of thyroid cancer and their clear clinical guidance value, covering more than 85% of known driver gene point mutation events in papillary carcinoma, follicular carcinoma, poorly differentiated carcinoma and undifferentiated carcinoma.

[0016] The design of allele-specific primers is one of the key steps in achieving highly specific mutation detection in this invention. For example... Figure 2 As shown, taking the BRAF V600E mutation site as an example, the penultimate nucleotide at the 3' end of the forward allele-specific primer is designed as a T base complementary to the mutant base A, enabling the primer to bind efficiently only to the mutant template and initiate extension. To further enhance the ability to distinguish between wild-type and mutant templates, a locked nucleic acid (LCA) modification is introduced at the penultimate nucleotide position of the 3' end of the primer. A LCA is a modified nucleotide that forms a rigid bicyclic structure by connecting the 2'-O and 4'-C positions of the ribose through a methylene bridge. This modification significantly enhances the binding affinity of oligonucleotides containing LCA to their complementary strands. In allele-specific primers, when the 3' end region contains both a mutation-specific base and a LCA modification, the binding affinity of the primer with a completely complementary mutant template is much higher than that with a single base mismatch with the wild-type template, with a measured Tm difference of 3°C to 8°C. The thermodynamic difference is further amplified by the exponential amplification effect of PCR cycles, so that under the same amplification conditions, the amplification efficiency of mutant templates can reach more than 500 times that of wild-type templates, thereby achieving effective enrichment of low-abundance mutant signals at the molecular level.

[0017] In the design of reverse primers, the reverse primers for each mutation site are located 100 to 200 bases downstream of the corresponding mutated base, with a primer length of 18 to 25 nucleotides. The Tm value is adjusted to be no more than 2°C different from the corresponding forward primer to ensure that all primer pairs can work in coordination at a uniform annealing temperature. All primer sequences underwent rigorous bioinformatics analysis, including whole-genome alignment using Primer-BLAST to eliminate the possibility of cross-binding with non-target regions, and calculation of the self-folding free energy and Gibbs free energy of primer dimer formation using OligoAnalyzer. Only primer combinations with an absolute free energy value below -6 kcal / mol were retained. In multiplex reaction systems, the formation of primer dimers consumes primer resources and generates non-specific amplification signals, which is one of the main factors affecting the sensitivity and specificity of multiplex PCR. This invention exhaustively calculated and screened the dimer formation tendencies of all primer pairings, ultimately determining a set of primers that do not interfere with each other at an annealing temperature of 60°C. Specifically, the amplicon length for the BRAF target is 145 bp, for the NRAS Q61 site it is 138 bp, for the HRAS Q61 site it is 128 bp, for the KRAS G12 site it is 115 bp, for the TERT C228T site it is 122 bp, and for the TERT C250T site it is 130 bp. The length of each amplicon is controlled within the range of 100 bp to 150 bp, a selection that balances the amplification of degraded DNA templates in paraffin-embedded tissues with the reliability of fluorescence signal detection. For paraffin-embedded tissue samples, DNA typically undergoes varying degrees of fragmentation during fixation and embedding. The degree of fragmentation is related to tissue fixation time and storage conditions. Limiting the length of amplicons to within 150 bp can effectively improve the detection success rate of such degraded samples. Furthermore, controlling the length difference between amplicons to within 30 bp is beneficial for achieving balanced amplification of each target under common annealing temperature and extension time conditions.

[0018] Regarding the design of dual-labeled fluorescent probes, this invention employs the TaqMan hydrolysis probe principle for fluorescence signal detection. Each probe has a fluorescent reporter group labeled at its 5' end and a fluorescent quencher group labeled at its 3' end. When the probe is intact, the quencher group inhibits the fluorescence emission of the reporter group through a fluorescence resonance energy transfer mechanism. During the PCR extension stage, the 5'→3' exonuclease activity of the hot-start DNA polymerase hydrolyzes the probe that has hybridized to the target sequence, releasing the reporter group and generating a fluorescent signal. The intensity of the fluorescent signal is directly proportional to the cumulative amount of amplified products. Figure 5As shown, for the configuration of the five fluorescence detection channels, the BRAFV600E mutation detection probe is labeled with the FAM fluorescent reporter group, with a maximum excitation wavelength of 495 nm and a maximum emission wavelength of 520 nm. The NRAS Q61R / K mutation detection probe is labeled with the HEX fluorescent reporter group, with a maximum excitation wavelength of 535 nm and a maximum emission wavelength of 556 nm. The HRAS Q61R / K and KRAS G12V / D mutation detection probes share the ROX fluorescent reporter group, with a maximum excitation wavelength of 575 nm and a maximum emission wavelength of 602 nm. Differential design of positive allele-specific primers is used to distinguish between different gene mutations. The Cy5 fluorescent reporter group is used to label the TERT C228T / C250T mutation detection probe, with a maximum excitation wavelength of 649 nm and a maximum emission wavelength of 670 nm. The housekeeping gene ACTB competitive internal control probe is labeled with the Quasar 705 fluorescent reporter group, with a maximum excitation wavelength of 690 nm and a maximum emission wavelength of 705 nm. Each probe is 20 to 30 nucleotides in length and has a Tm value of 65°C to 72°C, which is higher than the annealing temperature of 60°C for the amplification reaction, ensuring that the probe can stably bind to the target sequence during the annealing extension stage.

[0019] Regarding the specific implementation of sharing the ROX channel for HRAS and KRAS mutations, since HRAS Q61R / K and KRAS G12V / D mutations in thyroid cancer almost never occur simultaneously in the same patient sample, and their clinical co-occurrence rate is extremely low, classifying them into the same fluorescence channel does not affect clinical application. Specifically, the positive allele-specific primers for HRAS Q61R and Q61K are designed for mutations at positions 182 and 181, respectively, and the positive allele-specific primers for KRAS G12V and G12D are designed for mutations at position 35 (G>T and G>A, respectively). The amplicon lengths generated by each primer pair are 128 bp, 132 bp, 115 bp, and 119 bp, respectively. When a positive signal is observed in the ROX channel, melting curve analysis of the amplification products is performed after the amplification program to distinguish between HRAS and KRAS targets. Specifically, starting from 60℃, the temperature was continuously increased to 95℃ at a rate of 0.2℃ / step, and the fluorescence signal of the ROX channel was continuously acquired, and the negative first derivative curve of the fluorescence signal, i.e., the -dF / dT curve, was plotted. Since the HRASQ61 site amplicon is 128 to 132 bp in length and has a GC content of 48% to 52%, its melting peak temperature is 79℃ to 81℃; while the KRAS G12 site amplicon is 115 to 119 bp in length and has a GC content of 42% to 46%, its melting peak temperature is 75℃ to 77℃. The difference in melting peak temperature between the two types of amplicon is 3℃ to 5℃, which can be clearly distinguished in high-resolution melting curve analysis. Therefore, the identification of HRAS and KRAS mutation types can be completed in the same reaction tube without subsequent sequencing verification. This melting curve differentiation strategy utilizes the inherent differences in base composition and length of different gene amplicons, achieving non-destructive identification of multiple targets in a common channel, and is an important component of the "one-tube complete detection" system of this invention.

[0020] Applications of peptide nucleic acid clamping technology, such as Figure 3As shown, this invention introduces a peptide-nucleic acid clamp formulation as a selective inhibitor of wild-type template amplification in a multiplex reaction system. Peptide-nucleic acid is a synthetically produced nucleic acid analog whose backbone consists of repeating N-2-aminoethylglycine units, with amide bonds replacing the phosphodiester bonds of natural nucleic acids. Peptide-nucleic acid is non-negatively charged and exhibits significantly higher binding affinity to complementary DNA strands than DNA-DNA double strands. For the same length, the Tm value of a peptide-nucleic acid-DNA double strand is approximately 1°C / bp to 1.5°C / bp higher than that of a DNA-DNA double strand. Peptide-nucleic acid is not recognized or extended by DNA polymerase, thus serving as a highly efficient inhibitor of amplification reactions. Furthermore, the Tm value decreases by approximately 12°C to 18°C ​​when a single base mismatch exists between the peptide-nucleic acid and the DNA strand, far exceeding the 5°C to 8°C decrease caused by a single base mismatch in a DNA-DNA double strand. This significant mismatch penalty effect allows the peptide-nucleic acid clamp formulation to precisely distinguish between wild-type and mutant templates with only a single base difference. In the reaction system of this invention, a corresponding peptide nucleic acid clamp formulation is designed for each mutation site.

[0021] Taking BRAF V600E as an example, the peptide-nucleic acid clamp formulation has a sequence that is completely complementary to the wild-type sequence located at position 1799, T base of exon 15 of the BRAF gene. It is 15 bases long and has a Tm value of 72°C with the wild-type template. When the wild-type BRAF template is present in the reaction system, the peptide-nucleic acid clamp formulation preferentially binds to the primer binding site of the wild-type template or its adjacent region at an annealing temperature of 60°C, preventing primer annealing and extension, thereby effectively inhibiting the amplification of the wild-type template. When the V600E mutant template is present, because the peptide nucleic acid forms a single-base mismatch at position 1799 with the mutant template, its Tm value decreases by approximately 15°C to approximately 57°C. At an annealing temperature of 60°C, it cannot stably bind to the mutant template, and the amplification of the mutant template proceeds normally. For the NRAS Q61 site, a peptide-nucleic acid clamp formulation, 15 bases in length, is complementary to the wild-type sequence at codon 61 of exon 3 of the NRAS gene. Its Tm value with the wild-type template is 70°C. With the Q61R mutant template, the Tm value decreases to 56°C due to an A>G mismatch at position 182, and with the Q61K mutant template, the Tm value decreases to 54°C due to a C>A mismatch at position 181. For the HRAS Q61 site and KRAS G12 site, peptide-nucleic acid clamp formulations of 14 and 13 bases in length were designed, respectively, with Tm values ​​of 68°C and 69°C with the corresponding wild-type templates. The design of each peptide nucleic acid clamp formulation follows these principles: The length is selected to ensure that the Tm value with the wild-type template is within the range of 68℃ to 76℃. This guarantees stable binding at an annealing temperature of 60℃ while avoiding insufficient denaturation of the clamp formulation after the denaturation step due to excessively high Tm values, which could affect the amplification efficiency of the next cycle. Through this selective inhibition mechanism, the background interference of the wild-type template on the mutation signal is significantly eliminated, further reducing the mutation detection limit from 1% to 5% in conventional ARMS-PCR to an allele frequency of 0.5%.

[0022] The design of peptide-clamp formulations targeting TERT promoter mutation sites requires special consideration of the high GC content of the TERT promoter region. The GC content of the TERT promoter region is approximately 70% to 80%, and conventional primer and probe design faces challenges such as secondary structure formation and non-specific binding. This invention addresses this issue through the following strategies: the peptide-clamp formulation length for the TERT site is shortened to 14 bases to control the Tm value within a suitable range of 68°C to 74°C; simultaneously, a final concentration of 5% dimethyl sulfoxide is added to the reaction buffer to reduce the secondary structure stability of the GC-rich region. Furthermore, the positive allele-specific primer for the TERT site contains two locked nucleic acid modified nucleotides located at the third and fifth from the end of the 3' terminal to compensate for the loss of allele-specific discrimination ability in the context of high GC content. Figure 7As shown, it is worth noting that betaine, at a final concentration of 1.0 mol / L to 1.5 mol / L, is added to the reaction buffer as an isostabilizing agent. Betaine is an amphoteric compound, and its isostabilizing mechanism lies in selectively reducing the thermodynamic stability difference between AT base pairs and GC base pairs, thereby homogenizing the amplification efficiency of regions with different GC contents. Unlike dimethyl sulfoxide, which non-selectively reduces the Tm value of all double-stranded DNA, betaine selectively weakens the hydration stabilization effect of the guanine N7 position in GC base pairs by replacing water molecules in the major groove of the DNA double helix with hydrogen bonding sites. This effectively eliminates the secondary structural barrier in the high-GC region of the TERT promoter without significantly affecting the amplification efficiency of low-GC target regions. In the aforementioned multiple reaction system, the synergistic effect of betaine and low concentration of dimethyl sulfoxide (DMSO) improved the amplification efficiency of the TERT target to a level comparable to that of other targets. The coefficient of variation of Ct values ​​among the targets decreased from 8.5% when using DMSO alone to 3.2%. Such stabilization effects are an unexpected synergistic effect that cannot be achieved by using DMSO alone or betaine alone.

[0023] Regarding the design and function of the competitive internal control probe, this invention introduces a competitive internal control probe targeting the housekeeping gene ACTB into the multiplex reaction system. This is a crucial technical measure to ensure the accuracy of multiplex detection quantification. In multiplex PCR reactions, primer competition and amplification efficiency differences between targets may lead to excessively high or low detection signals for some targets, affecting the reliability of mutation abundance assessment. The introduction of the competitive internal control probe provides a reference standard for co-amplification with each mutation target in the same reaction tube. The ACTB gene is stably expressed in thyroid tissue cells, and its copy number reflects the total amount of genomic DNA input in the sample. By calculating the difference between the Ct value of each mutation target and the Ct value of the ACTB internal control, i.e., the ΔCt value, systematic biases caused by factors such as differences in sample DNA quality, the presence of PCR inhibitors, and pipetting errors can be eliminated. Specifically, when sample DNA undergoes partial degradation, the amplification efficiency of all targets and internal controls will be affected to a similar degree. Normalization using the ΔCt value yields more accurate mutation abundance assessment results. The final concentration of the internal control primers was set to be lower than that of the mutant target primers, specifically 100 nmol / L for the forward primer and 100 nmol / L for the reverse primer, in order to avoid excessive consumption of common reagent components in the reaction system by the internal control amplification, which would inhibit the detection of low-abundance mutant targets.

[0024] Regarding orthogonal optimization of multiple reaction systems, this invention employs orthogonal experimental design to systematically optimize the key parameters of the multiple reaction system. Orthogonal experimental design L 16 (4 5Five key factors were selected, with four levels for each factor. Factor 1 was the final concentration of the forward primers for each mutation target, with four levels of 100 nmol / L, 200 nmol / L, 300 nmol / L, and 400 nmol / L. Factor 2 was the final concentration of each double-labeled fluorescent probe, with four levels of 80 nmol / L, 120 nmol / L, 180 nmol / L, and 250 nmol / L. Factor 3 was the final concentration of each peptide nucleic acid clamp formulation, with four levels of 150 nmol / L, 250 nmol / L, 375 nmol / L, and 500 nmol / L. Factor 4 was the annealing extension temperature, with four levels of 58℃, 59℃, 60℃, and 61℃. Factor 5 was the final concentration of magnesium chloride, with four levels of 2.0 mmol / L, 2.5 mmol / L, 3.0 mmol / L, and 3.5 mmol / L. The optimal combination of factors was determined by using the lowest coefficient of variation (Ct) and highest signal intensity among the 1% allele frequency standards for each mutant target as the optimization index. Range and variance analyses were performed on 16 experimental results. The optimized reaction system parameters were as follows: the final concentration of the forward primer for each mutant target was 200 nmol / L to 300 nmol / L, adjusted according to the amplification efficiency of each target; the final concentration of each dual-labeled fluorescent probe was 150 nmol / L; the final concentration of each peptide nucleic acid clamp preparation was 375 nmol / L; the annealing extension temperature was 60℃; and the final concentration of magnesium chloride was 3.0 mmol / L. For BRAF V600E, the target with the highest detection rate in thyroid cancer, the final concentration of its forward primer was appropriately reduced to 200 nmol / L to avoid competitive inhibition of other low-abundance targets. For the TERT promoter mutant target, due to the relatively low amplification efficiency of the GC-rich region, the final concentration of its forward primer was increased to 300 nmol / L to ensure sufficient detection sensitivity.

[0025] Regarding the complete composition of the multiplex reaction premix, the 25 μL total reaction volume contains the following components: 12.5 μL of 2×PCR reaction buffer containing 100 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 8.3, 100 mmol / L potassium chloride and 6.0 mmol / L magnesium chloride, 0.5 μL of hot-start DNA polymerase at 2.5 U, 0.5 μL of dNTPs mixture with a final concentration of 200 μmol / L (dTTP is replaced by dUTP), 0.25 μL of uracil DNA glycosylase at a final concentration of 0.2 U / μL, 1.25 μL of dimethyl sulfoxide at a final concentration of 5%, 2.5 μL of betaine solution at a final concentration of 1.0 mol / L, 4.0 μL of primer-probe-peptide-nucleic acid mixture, and 1.0 μL of competitive internal control primer-probe mixture. After adding 5.0 μL of genomic DNA from the sample to be tested, the total reaction volume was 25 μL.

[0026] The detailed implementation of the sample nucleic acid amplification steps involves thoroughly mixing the prepared multiplex reaction premix with the genomic DNA of the sample to be tested, followed by brief centrifugation and transfer to a quantitative real-time PCR instrument for the following procedure: The contamination prevention pretreatment stage involves incubation at 37°C for 10 min. During this stage, uracil DNA glycosylation enzyme degrades any contaminants from previous amplification products containing uracil that may be present in the reaction system. The hot-start activation stage involves heating at 95°C for 10 min. This stage activates the hot-start DNA polymerase and simultaneously inactivates uracil DNA glycosylation enzyme, preventing it from degrading newly synthesized dUTP-containing products during subsequent amplification. The amplification and detection stage consists of 45 cycles. Each cycle includes denaturation at 95°C for 15 s to dissolve the double-stranded DNA template and annealing and extension at 60°C for 60 s to anneal the primers, bind to the template, and complete the extension reaction. At the end of the annealing and extension stage of each cycle, the quantitative real-time PCR instrument simultaneously acquires fluorescence signal intensity data from five fluorescence channels: FAM, HEX, ROX, Cy5, and Quasar 705. In a preferred embodiment of the invention, as shown... Figure 4As shown, the annealing extension stage further employs a two-stage temperature gradient mode to achieve temporal separation of peptide nucleic acid clamping and primer annealing. Specifically, the annealing extension stage of each cycle consists of two temperature steps: The first stage is held at 65°C for 15 seconds. This temperature is higher than the Tm values ​​of each allele-specific primer (58°C to 63°C) but lower than the Tm values ​​of each peptide nucleic acid clamp preparation and the wild-type template (68°C to 76°C). Therefore, at this temperature, the peptide nucleic acid clamp preparation can stably bind to the wild-type template and occupy the target region, while the allele-specific primers cannot form a stable hybrid with the template due to the temperature being higher than their Tm values. The second stage is held at 60°C for 45 seconds. After the temperature decreases, the allele-specific primers begin to anneal. At this time, the primer binding sites of the wild-type template have been pre-occupied by the peptide nucleic acid clamp preparation and cannot be competitively replaced by primers. However, due to the low Tm value caused by the single base mismatch between the mutant template and the peptide nucleic acid, the peptide nucleic acid cannot bind stably in the first stage at 65°C. Therefore, the primer binding sites of the mutant template are always available. This two-stage temperature gradient mode eliminates wild-type leakage amplification caused by insufficient kinetic differences when peptide-nucleotides and primers simultaneously compete for template binding in the traditional single-temperature annealing mode by separating the two competing events of peptide-nucleotide clamping and primer annealing in the temporal dimension. Experimental results show that after adopting the two-stage temperature gradient mode, the wild-type background suppression efficiency is increased from 500 times to more than 2000 times that of the single-temperature mode, and the mutation detection limit is further reduced from 0.5% to 0.1% allele frequency. This non-linear improvement in detection sensitivity far exceeds the improvement that can be achieved by simply extending the annealing time or adjusting the single annealing temperature, confirming that the temporal separation strategy produces unexpected technical effects.

[0027] For detailed implementation of the result determination steps, sample quality is first assessed after amplification. The amplification curve of the Quasar 705 channel (ACTB internal control channel) is examined. A sample is considered acceptable if the internal control Ct value is no more than 28, indicating sufficient amplifiable genomic DNA template. A sample is considered low-quality if the internal control Ct value is greater than 28 but no more than 32, and the result is for reference only. A sample is considered unacceptable if the internal control Ct value is greater than 32 or no amplification curve appears, requiring DNA re-extraction or sample replacement. For acceptable samples, the amplification curves of the four mutation detection channels (FAM, HEX, ROX, and Cy5) are examined. When an S-shaped amplification curve appears in a channel and the Ct value is no more than 38, the ΔCt value for that channel is calculated (target mutation Ct value minus internal control Ct value). The positive threshold for each mutation site is determined by establishing a standard curve using serially diluted standard samples. In a preferred embodiment of the present invention, the positive determination ΔCt threshold for BRAF V600E is 14, for NRAS Q61R / K it is 13, for HRAS Q61R / K and KRAS G12V / D it is 13, and for TERT C228T / C250T it is 14. A positive result is determined when the ΔCt value does not exceed the corresponding threshold. Figure 6 As shown, for samples that tested positive in the ROX channel, a melting curve analysis procedure was immediately performed after 45 amplification cycles. The melting curve analysis procedure was as follows: the reaction system temperature was first lowered to 60℃ and held for 30 s to allow all amplification products to form a double-stranded state. Then, the temperature was continuously increased from 60℃ to 95℃ at a rate of 0.2℃ / step, with each temperature step held for 5 s while acquiring the ROX fluorescence channel signal. After acquisition, the negative first derivative curve of the fluorescence signal, i.e., the -dF / dT curve versus temperature T, was plotted. The amplicons at the HRAS Q61 site, due to their length of 128 bp to 132 bp and GC content of 48% to 52%, had melting peak temperatures of 79℃ to 81℃ for their -dF / dT curves. The amplicons at the KRAS G12 site, due to their length of 115 bp to 119 bp and GC content of 42% to 46%, had melting peak temperatures of 75℃ to 77℃ for their -dF / dT curves. A single melting peak in the -dF / dT curve within the 79℃ to 81℃ range indicates a positive HRAS mutation, while a single melting peak within the 75℃ to 77℃ range indicates a positive KRAS mutation. This melting curve differentiation method utilizes the inherent thermodynamic differences in the base composition of different gene amplicones. It allows for the identification of mutant gene types within the same reaction tube without additional sequencing verification, seamlessly integrating with quantitative real-time PCR detection, increasing the total detection time by no more than 15 minutes.

[0028] Regarding the implementation method of semi-quantitative grading assessment of mutation abundance, this invention divides ΔCt values ​​into three levels for semi-quantitative assessment of mutation abundance. When the ΔCt value is no more than 6, it is considered a high-abundance mutation, corresponding to a mutation allele frequency greater than 20%. This type of mutation signal is strong, suggesting a high proportion of tumor cells in the sample or a homozygous mutation. When the ΔCt value is greater than 6 but no more than 10, it is considered a medium-abundance mutation, corresponding to a mutation allele frequency of 5% to 20%. Mutation signals within this range are of moderate intensity and are commonly found in fine-needle aspiration samples. When the ΔCt value is greater than 10 but does not exceed the mutation positivity threshold, it is considered a low-abundance mutation, corresponding to a mutation allele frequency of 0.5% to 5%. This type of mutation signal is close to the detection limit and may be found in samples with low tumor cell content or subclonal mutations. The mutation positivity threshold ranges from 12 to 16, and the specific value is determined based on the amplification efficiency of each mutation site and the background noise level through a standard curve established by detecting a series of gradient-dilution standards. In a preferred embodiment, the mutation positivity threshold for BRAF V600E is 14, for NRAS Q61R / K it is 13, for HRAS Q61R / K and KRAS G12V / D it is 13, and for TERTC228T / C250T it is 14. The thresholds are selected based on the allele frequency corresponding to the ΔCt value being approximately 0.5% of the method's detection limit, while also ensuring a false positive rate of no more than 1%. When the laboratory changes to different batches of reagents or different instrument models, the above thresholds can be fine-tuned and calibrated by retesting the standard series; the adjustment range is typically no more than ±1 cycle number.

[0029] Regarding the construction of the quality control system, positive control samples were prepared using gene synthesis and plasmid cloning techniques. DNA fragments containing the mutant sequences BRAF V600E, NRAS Q61R, NRAS Q61K, HRAS Q61R, HRAS Q61K, KRAS G12V, KRASG12D, TERT C228T, and TERT C250T were synthesized, each fragment ranging from 200 bp to 300 bp in length and containing the corresponding primer binding sites and probe hybridization regions. Each mutant fragment was cloned into the pUC19 vector, transformed, screened, and sequenced for verification before plasmid DNA was extracted. The mutant plasmid DNAs were mixed at equal copy numbers, and quantified using a combination of UV spectrophotometry and digital PCR to adjust the final concentration of each mutant plasmid to 2 × 10³ copies / μL. The mixed plasmid solution was aliquoted for single use and stored at -20°C. The negative control was genomic DNA extracted from peripheral blood of healthy volunteers, and Sanger sequencing confirmed that all nine mutation sites were wild-type. At least one positive control and one negative control were included in each batch of testing. The batch test results were only valid if the positive control showed positive signals in all mutation detection channels and the negative control showed negative results in all channels.

[0030] The following are verification examples of the present invention.

[0031] Example 1 demonstrates the validation of detection sensitivity. Positive control quality control DNA and negative control genomic DNA were mixed in different proportions to simulate mutation samples with different allele frequencies. A series of diluted standards with allele frequencies of 50%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0.1% were prepared, using 50 ng of total genomic DNA as background, with three replicates for each concentration gradient. Detection was performed using the multiplex fluorescent PCR method of this invention. The results showed that all nine mutation sites could be accurately detected within the allele frequency range of 50% to 0.5%, and the amplification curves of each channel exhibited a typical S-shape with the coefficient of variation of Ct values ​​between the three replicates not exceeding 5%. At the 0.5% allele frequency level, the detection rates for BRAF V600E, NRAS Q61R / K, HRAS Q61R / K, KRASG12V / D, and TERT C228T / C250T were all 100% (3 / 3). At the 0.1% allele frequency level, the detection rates for each mutation site decreased to 33% to 67% (1 / 3 to 2 / 3), indicating that 0.5% is the reliable detection limit for this method. No false positives were observed in the negative controls at all allele frequency levels, with a specificity of 100%.

[0032] Example 2 demonstrates the specificity validation of the multiplex reaction. To verify whether there is cross-reactivity between targets in the multiplex reaction system, nine standards containing only a single mutation type were prepared, with each standard having an allele frequency of 10%. Each single-mutation standard was detected using the multiplex fluorescence PCR method of this invention, and the signal characteristics of each fluorescence channel were observed. The results showed that when a standard containing only the BRAF V600E mutation was added, only the FAM channel showed a positive signal, while the HEX, ROX, and Cy5 channels were negative. When a standard containing only the NRAS Q61R mutation was added, only the HEX channel showed a positive signal. The detection results of each single-mutation standard were completely consistent with expectations, and no cross-reactivity was found between the nine mutation types, confirming the specificity of the multiplex reaction system.

[0033] Example 3 is a clinical sample testing validation. Paraffin-embedded tissue samples were collected from 120 patients with surgically diagnosed thyroid cancer, including 85 papillary carcinomas, 20 follicular carcinomas, 10 poorly differentiated carcinomas, and 5 undifferentiated carcinomas, as well as 30 benign thyroid nodule samples as controls. All tissue samples were independently reviewed and confirmed by two senior pathologists with thyroid pathology diagnostic qualifications. Genomic DNA was extracted using a paraffin-embedded tissue DNA extraction kit. Before extraction, each sample was serially sectioned to a thickness of 10 μm, and 5 to 10 sections were used for DNA extraction. Before extraction, the pathologist marked the tumor region on adjacent HE-stained sections and estimated the proportion of tumor cells, including only samples with a tumor cell proportion of not less than 30%. The concentration of extracted DNA was determined using the Qubit quantitative PCR method, and DNA integrity was assessed using agarose gel electrophoresis. Samples that passed the initial testing were tested in parallel using the method of this invention and Sanger sequencing. Sanger sequencing designed primers for PCR amplification and forward and reverse sequencing of exon 15 of BRAF, exon 3 of NRAS, exon 3 of HRAS, exon 2 of KRAS, and the TERT promoter region.

[0034] The detection results of the method of this invention are as follows: Among 85 cases of papillary thyroid carcinoma, 52 cases (61.2%) showed BRAF V600E mutation, 7 cases (8.2%) showed NRAS Q61R / K mutation, 9 cases (10.6%) showed TERT C228T / C250T mutation, and 6 cases (7.1%) showed co-mutation of BRAF V600E and TERT. The detection rate of BRAF V600E is consistent with the range of BRAF mutation rates reported in large-scale epidemiological studies of thyroid papillary carcinoma both domestically and internationally. Among 20 cases of follicular carcinoma, 6 cases (30.0%) showed NRAS Q61R / K mutation, 3 cases (15.0%) showed HRAS Q61R / K mutation, and 2 cases (10.0%) showed KRAS G12V / D mutation. The overall detection rate of RAS family gene mutations in follicular carcinoma was 55.0%, which is also within the expected range reported in the literature. In 10 cases of poorly differentiated carcinoma, BRAF V600E mutation was detected in 3 cases, TERT C228T mutation in 4 cases, and NRAS Q61R mutation in 2 cases. Among these, 2 cases carried co-mutations of BRAF V600E and TERT C228T, verifying the enrichment of BRAF and TERT co-mutations in disease progression. In 5 cases of undifferentiated carcinoma, BRAF V600E mutation was detected in 2 cases and TERT C228T / C250T mutation in 3 cases. No mutations were detected in 30 benign nodule samples, with a specificity of 100%. Compared with Sanger sequencing results, the overall concordance rate of the method of this invention was 97.3%, or 146 / 150. The method of this invention additionally detected 4 low-abundance mutation samples that were missed by Sanger sequencing due to insufficient sensitivity. These were all low-abundance mutations with allele frequencies ranging from 1% to 5%. The four differentially expressed samples were further validated using digital PCR. The results confirmed that the detection results of the method of the present invention were true positives, indicating that the method of the present invention has a higher ability to detect low-abundance mutations than Sanger sequencing.

[0035] Example 4 verifies repeatability and batch-to-batch consistency. Three BRAF V600E mutant standards with different allele frequencies (high abundance 30%, medium abundance 10%, and low abundance 1%) were selected. Intra-batch repeatability was assessed by the same operator performing 10 consecutive tests on the same quantitative PCR instrument. Batch-to-batch consistency was assessed by three different operators performing 5 tests each on three different models of quantitative PCR instruments. Intra-batch repeatability results showed that the coefficient of variation (Ct) for the high abundance standard was 1.2%, for the medium abundance standard it was 2.3%, and for the low abundance standard it was 3.8%. Batch-to-batch consistency results showed that the Ct coefficient of variation for the high abundance standard was 2.5%, for the medium abundance standard it was 3.6%, and for the low abundance standard it was 5.2%. All test results showed complete consistency in positive / negative qualitative interpretations, with no batch-to-batch differences, indicating that this method has good repeatability and reproducibility.

[0036] Example 5 validates the suitability of different sample types. Twenty fine-needle aspiration biopsy samples of thyroid nodules, 15 fresh tissue samples from thyroid surgical resection, and 30 paraffin-embedded tissue samples were collected. Genomic DNA was extracted using DNA extraction kits matched to each sample type. The method of this invention was used to detect all three sample types. Results showed that for fine-needle aspiration biopsy samples, the DNA extraction amount ranged from 5 ng to 200 ng, with a median extraction amount of 35 ng. The median ACTB internal control Ct value was 24.5, and the proportion of samples meeting the detection requirements was 95%, or 19 / 20. For fresh tissue samples, the DNA extraction amount ranged from 200 ng to 5000 ng, with a median internal control Ct value of 19.8, and all samples met the detection requirements. The DNA extraction volume of paraffin-embedded tissue samples ranged from 20 ng to 500 ng, with a median internal control Ct value of 23.2. The proportion of samples meeting the detection requirements was 93.3%, or 28 / 30. The two samples that did not meet the requirements were old paraffin tissues preserved for more than 5 years. Known mutation-positive samples from all three sample types were correctly detected by the method of this invention, and the detection sensitivity and specificity showed no significant difference among the three sample types. It is worth noting that in fine-needle aspiration biopsy samples, due to the limited sample volume and the mixture of tumor cells and normal thyroid follicular cells, the frequency of mutated alleles is usually lower than in surgically removed tissue samples. The 0.5% detection limit of this invention's method enables reliable detection of driver gene mutations carried by a low proportion of tumor cells in fine-needle aspiration samples, which has significant application value in clinical scenarios of preoperative molecular diagnosis via fine-needle aspiration biopsy. Especially for Bethesda classification III and IV cytologically uncertain nodules, this invention's method can provide multi-gene mutation information under limited sample volume conditions, effectively assisting clinicians in making more accurate preoperative assessment decisions. Furthermore, the detection of coexistence of BRAF V600E and TERT promoter mutations is of particular value in identifying highly aggressive papillary carcinoma subgroups. Literature reports that patients with double mutations have significantly higher disease-specific recurrence and distant metastasis rates than those with single mutations or no mutations. The method of this invention can simultaneously obtain BRAF and TERT mutation information in a single tube reaction, without the need for additional detection steps, providing clinicians with an efficient tool for assessing combined prognostic biomarkers.

[0037] Example 6 compares the performance with existing technologies. Using 120 clinical samples of thyroid cancer and 30 benign control samples from Example 3 as the research subjects, parallel detection was performed using the method of this invention, conventional ARMS-PCR single-sample detection, and next-generation sequencing. The detection sensitivity, specificity, sample volume, detection time, and cost of the three methods were comprehensively compared. The detection sensitivity of the method of this invention is 0.5% allele frequency, while that of conventional ARMS-PCR is 1% to 5%, and that of next-generation sequencing is 2% to 5%. The DNA volume used per sample in the method of this invention is 20 ng to 100 ng, while the total DNA volume used per sample in conventional ARMS-PCR is 100 ng to 500 ng due to the need for multiple tubes, and the DNA volume used in next-generation sequencing is 50 ng to 200 ng. The total time from sample loading to result interpretation for the method of this invention is 2.5 hours, while the total time for conventional ARMS-PCR multi-tube sorting is 4 to 6 hours, and the total time for next-generation sequencing from library preparation to data analysis is 2 to 3 working days. The method of this invention can be run on any real-time PCR instrument equipped with five-channel fluorescence detection capability, without the need for additional specialized equipment. The above comparative results show that the method of this invention maintains detection sensitivity comparable to or even better than next-generation sequencing, while offering significant advantages in terms of operational timeliness and equipment requirements, making it particularly suitable for routine applications in clinical molecular pathology laboratories.

[0038] Regarding the compatibility of the method of this invention with different PCR instrument platforms, the method was validated on three mainstream real-time PCR instruments: Applied Biosystems QuantStudio 5, Bio-Rad CFX96, and Roche LightCycler 480. All three instrument platforms are equipped with five or more fluorescence detection channels, capable of simultaneously acquiring fluorescence signals from five channels: FAM, HEX, ROX, Cy5, and Quasar 705. Using the same reaction premix and standards on all three instruments, the results showed consistent detection sensitivity and specificity across all platforms, with 0.5% allele frequency standards reliably detected on all three instruments. The absolute difference in Ct values ​​between different instruments did not exceed 1.5 cycles, and the difference in ΔCt values ​​did not exceed 0.8 cycles, indicating that the result interpretation strategy based on ΔCt values ​​has good inter-instrument portability. This characteristic eliminates the need for re-optimization of reaction parameters for different instrument platforms, significantly lowering the barrier to widespread application of the method across different laboratories.

[0039] Regarding the stability assurance measures of the reaction system of this invention, in multiplex PCR reactions, degradation of primers and probes during storage and decrease in enzyme activity may affect the stability of detection results. This invention ensures the long-term stability of the reaction system through the following measures: the primer-probe-peptide-nucleic acid mixture is aliquoted and frozen after preparation, with each tube containing only the amount needed for a single experiment, avoiding oligonucleotide degradation caused by repeated freeze-thaw cycles. Trehalose is added to the reaction buffer as an enzyme stabilizer at a final concentration of 50 mmol / L. The polyhydroxy structure of trehalose can form a protective hydration layer on proteins during freeze-drying and freeze-thaw cycles, maintaining the catalytic activity of hot-start DNA polymerase. The prepared multiplex reaction premix is ​​stable for 7 days at 4°C and for 6 months at -20°C. Within the shelf life, the premix used shows no significant difference in detection sensitivity and specificity compared to freshly prepared premix, ensuring the reproducibility of clinical test results.

[0040] The technical solution of this invention achieves highly sensitive single-tube multiplex detection of thyroid cancer driver gene mutations through a triple synergistic mechanism. At the first level, locked nucleic acid modification introduces a rigid double-loop structure into the 3' end region of the primer. This structure restricts the conformational freedom of the ribosome, locking the modified nucleotide in the C3'-endo conformation. This conformation highly matches the glycosidic bond configuration of the A-type double helix, thus enhancing the stacking effect of the locked nucleic acid modified nucleotide with complementary bases and improving the thermodynamic stability of the hybrid. In allele-specific primers, the locked nucleic acid modification is located near the 3' end and synergizes with the mutation-specific base at the 3' end, increasing the difference in binding affinity between the mutant and wild-type templates at the thermodynamic level. When a single-base mismatch occurs between the 3' end of the primer and the wild-type template, the rigid structure of the locked nucleic acid not only fails to compensate for the thermodynamic loss caused by the mismatch but also further amplifies the mismatch penalty effect by limiting the flexibility of local conformational adjustments. Experimental data show that the Tm value difference between allele-specific primers containing locked nucleic acid modifications and mutant and wild-type templates ranges from 3°C to 8°C, while the corresponding Tm value difference between ordinary allele-specific primers without locked nucleic acid modifications is only 1°C to 3°C. This thermodynamic difference is further amplified by the exponential amplification effect of PCR cycles, resulting in an increase in amplification efficiency advantage of mutant templates from several times to hundreds of times under the same amplification conditions.

[0041] At the second mechanism level, peptide-nucleotide clamping kinetically blocks primer annealing and extension of wild-type templates. Because the peptide-nucleotide backbone is uncharged, its binding to the DNA template is unaffected by changes in ionic strength, allowing peptide-nucleotide clamp formulations to maintain stable blocking effects in PCR buffers containing varying concentrations of divalent cations. The binding rate of peptide-nucleotide clamp formulations is faster than that of allele-specific primers, thus preferentially occupying the target region of the wild-type template at annealing temperatures. The resulting peptide-nucleotide-DNA hybrid is not replaced by DNA polymerase at extension temperatures, fundamentally preventing the amplification of the wild-type template. The synergistic effect of these two mechanisms allows the thermodynamic selectivity provided by the locked nucleic acid to work synergistically with the kinetic blocking effect of the peptide-nucleotide, achieving a lower detection limit than either technique alone.

[0042] At the third mechanism level, the competitive internal control probe provides a normalized calibration benchmark at the system level, eliminating systematic biases caused by substrate competition and template quality differences in multiplex reactions. The ACTB internal control amplicon and each mutant target amplicon undergo the same thermal cycling conditions and enzyme kinetics environment in the same reaction tube. Any systematic factors affecting amplification efficiency, such as PCR inhibitor concentration, DNA degradation degree, and pipetting error, will have a consistent impact on both the internal control and the target. Normalization achieved by calculating the ΔCt value effectively eliminates these systematic biases, making the mutation abundance assessment results between different samples comparable. The synergistic effect of the three mechanisms enables the method of this invention to achieve high sensitivity, high specificity, and quantitative accuracy while maintaining clinical operability. Its detection performance is 2 to 10 times better than conventional ARMS-PCR in sensitivity, more than 5 times better than digital PCR in throughput, and better than next-generation sequencing in terms of ease of operation and cost. Figure 8As shown, based on the aforementioned triple molecular-level synergy, this invention further generates unexpected synergistic effects at the reaction kinetics and multiple equilibrium levels through a fourth mechanism—a two-stage temperature gradient temporal separation mechanism—and a fifth mechanism—a betaine-based stabilization mechanism. At the fourth mechanism level, the two-stage temperature gradient mode separates the annealing phase of each PCR cycle into two non-overlapping kinetic events in the temporal dimension: the peptide-nucleic acid clamping establishment window and the primer annealing extension window. In traditional single-temperature annealing schemes, the peptide-nucleic acid clamp formulation and allele-specific primers simultaneously compete for binding to the template target region at the same temperature. Due to spatial overlap or proximity effects at their binding sites, rapid primer annealing may partially occupy the wild-type template binding site before the peptide-nucleic acid clamping is complete, resulting in wild-type leakage amplification signals. The two-stage temporal separation establishes a peptide-nucleic acid-exclusive kinetic window in the first stage, ensuring that the peptide-nucleic acid clamp formulation completely occupies all wild-type template target regions before the primers participate in the competition. The improvement in wild-type inhibition efficiency resulting from this temporal segregation is non-linear, jumping from approximately 500-fold to approximately 2000-fold in the single-temperature mode. This 4-fold non-linear jump surpasses the theoretically achievable linear improvement through increasing peptide nucleic acid concentration or extending annealing time, confirming a non-additive synergistic effect between temporal segregation and chemical clamping. At the fifth mechanism level, the isostabilizing effect of betaine, by selectively reducing the thermodynamic stability difference between GC and AT base pairs, homogenizes the amplification efficiency of nine mutant targets and one internal control target in the same reaction system. In traditional reaction systems containing only dimethyl sulfoxide, the amplification efficiency of TERT promoter targets with GC content as high as 70% to 80% is significantly lower than that of RAS family gene targets with GC content of 45% to 55%, making the detection sensitivity of TERT targets a bottleneck in the entire multiplex reaction system. The addition of betaine enhances the amplification efficiency of the TERT target to a level comparable to other targets, enabling reliable detection of all nine mutant targets at an allele frequency of 0.5%, thus achieving uniformity in detection sensitivity in nine-target multiplex detection. This uniformity effect is a synergistic result of the stabilizing effect of betaine and the destabilizing effect of dimethyl sulfoxide at specific concentration ratios. Using betaine alone cannot achieve the same effect because it cannot sufficiently eliminate the strong secondary structure of the TERT region, while using dimethyl sulfoxide alone impairs the amplification specificity of low-GC content targets due to the non-selective reduction of the Tm values ​​of all targets. The five-fold synergistic mechanism works together from five dimensions: thermodynamic selectivity, kinetic blocking, system normalization, temporal separation, and amplification uniformity, enabling the method of this invention to achieve comprehensive detection performance that cannot be achieved by any single technology or simple combination of technologies.

[0043] The embodiments of the present invention are not limited to the specific embodiments described above. Those skilled in the art can make various equivalent changes or substitutions based on the technical solutions of the present invention, and all such changes or substitutions should be included within the protection scope of the present invention.

Claims

1. A method for detecting driver gene mutations in thyroid cancer based on multiplex fluorescent PCR, characterized in that, Includes the following steps: Primer-probe system construction steps: Allele-specific primers and dual-labeled fluorescent probes were designed for high-frequency mutation sites of thyroid cancer driver genes. The high-frequency mutation sites include BRAF V600E, NRAS Q61R, NRAS Q61K, HRASQ61R, HRAS Q61K, KRAS G12V, KRAS G12D, TERT C228T, and TERT C250T. The penultimate nucleotide at the 3' end of the allele-specific primer corresponds to the mutation base site, and at least one nucleotide from the penultimate to the fourth-to-last position at the 3' end of the allele-specific primer is a locked nucleic acid modified nucleotide. The 5' end of the dual-labeled fluorescent probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. Different target probes in the same reaction tube are labeled with different fluorescent reporter groups from FAM, HEX, ROX, Cy5, and Quasar 705, respectively. Multiplex reaction system optimization steps: The allele-specific primers, the dual-labeled fluorescent probes, the competitive internal control probes, the peptide-clamp formulation, the hot-start DNA polymerase, dNTPs, and PCR buffer are mixed to form a multiplex reaction premix. The competitive internal control probes target the conserved region of the housekeeping gene ACTB and are labeled with an independent fluorescent channel. The sequence of the peptide-clamp formulation is complementary to the wild-type template sequence corresponding to each mutation site and has a length of 13 to 17 bases. During the PCR annealing stage, the peptide-clamp formulation preferentially binds to the wild-type template, thereby selectively inhibiting the amplification of the wild-type template. Sample nucleic acid amplification steps: Add the genomic DNA of the sample to be tested to the multiplex reaction premix, and perform the amplification program on the real-time PCR instrument. The amplification program includes a hot start phase of 95°C pre-denaturation for 10 min, followed by an amplification phase of 45 cycles. Each cycle includes 95°C denaturation for 15 s and 60°C annealing extension for 60 s. During the annealing extension phase, the fluorescence signal of each fluorescence channel is collected. The annealing extension stage adopts a two-stage temperature gradient mode. The first stage is a peptide nucleic acid clamp pre-annealing stage held at 65°C for 15 s. At this temperature, the peptide nucleic acid clamp formulation preferentially binds to the wild-type template, while the allele-specific primers cannot be stably annealed. The second stage is a primer annealing extension stage held at 60°C for 45 s. At this temperature, the allele-specific primers bind to the mutant template and complete the extension reaction. Result determination steps: The Ct value of the fluorescent channel corresponding to the competitive internal reference probe is used as the sample quality assessment index. When the internal reference Ct value does not exceed 28, the sample is considered valid. The amplification curves of each mutation target fluorescent channel are analyzed. The ΔCt value, i.e., the difference between the mutation target Ct value and the internal reference Ct value, is used as the determination basis. When the ΔCt value does not exceed the mutation positive determination threshold, the corresponding mutation site is determined to be positive. The mutation abundance is semi-quantitatively graded and evaluated according to the ΔCt value. When the ROX fluorescent channel is determined to be positive, the melting curve analysis step of the amplification product is performed after the amplification program is completed. The melting curve analysis step includes starting at 60℃ and continuously heating to 95℃ at a heating rate of 0.2℃ / step. The fluorescence signal intensity of the ROX channel is continuously collected at each temperature step. The melting peak temperature of the negative first derivative curve of the fluorescence signal is used to distinguish between HRAS target amplicon and KRAS target amplicon.

2. The method according to claim 1, characterized in that, The locked nucleic acid modified nucleotide is located at the third-to-last position of the 3' end of the allele-specific primer. The 2'-O and 4'-C of the locked nucleic acid modified nucleotide are connected by a methylene bridge to form a double-loop structure, which increases the binding affinity of the allele-specific primer to the mutant template by 3 to 8 °C compared to the binding affinity to the wild-type template.

3. The method according to claim 1, characterized in that, The peptide-nucleic acid clamp formulation targets a 15-meric peptide nucleic acid complementary to the wild-type sequence at the BRAF V600E site, which is located in the region containing the T base at position 1799 of exon 15 of the BRAF gene. The peptide-nucleic acid clamp formulation targets a 14-meric peptide nucleic acid complementary to the wild-type sequence at the C base at position 228 of the TERT promoter region. The Tm values ​​of each peptide-nucleic acid clamp formulation with the corresponding wild-type template are 68°C to 76°C, and the Tm values ​​with the corresponding mutant template are reduced by 12°C to 18°C ​​due to single base mismatches.

4. The method according to claim 1, characterized in that, In the optimization step of the multiplex reaction system, the final concentration of each allele-specific primer was 100 nmol / L to 400 nmol / L, the final concentration of each double-labeled fluorescent probe was 80 nmol / L to 250 nmol / L, the final concentration of each peptide-nucleic acid clamp formulation was 150 nmol / L to 500 nmol / L, and the final concentration of the competitive internal control probe was 100 nmol / L to 200 nmol / L. The concentration ratio of each component was determined by orthogonal experimental design. 16 (4 5 (Screening) 5. The method according to claim 1, characterized in that, The FAM fluorescent reporter group is used to label probes for detecting BRAFV600E mutations, the HEX fluorescent reporter group is used to label probes for detecting NRAS Q61R / K mutations, the ROX fluorescent reporter group is used to label probes for detecting HRAS Q61R / K and KRAS G12V / D mutations, the Cy5 fluorescent reporter group is used to label probes for detecting TERT C228T / C250T mutations, and the Quasar 705 fluorescent reporter group is used to label the competitive internal control probe.

6. The method according to claim 1, characterized in that, The sample to be tested is genomic DNA extracted from thyroid nodule fine-needle aspiration biopsy samples, thyroid surgical resection tissue samples, or paraffin-embedded tissue samples, wherein the concentration of the genomic DNA is not less than 5 ng / μL and the total input amount is 20 ng to 100 ng.

7. The method according to claim 1, characterized in that, The semi-quantitative grading assessment includes: when the ΔCt value is no more than 6, it is judged as a high abundance mutation, with a corresponding mutation allele frequency greater than 20%; when the ΔCt value is greater than 6 and no more than 10, it is judged as a medium abundance mutation, with a corresponding mutation allele frequency of 5% to 20%; when the ΔCt value is greater than 10 and does not exceed the mutation positive judgment threshold, it is judged as a low abundance mutation, with a corresponding mutation allele frequency of 0.5% to 5%.

8. The method according to claim 1, characterized in that, The multiplex reaction premix also contains uracil DNA glycosylation enzyme and dUTP. The final concentration of the uracil DNA glycosylation enzyme is 0.2 U / μL to 0.5 U / μL. An anti-contamination treatment stage of incubation at 37°C for 10 min is added before the hot start stage to degrade the previous amplification product containing uracil.

9. The method according to claim 1, characterized in that, The method further includes a quality control system construction step, wherein the quality control system includes a positive control and a negative control. The positive control is a mixture of recombinant plasmids containing mutant sequence fragments corresponding to each mutation site, and the copy number concentration of each mutant plasmid is 1×10³ copies / μL to 5×10³ copies / μL. The negative control is genomic DNA from peripheral blood of normal individuals. At least one positive control and one negative control are set in each batch of testing.

10. The method according to claim 1, characterized in that, The mutation detection limit of the method is 0.5% allele frequency. The detection sensitivity for BRAF V600E mutation is not less than 99% and the specificity is not less than 98%. The detection sensitivity for NRAS Q61R / K mutation is not less than 97% and the specificity is not less than 98%. The detection sensitivity for TERT C228T / C250T mutation is not less than 96% and the specificity is not less than 97%.

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