Detection method for low-frequency mutation in cfDNA and application thereof

Abstract

The invention relates to a method for detecting low-frequency mutation in cfDNA and application of the method, a mutation detection method with high sensitivity and high specificity is established through a TARGET directional enrichment technology, and the problems that traditional NGS is high in false negative rate, inaccurate in quantification and the like in ultralow-frequency mutation detection are solved. The technical breakthrough not only provides a new tool for early screening and drug resistance monitoring of lung cancer, but also lays a methodological foundation for liquid biopsy application of other tumors.

Description

Technical Field

[0001] The present application relates to the field of molecular diagnostic technology, and in particular to a method for detecting low-frequency mutations in cfDNA and its application. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality rates worldwide. In recent years, breakthroughs in targeted therapies and immunotherapy have enabled lung cancer treatment to become increasingly targeted. The key to achieving this goal lies in the accurate identification of tumor genetic mutations. Epidermal growth factor receptor (EGFR) mutations, in particular, are key driver mutations in non-small cell lung cancer (NSCLC). Their detection directly impacts the use of targeted drugs such as tyrosine kinase inhibitors, significantly improving patients' quality of life. Therefore, the development of highly sensitive and specific gene mutation detection technologies is crucial for early diagnosis and personalized treatment of lung cancer.

[0003] Traditional tumor genetic testing relies on tissue biopsies, which are subject to challenges such as high invasiveness, difficulty obtaining samples, and detection bias due to tumor heterogeneity. Against this backdrop, liquid biopsy techniques—particularly those based on cell-free DNA (cfDNA)—have become a hot topic of research due to their non-invasive and dynamic monitoring capabilities. cfDNA released by tumor cells typically accounts for less than 1% of plasma and is heavily fragmented (approximately 160-180 bp), placing extremely high demands on detection sensitivity. While next-generation sequencing (NGS) can comprehensively analyze the spectrum of genetic mutations, detecting ultra-low-frequency mutations still faces bottlenecks such as sequencing noise, high costs, and complex data analysis. Summary of the Invention

[0004] The purpose of this application is to provide a method for detecting low-frequency mutations in cfDNA. Through TARGET targeted enrichment technology, a highly sensitive and specific mutation detection system is established to address the problems of high false negative rate, inaccurate quantification and limitations of traditional NGS in ultra-low-frequency mutation detection. This technological breakthrough not only provides a more powerful and convenient new tool for early screening of lung cancer (especially suitable for large-scale population screening and remote areas) and long-term dynamic monitoring of drug resistance (facilitating regular sampling by patients at home), but also lays a solid methodological foundation for expanding the application of liquid biopsy to other tumors.

[0005] In a first aspect, the present application provides a method for detecting low-frequency mutations in cfDNA, comprising the following steps: (1) cfDNA library construction; (2) TARGET detection: Quantitative PCR is performed on the cfDNA library obtained in step (1) using blocking probes and enrichment primers that detect gene mutation sites to obtain the Ct value; (3) Data analysis: Calculate the difference ΔCt between the standard (wild-type DNA) and the sample Ct. When the ΔCt value exceeds the preset threshold, it is considered that a mutation exists in the sample; The blocking probe precisely matches the wild-type (WT) template sequence, and has a Tm of 60°C to 72°C.

[0006] Optionally, the cfDNA is derived from plasma or dried blood spots.

[0007] Optionally, the cfDNA library construction includes the following steps: sample collection; extraction and quantification of cfDNA; end modification; linker ligation; enrichment of the cfDNA library; and quality control of the cfDNA library.

[0008] Optionally, the sample volume is 50 μL-200 μL, and the input amount of the cfDNA library is 20 ng-30 ng.

[0009] Optionally, the preset threshold is determined by the Youden index calculated by the ROC curve.

[0010] Optionally, the 3' end of the blocking probe is modified with a phosphorylation group or Spacer C3.

[0011] Optionally, the gene mutation sites include EGFR L858R, EGFR T790M, EGFR E746_A750del, EGFR G719S and EGFR L747_T751del, KRAS G12D / 13D, BRAF V600E and PIK3CA E545K.

[0012] Optionally, the nucleotide sequence of the primer pair for detecting the EGFR L858R site is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12; the nucleotide sequence of the primer pair for detecting the EGFR T790M site is shown in SEQ ID NO: 3-5, and the nucleotide sequence of the probe is shown in SEQ ID NO: 13; the nucleotide sequence of the primer pair for detecting the EGFR E746_A750del site is shown in SEQ ID NO: 6-7, and the nucleotide sequence of the probe is shown in SEQ ID NO: 14; the nucleotide sequence of the primer pair for detecting the EGFR G719S site is shown in SEQ ID NO: 8-9, and the nucleotide sequence of the probe is shown in SEQ ID NO: 15; the nucleotide sequence of the primer pair for detecting the EGFR L747_T751del site is shown in SEQ ID NO: 10-11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 16; the nucleotide sequence of the primer pair for detecting the KRASG12D / 13D site is shown in SEQ ID NO: NO: 17-18, the nucleotide sequence of the probe is shown in SEQ ID NO: 23; the nucleotide sequence of the primer pair for detecting the BRAF V600E site is shown in SEQ ID NO: 19-20, and the nucleotide sequence of the probe is shown in SEQ ID NO: 24; the nucleotide sequence of the primer pair for detecting the PIK3CA E545K site is shown in SEQ ID NO: 21-22, and the nucleotide sequence of the probe is shown in SEQ ID NO: 25.

[0013] In a second aspect, the present application provides an application of the method of the first aspect in lung cancer detection.

[0014] In summary, this application includes at least one of the following beneficial technical effects: 1. TARGET technology effectively inhibits the amplification of wild-type sequences, significantly enriches mutant sequences, and effectively improves detection accuracy. It is particularly suitable for dynamic tracking of low-frequency mutations in efficacy monitoring; 2. The standardized cfDNA extraction and library construction process significantly reduces sample batch variability, providing reliability for clinical translation; 3. The stability of cfDNA in dried blood spots is significantly higher than that in plasma, which allows the storage period of blood samples at room temperature to be extended to more than 30 days, breaking through the limitations of cold chain transportation and providing new possibilities for tumor mutation detection and dynamic disease monitoring. This study innovatively integrated it into a high-sensitivity cfDNA detection platform - ultra-early monitoring of molecular residual disease (MRD) in cancer patients can be achieved through two drops of blood samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Plasma-target ROC curve for plasma samples; Figure 2 This is the DBS-target ROC curve of dried blood spot samples. DETAILED DESCRIPTION

[0016] In this application, the forward primer (abbreviated as F), reverse primer (abbreviated as R) and blocking probe (abbreviated as B) information of the detected mutant gene are shown in Table 1 below.

[0017] Table 1

[0018] Example 1: A method for detecting low-frequency mutations in cfDNA

[0019] 1.1 cfDNA library construction

[0020] Step 1: Collection of plasma samples from lung cancer patients.

[0021] Specifically, venous blood samples freshly collected from four lung cancer patients (20 mL each) were used for testing. Immediately after blood collection, the EDTA anticoagulant tubes were gently inverted 8-10 times to fully anticoagulate. The tubes were then centrifuged at 1600 g for 10 minutes at 4°C, and the upper plasma layer was transferred to a 15 mL sterile centrifuge tube. Residual cell debris was removed by centrifugation at 16,000 g for 10 minutes at 4°C, and finally, ≥8 mL of clarified plasma was dispensed into 4 mL cryogenic tubes.

[0022] Step 2: Extraction and quantification of cfDNA.

[0023] Specifically, cfDNA was extracted from a 4 mL plasma sample. The concentration of the extracted cfDNA was measured using a Qubit Fluorometer, and the fragment size distribution of the extracted cfDNA was analyzed using an Agilent 2100 Bioanalyzer. Qualified cfDNA was stored at -20°C.

[0024] Step three: end repair.

[0025] The details are as follows: 10 ng of cfDNA was taken and end-repaired with dA tailing using the VAHTS Universal DNA Library Prep Kit, and the volume was filled to 65 μL with ddH2O. The reaction was performed in a PCR instrument and the end-repair program was run: hot cover temperature 105°C, 20°C for 15 min, 65°C for 15 min, and 4°C hold.

[0026] Step 4: Adapter ligation: DNA is ligated to Dual UMI UDI Adapters using DNA ligase. The ligation product is purified using magnetic beads and screened for fragments of appropriate size. Dual UMI UDI Adapters are specialized adapters used for high-throughput sequencing (NGS) library construction. Their design combines dual-end unique molecular identifiers (UMIs) and unique dual-end indexes (UDIs) to improve sequencing data accuracy and the ability to distinguish diverse samples.

[0027] To do this, dilute the adapter 15-fold to 1 μM. Perform the adapter ligation reaction using the reaction system in Table 2 below. Use the following protocol in a PCR instrument: heated lid at 105°C, incubate at 20°C for 15 minutes, and hold at 4°C. Pipette 60 μL of VAHTS DNAClean Beads into 100 μL of ligation product, mix thoroughly, and incubate at room temperature for 5 minutes. Remove the supernatant and rinse the beads with 200 μL of freshly prepared 80% ethanol. Incubate at room temperature for 30 seconds, remove the supernatant, and repeat the rinse twice. Elute with 22.5 μL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5), mix thoroughly, and incubate at room temperature for 2 minutes. Transfer 20 μL of the purified product to a fresh microcentrifuge tube.

[0028] Table 2

[0029] Step 5: Library enrichment.

[0030] The details are as follows: The PCR amplification reaction system and PCR program are shown in Tables 3 and 4. The amplified library product was purified using VAHTS DNA CleanBeads (0.9×) magnetic beads. After purification, 22.5 μL of 10 mM Tris-HCl, pH 8.0-8.5, was added for elution. 20 μL of the purified product was transferred to a new 1.5 mL centrifuge tube to obtain the sample DNA prelibrary.

[0031] Table 3 Reaction system

[0032] Table 4 PCR program

[0033] Step 6: Quality control of the library.

[0034] Specifically, the library concentration and fragment size distribution were detected using Qubit 3.0 Fluorometer and Agilent 2100 Bioanalyzer, respectively.

[0035] 1.2 Plasma-TARGET Detection CFDNA libraries derived from plasma samples were tested using the newly developed plasma-TARGET technology for quantitative PCR (qPCR) analysis. The core breakthrough of plasma-TARGET technology lies in the targeted enrichment of mutant sequences through molecular blocking and highly specific amplification.

[0036] To ensure the specificity, sensitivity, and efficiency of mutation detection, the design principles of the amplification primers and blocking probes used in plasma-TARGET are based on several key guidelines: the primers, also called enrichment primers, are located near the target mutation locus and are designed with a melting temperature (Tm) between 55°C and 63°C, enabling robust amplification under standard PCR conditions; on the other hand, the blocking probes precisely match the wild-type (WT) template sequence, with their Tm ranging from 60°C to 72°C. Their main function is to compete with the upstream enrichment primers for binding to the WT template, thereby preventing the amplification of non-mutant sequences; to further enhance specificity, the 3' end of the blocking probe is modified with a phosphorylated group or Spacer C3 (3SpC3), which effectively inhibits extension during PCR and reduces nonspecific amplification; in addition, the overlapping region between the enrichment primers and the blocking probe spans 6 to 14 bases, and the Tm of the non-overlapping fragment of the blocking probe is intentionally designed to be higher than the Tm of the non-overlapping fragment of the enrichment primer, ensuring effective discrimination between wild-type and mutant sequences.

[0037] The details of the forward primer (F), reverse primer (R), and blocking probe (B) for EGFR gene mutations are shown in Table 1. Standard Taq polymerase reactions were performed using PowerUp SYBR Green Master Mix, and qPCR was performed for mutant and wild-type samples using the SLAN-96P Real-Time PCR Detection System. The qPCR reaction system and protocol are shown in Tables 5 and 6.

[0038] Table 5

[0039] Table 6

[0040] 1.3 Data Analysis Calculate the Ct difference (ΔCt value) between the sample and the standard (wild type). The calculation formula for the ΔCt value is: ΔCt=WT_Ct−MT_Ct, where MT_Ct is the Ct value of the sample and WT_Ct is the Ct value of the standard (wild type).

[0041] Determination of the Preset Threshold: High-throughput sequencing was performed using an Illumina HiSeq X Ten NGS platform to obtain detailed gene mutation information. The mutation types and allele frequencies (VAF) in plasma cfDNA samples were analyzed. A VAF value > 0.5% was considered positive by NGS. The results of NGS were used to construct a plasma-target receiver operating characteristic (ROC) curve, and the area under the curve (AUC) was calculated to measure the overall detection performance. The Youden Index (Yoden Index) was calculated from the ROC curve. The optimal detection threshold (ΔCT) was determined based on the Youden Index and used as the pre-set threshold.

[0042] When the ΔCt value exceeds the preset threshold, it is considered that a mutation exists in the sample.

[0043] Example 2: Plasma sample test results 2.1 Minimum detection limit of plasma-TARGET technology The minimum detection limit test results of plasma-TARGET technology are shown in Table 7.

[0044] For a mutation frequency of 0% (such as EGFR L858R-0%), the CT value of the probe without blocking was 21.2, while the CT value of the probe with blocker increased to 35.6, indicating that wild-type amplification was effectively inhibited and the specificity was significantly improved.

[0045] Without the blocking probe, the Ct values ​​for mutation frequencies of 1%, 0.10%, and 0% were essentially identical. However, with the introduction of the blocking probe, the Ct values ​​for mutation frequencies of 1% and 0.1% compared to 0% significantly increased, indicating that the method's minimum detection limit (LOD) is lowered and detection capability is enhanced after the blocking probe is introduced. This also demonstrates that the plasma-TARGET technology described in this application can achieve a minimum detection limit of 0.10% for mutation frequencies. In liquid biopsies, early cancer screening, or MRD monitoring, the mutant allele frequency (VAF) can be extremely low (e.g., <0.5%). If the LOD of the detection method is higher than this value, many low-frequency mutations will be misclassified as "negative" (increasing FN and reducing sensitivity). The LOD of this method, 0.10%, significantly improves sensitivity. Mutation frequencies are determined by NGS.

[0046] Table 7

[0047] 2.2 Test results of clinical samples The test results for 131 clinical samples showed that the AUC value of the method described in Example 1 reached 0.97. Figure 1 As shown, it shows that the detection method has high robustness and reliability. The Youden Index was calculated by the ROC curve, and the optimal detection threshold (ΔCT) was determined based on the Youden Index, and the optimal threshold was finally obtained as 3.24. At this threshold, the positive consistency of the detection of the method described in Example 1 was 90.79%, the negative consistency was 89.1%, and the overall consistency was 90%, which shows that this method can effectively balance the false positive and false negative rates in mutation determination, ensuring the reliability and accuracy of the detection. The representative clinical sample test results are shown in Table 8. The ΔCt values ​​of all samples are significantly greater than the preset threshold, and the higher the mutation frequency, the greater the ΔCt value. This shows that the plasma-TARGET technology can achieve semi-quantitative analysis of mutation abundance through ΔCt values, which is suitable for dynamic monitoring of the progression of drug-resistant mutations.

[0048] Therefore, we believe that the method described in Example 1 demonstrates excellent performance in plasma cfDNA mutation detection. It can not only effectively identify low-frequency mutations, but also maintain high accuracy and sensitivity in the detection of different types of mutations, demonstrating its application potential in clinical tumor gene detection.

[0049] Table 8

[0050] Example 3: A method for detecting low-frequency mutations in dried blood spot cfDNA 3.1 cfDNA library construction Step 1: Collection of dried blood spot samples from lung cancer patients.

[0051] Freshly collected fingerstick blood samples from 68 lung cancer patients were used to prepare dried blood spot specimens. Approximately 100 µL (approximately two drops) of fingerstick blood was dispensed from each sample and applied to the QIAcard FTA Classic (QIAGEN). All samples were allowed to rest at ambient temperature for at least four hours before being transferred to sealed plastic bags and stored at room temperature to ensure sample integrity and prevent degradation.

[0052] Step 2: Extraction and quantification of cfDNA from dried blood spots.

[0053] The specific procedure is as follows: Based on the experimental requirements, a 3mm diameter hole punch is used to punch out a dried blood spot of the specified size. This spot is then placed in a 1.5ml centrifuge tube and eluted with a predetermined volume of eluent. The resulting dried blood spot eluate, the sample to be tested, is then used for extraction. The concentration of the extracted cfDNA is measured using a Qubit Fluorometer, and the size distribution of the extracted cfDNA is analyzed using an Agilent 2100 Bioanalyzer. Qualified cfDNA is stored at -20°C.

[0054] Step three: end repair.

[0055] The details are as follows: 100 μL of DNA extracted from dried blood spot samples was end-repaired with dA tailing using the VAHTS Universal DNA LibraryPrep Kit, and the volume was filled to 65 μL with ddH2O. The DNA was placed in a PCR instrument and the following reaction was performed. The end-repair program was run: hot cover temperature 105°C, 20°C for 15 min, 65°C for 15 min, and 4°C hold.

[0056] Step 4: Adapter ligation: DNA is ligated to Dual UMI UDI Adapters using DNA ligase. The ligation product is purified using magnetic beads and screened for fragments of appropriate size. Dual UMI UDI Adapters are specialized adapters used for high-throughput sequencing (NGS) library construction. Their design combines dual-end unique molecular identifiers (UMIs) and unique dual-end indexes (UDIs) to improve sequencing data accuracy and the ability to distinguish diverse samples.

[0057] To do this, dilute the adapter 5-fold. Perform the adapter ligation reaction using the reaction system in Table 9 below. Use the following cycler program: heated lid at 105°C, incubate at 20°C for 15 minutes, and hold at 4°C. Pipette 60 μL of VAHTS DNA Clean Beads into 100 μL of ligation product, mix thoroughly, and incubate at room temperature for 5 minutes. Remove the supernatant and rinse the beads with 200 μL of freshly prepared 80% ethanol. Incubate at room temperature for 30 seconds, remove the supernatant, and repeat the rinse twice. Elute with 22.5 μL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5), mix thoroughly, and let stand at room temperature for 2 minutes. Transfer 20 μL of the purified product to a fresh microcentrifuge tube.

[0058] Table 9

[0059] Step 5: Library enrichment.

[0060] The details are as follows: The PCR amplification reaction system and PCR program are shown in Tables 10 and 11. The amplified library product was purified using VAHTS DNAClean Beads (0.9×) magnetic beads. After purification, 22.5 μL of 10 mM Tris-HCl, pH 8.0-8.5, was added for elution. 20 μL of the purified product was transferred to a new 1.5 mL centrifuge tube to obtain the sample DNA prelibrary.

[0061] Table 10 Reaction system

[0062] Table 11 PCR program

[0063] Step 6: Quality control of the library.

[0064] Specifically, the library concentration and fragment size distribution were detected using Qubit 3.0 Fluorometer and Agilent 2100 Bioanalyzer, respectively.

[0065] 3.2 DBS-TARGET Detection Dried blood spot samples were tested using the newly developed DBS-TARGET technology for quantitative PCR (qPCR) analysis. The core breakthrough of DBS-TARGET technology lies in the targeted enrichment of mutant sequences through molecular blocking and highly specific amplification.

[0066] To ensure the specificity, sensitivity, and efficiency of mutation detection, the design principles of the amplification primers and blocking probes used in DBS-TARGET are consistent with those of plasma-TARGET technology.

[0067] Details are as follows: The forward primer (F), reverse primer (R), and blocking probe (B) for the gene mutation are shown in Table 1. Standard Taq polymerase reactions were performed using PowerUp SYBR Green Master Mix, and qPCR was performed for mutant and wild-type samples using the SLAN-96P Real-Time PCR Detection System. The qPCR reaction system and protocol are shown in Tables 12 and 13.

[0068] Table 12

[0069] Table 13

[0070] 3.4 Data Analysis Calculate the Ct difference (ΔCt value) between the sample and the standard (wild type). The calculation formula for the ΔCt value is: ΔCt=WT_Ct−MT_Ct, where MT_Ct is the Ct value of the sample and WT_Ct is the Ct value of the standard (wild type).

[0071] Determination of the Preset Threshold: High-throughput sequencing was performed using an Illumina HiSeq X Ten NGS platform to obtain detailed gene mutation information. The mutation types and allele frequencies (VAF) in plasma cfDNA samples were analyzed. A VAF value > 0.5% was considered positive by NGS. The results of NGS were used to construct a plasma-target receiver operating characteristic (ROC) curve, and the area under the curve (AUC) was calculated to measure the overall detection performance. The Youden Index (Yoden Index) was calculated from the ROC curve. The optimal detection threshold (ΔCT) was determined based on the Youden Index and used as the pre-set threshold.

[0072] When the ΔCt value exceeds the preset threshold, it is considered that a mutation exists in the sample.

[0073] Example 4: Dried blood spot sample test results The test results for 68 clinical samples showed that the AUC value of the method described in Example 3 reached 0.96. Figure 2 As shown, this indicates that the detection method has high robustness and reliability. The Youden Index was calculated by the ROC curve, and the optimal detection threshold (ΔCT) was determined based on the Youden Index, and the optimal threshold was finally obtained as 1.39. At this threshold, the specificity of the detection method described in Example 3 was 93.00%, the sensitivity was 92.00%, the positive consistency was 86.84%, the negative consistency was 88%, and the overall consistency was 83.82%, which shows that this method performs well in mutation determination. The representative clinical sample test results are shown in Table 14. The ΔCt values ​​of all samples are significantly greater than the preset threshold, and the higher the mutation frequency, the greater the ΔCt value. This shows that the DBS-TARGET technology can achieve semi-quantitative analysis of mutation abundance through the ΔCt value, which is suitable for dynamic monitoring of the progression of drug-resistant mutations.

[0074] Therefore, we believe that the method described in Example 3 also exhibits excellent performance in the detection of cfDNA mutations in dried blood spots. It can not only effectively identify low-frequency mutations, but also maintain high accuracy and sensitivity in the detection of different types of mutations, demonstrating its application potential in clinical tumor gene detection.

[0075] Table 14

[0076] Example 5 Optimization of Dried Blood Spot Samples in the TARGET Technology Detection Process 5.1 Extraction of cfDNA from Dried Blood Spot Samples DNA was extracted using the TIANamp Micro DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) according to the manufacturer's instructions for FTA cards, with several key optimizations performed to improve cfDNA recovery. First, three 3 mm discs were punched from the dried blood spot sample, and carrier RNA was added to Buffer AL according to the manufacturer's instructions. Optimization experiments evaluated different incubation times and reaction temperatures, ultimately determining that incubation at 56°C for 1 hour yielded the highest cfDNA yield and quality, as shown in Table 15.

[0077] Table 15

[0078] 5.2 Library input optimization results In an experiment to optimize the library input, this study evaluated the effects of cfDNA library inputs of 10 ng, 20 ng, 30 ng, and 50 ng on the sensitivity and specificity of the TARGET assay. Three replicates were performed for each condition. Each reaction had a total volume of 10 μL, and a standardized qPCR protocol was used. The experiment began with a polymerase activation step at 95°C for 3 minutes, followed by 66 amplification cycles, each consisting of a DNA denaturation step at 95°C for 10 seconds and an annealing / extension step at 60°C for 30 seconds.

[0079] Experimental results showed that an input of 20 ng demonstrated optimal performance in low-frequency mutation detection, significantly enhancing signal intensity and effectively suppressing background noise. Although inputs of 30 ng and 50 ng demonstrated similar detection results to 20 ng, due to the larger sample size requirements, these inputs may not be suitable for samples where sufficient DNA cannot be obtained. Furthermore, an input of 10 ng exhibited poor reproducibility between replicates, resulting in unstable test results. Therefore, considering both sample size requirements and detection accuracy, this study determined that 20 ng was the most appropriate input, ensuring high sensitivity and accuracy while also meeting the sample size requirements in practical applications.

[0080] 5.3 Sample testing input optimization results To further verify the clinical applicability of dried blood spot samples, this study evaluated the detection effect of different blood input volumes (50 μL, 100 μL, and 200 μL). EGFR L858R and EGFR The two frequently occurring mutation sites, E746 and A750del, represent both point and deletion mutations. This study specifically selected two samples with low mutation frequencies, 1.11% and 1.17%, to test the sensitivity of the DBS-TARGET technique for these low-frequency mutations. The results showed that with a DBS input of 100 μL (2 x 50 μL, approximately two drops of fingerstick blood), the Ct values ​​of positive samples were significantly lower than those of negative samples, and a significant difference in ΔCt values ​​was observed, effectively distinguishing mutation-positive from negative samples. Although a positive result can also be obtained with an input of four dried blood spots (4 x 50 μL, approximately four drops of fingerstick blood), the 100 μL input offers a better balance between performance and cost-effectiveness. Compared to larger sample volumes, the 100 μL sample volume is not only simpler to use but also more practical for routine clinical collection and analysis. Therefore, based on these results, the 100 μL DBS sample input was used as the standard input for subsequent experiments and clinical trials.

[0081] These results highlight the potential of TARGET technology for DBS samples, demonstrating its ability to efficiently and sensitively detect low-frequency cfDNA mutations, particularly in limited sample sizes. This approach offers a practical and feasible solution for sensitive and noninvasive detection of low-frequency mutations, with strong clinical application prospects, particularly in settings with small sample sizes, low mutation frequencies, and resource constraints.

[0082] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for detecting low-frequency mutations in cfDNA, characterized in that: The following steps are involved: (1) cfDNA library construction; (2) TARGET detection: Quantitative PCR is performed on the cfDNA library obtained in step (1) using blocking probes and enrichment primers that detect gene mutation sites to obtain the Ct value; (3) Data analysis: Calculate the difference between wild-type DNA and sample Ct, ΔCt. When the ΔCt value exceeds the preset threshold, it is considered that a mutation exists in the sample; The gene mutation site is selected from one or more of EGFR L858R, EGFR T790M, EGFR E746_A750del, EGFR G719S, EGFR L747_T751del, KRAS G12D / 13D, BRAF V600E, and PIK3CA E545K; the blocking probe precisely matches the wild-type template sequence, and has a Tm of 60°C to 72°C; The nucleotide sequence of the enrichment primer pair for detecting the EGFR L858R site is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 12; The nucleotide sequences of the enrichment primer pair for detecting the EGFR T790M site are shown in SEQ ID NOs: 3-5, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 13; The nucleotide sequences of the enrichment primer pair for detecting the EGFR E746_A750del site are shown in SEQ ID NOs: 6-7, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 14; The nucleotide sequences of the enrichment primer pair for detecting the EGFR G719S site are shown in SEQ ID NOs: 8-9, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 15; The nucleotide sequences of the enrichment primer pair for detecting the EGFR L747_T751del site are shown in SEQ ID NOs: 10-11, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 16; The nucleotide sequence of the enrichment primer pair for detecting the KRAS G12D / 13D site is shown in SEQ ID NOs: 17-18, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 23; The nucleotide sequences of the enrichment primer pair for detecting the BRAF V600E site are shown in SEQ ID NOs: 19-20, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO: 24; The nucleotide sequences of the enrichment primer pair for detecting the PIK3CA E545K site are shown in SEQ ID NOs: 21-22, and the nucleotide sequence of the blocking probe is shown in SEQ ID NO:

25.

2. The method for detecting low-frequency mutations in cfDNA according to claim 1, characterized in that: The cfDNA is derived from plasma or dried blood spots.

3. The method for detecting low-frequency mutations in cfDNA according to claim 1, characterized in that: The construction of the cfDNA library includes the following steps: sample collection; extraction and quantification of cfDNA; end repair; adapter ligation; enrichment of the cfDNA library; and quality control of the cfDNA library.

4. The method for detecting low-frequency mutations in cfDNA according to claim 3, characterized in that: The sample collection volume is 50 μL-200 μL, and the input amount of the cfDNA library is 20 ng-30 ng.

5. The method for detecting low-frequency mutations in cfDNA according to claim 1, characterized in that: The preset threshold is determined by the Youden index calculated by the ROC curve.

6. The method for detecting low-frequency mutations in cfDNA according to claim 1, characterized in that: The 3' end of the blocking probe is modified with a phosphorylation group or Spacer C3.

7. Use of the method according to any one of claims 1 to 6 in detecting lung cancer.

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