Method for detecting EGFR (epidermal growth factor receptor) mutation through digital PCR (polymerase chain reaction) based on micro-fluidic chip
By optimizing the reaction system and fluorescence signal interpretation standards of the microfluidic chip platform, the accuracy and sensitivity issues of EGFR mutation detection in existing technologies have been resolved, achieving highly sensitive and standardized EGFR mutation detection, which is suitable for the precision diagnosis and treatment of non-small cell lung cancer.
Patent Information
- Application Number
- CN202511593664.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2026-01-16
AI Technical Summary
Existing digital PCR technology has a critical point for the maximum loading amount of the reaction system when detecting EGFR mutations. The wild-type template background and the mutant template competitively inhibit each other, resulting in a decrease in the signal-to-noise ratio and affecting the accuracy and sensitivity of the detection. The lack of unified standards for operation procedures and signal interpretation criteria affects the specificity and sensitivity of the detection. The lack of rigorous performance verification leads to unstable detection results.
Using a microfluidic chip platform, the sample loading volume of the reaction system was optimized to no more than 1×10⁵ copies/reaction. Specific primers and probes were designed, fluorescence signal interpretation criteria were set, and a scientific positive interpretation threshold (≥3 positive droplets) was established. EGFR mutations were detected through thermal cycling amplification and fluorescence signal acquisition.
It achieves highly sensitive EGFR mutation detection with a detection limit as low as 0.30%, exhibits excellent linearity in the mutation rate range of 0.01% to 50%, is accurate in quantification, and has good standardization and reproducibility, making it suitable for stable application in clinical laboratories.
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Figure CN121344167A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of digital PCR detection technology, and more specifically, to a method for detecting EGFR mutations using digital PCR based on a microfluidic chip. Background Technology
[0002] Precision treatment of non-small cell lung cancer (NSCLC) heavily relies on the accurate detection of epidermal growth factor receptor (EGFR) gene mutations. Exon 19 deletion (19del) and exon 21 L858R point mutations are the most important targets. Although tissue biopsy is the gold standard for genetic testing, its limitations, such as invasiveness, tumor heterogeneity, and difficulty in repeating sampling, have led to liquid biopsy becoming an important supplementary or even alternative solution. Genotyping by detecting circulating tumor DNA (ctDNA) in the patient's plasma offers the advantages of being non-invasive and allowing for dynamic monitoring.
[0003] Digital PCR (dPCR) technology is particularly suitable for detecting low-frequency mutations in ctDNA due to its ability to achieve absolute quantification without relying on a standard curve and its extremely high sensitivity. This technology achieves PCR amplification of "single-molecule templates" by dividing the reaction system into a large number of independent microreaction units, thereby enabling the precise identification of low-frequency mutation signals in a large background of wild-type sequences.
[0004] Microfluidic chip technology is key to achieving efficient and stable sample segmentation. Among them, microfluidic array chip-based digital PCR integrates tens of thousands of microchambers on a chip through precise microfabrication technology, enabling automated and uniform sample segmentation. Compared with manual operation or other droplet generation techniques, it has better repeatability and throughput.
[0005] However, the successful application of digital PCR technology in clinical ctDNA detection relies not only on highly sensitive primers and probes or advanced microarray platforms, but also on a complete set of fully optimized and validated standardized operating procedures. Currently, although the principles of digital PCR are well understood, a complete methodological system for specific detection targets (such as EGFR mutations) and specific platforms (such as microfluidic array chips) remains incomplete. This is specifically reflected in: First, for microfluidic chip platforms, there is a critical point for the maximum loading capacity of the reaction system. Exceeding this limit, excessively high wild-type template background will competitively inhibit the mutant template, leading to a sharp drop in the signal-to-noise ratio and even making it impossible to effectively distinguish between positive and negative signals, severely affecting the accuracy and sensitivity of detection. Therefore, determining the optimal nucleic acid loading capacity range for this platform is a prerequisite for ensuring detection performance.
[0006] Second, existing detection methods lack unified standards in terms of operational procedures and signal interpretation criteria. For example, how to set the threshold for a positive signal (how many positive micro-units are considered positive) directly affects the sensitivity and specificity of the detection. Overly lenient standards may lead to false positives, while overly strict standards will reduce sensitivity and cause false negatives.
[0007] Third, a reliable clinical testing method requires rigorous performance validation, including linear range, precision, accuracy, and limit of detection. These performance parameters are closely related to specific chip structures, hydrodynamic characteristics, and thermal cycling conditions, and must be specifically established and confirmed through extensive experiments.
[0008] Therefore, there is an urgent need in this field to establish a standardized and reproducible method for detecting EGFR mutations specifically for microfluidic chip-based digital PCR platforms. This method should clearly define key operational steps, optimized reaction parameters, and scientific interpretation criteria to ensure stable, sensitive, and accurate detection of low-frequency EGFR mutations from limited ctDNA samples in clinical applications, providing reliable technical support for the precision diagnosis and treatment of NSCLC. Summary of the Invention
[0009] In view of this, the present invention proposes a method for detecting EGFR mutations by digital PCR based on microfluidic chips, in order to solve the problems existing in the prior art.
[0010] To achieve the above objectives, this invention proposes a method for detecting EGFR mutations using digital PCR based on a microfluidic chip, comprising the following steps: a. Provide sample DNA: Extract genomic DNA or circulating cell-free DNA from the sample to be tested; b. Preparation of reaction system: Mix the sample DNA with digital PCR reaction premix, which contains specific primers and probes for detecting the EGFR gene exon 19 deletion mutation 19del and exon 21 L858R point mutation; c. Droplet preparation: The reaction system is added to a microfluidic array chip; d. PCR amplification: The microfluidic chip was subjected to thermal cycling amplification; e. Fluorescence signal acquisition: Detect the signal of each microreaction unit in four fluorescence channels, the four fluorescence channels corresponding to FAM, HEX, ROX and CY5 fluorescent groups respectively; f. Result Interpretation: Determine the mutation status based on the fluorescence signal; where, When the number of positive microreaction units in the HEX channel corresponding to the L858R mutant probe is ≥3 and the number of positive microreaction units in the ROX channel corresponding to the L858R wild-type probe is ≥10, it is judged as L858R mutation positive. A 19del mutation is identified as positive when the number of positive microreaction units in the FAM channel corresponding to the 19del mutant probe is ≥3 and the number of positive microreaction units in the CY5 channel corresponding to the 19del wild-type probe is ≥10.
[0011] Further, the primer pair sequences for detecting 19del described in step b are shown in SEQ ID NO: 3 and SEQ ID NO: 4; the primer pair sequences for detecting L858R are shown in SEQ ID NO: 1 and SEQ ID NO: 2.
[0012] Furthermore, the probe used to detect 19del in step b includes: The wild-type probe has the sequence shown in SEQ ID NO: 7, with the 5' end modified with a CY5 fluorescent group and the 3' end modified with a BHQ2 quenching group; At least one mutant probe, the sequence of which is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, the 5' end of which is modified with a FAM fluorescent group, and the 3' end of which is modified with a BHQ1 quencher group; The probe used to detect L858R includes: The wild-type probe has the sequence shown in SEQ ID NO: 5, with a ROX fluorescent group modified at the 5' end and a BHQ2 quencher group modified at the 3' end. The mutant probe, whose sequence is shown in SEQ ID NO: 6, has a HEX fluorescent group modified at the 5' end and an MGB quencher group modified at the 3' end.
[0013] Furthermore, the mutation probe for detecting 19del comprises all five sequences shown in SEQ ID NO: 8 to SEQ ID NO: 12.
[0014] Further, in step b, the amount of sample DNA loaded into the reaction system, calculated as wild-type genome equivalent, does not exceed 1 × 10⁻⁶. 5 Copy / reaction.
[0015] Further, in step d, the thermal cycling amplification procedure includes: pre-denaturation at 95°C for 10 minutes; followed by 45 cycles of denaturation at 95°C for 20 seconds and annealing / extension at 60°C for 40 seconds.
[0016] Furthermore, the sample to be tested in step a is a body fluid sample, preferably a plasma or serum sample.
[0017] The present invention also provides a kit for implementing the above method, the kit comprising: Primers and probes for detecting EGFR gene 19del and L858R mutations; Digital PCR reaction buffer and enzymes; Components or finished chips used to build microfluidic array chips.
[0018] The present invention also provides the application of the above-described method in the preparation of medical devices or companion diagnostic reagents for the diagnosis, prognostic assessment, or targeted drug use guidance of tyrosine kinase inhibitors for non-small cell lung cancer.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Extremely high detection sensitivity: By optimizing the sample loading amount and establishing scientific interpretation criteria (≥3 positive droplets), the method of this invention has a limit of detection (LoD) of 0.30% for EGFR 19del and L858R mutations (with 15 ng genomic DNA as background), which can effectively capture mutations with extremely low abundance in plasma cfDNA and is suitable for early drug resistance monitoring.
[0020] 2. Accurate and reliable quantification: Performance verification shows that the method exhibits excellent linearity (R²>0.99) over a wide mutation rate range of 0.01% to 50%, high quantitative accuracy, and good precision (e.g., the total imprecision at the 5% mutation rate level is 11.38%), achieving absolute quantification independent of the standard curve.
[0021] 3. Good standardization and reproducibility: The method clarifies the key parameters of the entire process from sample processing, system preparation, chip loading, amplification to result interpretation, especially the determination of the maximum loading amount and the positive interpretation threshold. This makes the method standardized and can obtain stable and consistent results between different operators and different batches, making it easy to promote in clinical laboratories.
[0022] 4. Strong clinical concordance: In comparison with the gold standard (tissue NGS), the method of this invention showed high concordance (overall concordance rate of 96%) for mutations with an abundance of ≥1%. Furthermore, this method can detect low-frequency mutations (<1%) that are difficult for NGS to detect, demonstrating its unique application value in liquid biopsy and providing a powerful tool for the accurate diagnosis and dynamic monitoring of NSCLC. Attached Figure Description
[0023] Figure 1The results are for digital PCR negative samples; where A is the signal graph of the FAM mutation channel negative sample test result; B is the signal graph of the HEX mutation channel negative sample test result; C is the signal graph of the ROX wild-type channel negative sample test result; and D is the signal graph of the CY5 wild-type channel negative sample test result. Figure 2 The results are for digital PCR positive samples; where A is the signal graph of the FAM mutation channel positive sample detection result; B is the signal graph of the HEX mutation channel positive sample detection result; C is the signal graph of the ROX wild-type channel positive sample detection result; and D is the signal graph of the CY5 wild-type channel positive sample detection result. Figure 3 The images above show the original results of digital PCR four-channel FAM, HEX, ROX, and CY5 detections; the original images are monochrome photographs, while the images above are manually colored and then resolved. Figure 4 The figures show the maximum loading results for the 19del system; where A and B in the figure represent concentration gradients of 10... 1 10 2 10 3 10 4 10 5 10 6 10 7 The results of the copy / reaction are shown in Figure A, which shows the positive signal results at different concentration gradients; Figure B shows the negative signal results at different concentration gradients; and Figure C shows the change in average signal-to-noise ratio as the wild template concentration increases. Figure 5 The results of linearity testing of the 19del & L858R system are shown in one-dimensional scatter plots and linearity analysis. Figures A, B, C, and D represent the number of signal points in the four channels at different concentration gradients. Figures A and B show the signal maps of the 19del and L858R mutant channels, respectively; Figures C and D show the signal maps of the 19del and L858R wild-type channels, respectively; Figures E and F show the linear relationship between the theoretical mutation rate of 19del & L858R and the actual mutation rate detected by digital PCR. Figure 6 The results show a comparison between ultraviolet spectrophotometry (OD260) and digital PCR detection; where A represents the ratio between DNA concentration dilution and OD value; B, C, and D are graphs showing the relationship between DNA concentration and copy number measured by digital PCR. Figure 7The diagrams show the results of digital PCR testing for patients positive for the L858R site. A, B, and C represent the results of digital PCR testing for the T790M and C797S sites. No positive spots were found in A and B, with a mutation rate of 0.00%, indicating negative results for both sites. D, E, and F represent the results of digital PCR testing for the 19del and L858R sites. Positive spots were observed in the HEX channel of the L858R mutation site, with a mutation rate of 17%, defined as positive. The 19del test result was negative. Detailed Implementation
[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0025] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.
[0026] Example 1: Design, synthesis and validation of specific primer and probe sets 1.1 Target selection and sequence design Based on clinical practice in non-small cell lung cancer (NSCLC), this invention selects two of the most common mutation sites in the EGFR gene with clear guidance for drug use: exon 19 deletion mutation (19del) and exon 21 L858R point mutation. By searching the COSMIC database, five common deletion subtypes of 19del (such as E746_A750del) and one point mutation of L858R were identified. Using professional software such as Primer Express and following primer and probe design principles, primers and probes with high specificity and amplification efficiency were designed. All sequences were synthesized by Shanghai Jierui Biotechnology Co., Ltd.
[0027] 1.2 Primer and probe sequences and modifications The primer and probe sequences and fluorescence modifications used in this invention are shown in Table 1 below. The probes are dual-labeled (fluorescent reporter group and quencher group), and multiple mutant probes were designed for different deletion forms of 19del to ensure comprehensive detection.
[0028] Table 1. Detailed list of primer and probe sequences for EGFR gene mutation detection according to the present invention.
[0029] 1.3 Construction and Quantitative Validation of Plasmid Templates To verify the effectiveness of the primers and probes, a pUC57 plasmid containing the 19del and L858R mutations and their corresponding wild-type sequences was constructed by a synthetic company. The synthesized plasmid was linearized with restriction endonucleases (HindIII), and absolute quantification was performed using digital PCR. The quantification results (Table 2) showed that the wild-type plasmid showed signal only in the wild-type channel, while the mutant plasmid showed signal in both the mutant and wild-type channels, and the quantification concentrations were as expected, proving that the plasmid template was successfully constructed and could be used for subsequent system validation.
[0030] Table 2. Results of plasmid quantification
[0031] 1.4 Preliminary Validation of the Detection System The constructed detection system was tested using human genomic DNA (negative control) and a synthetic mutant plasmid (positive control). A one-dimensional scatter plot of the digital PCR detection results is shown. Figure 1 The results showed that human genome samples exhibited no signal points in the FAM and HEX mutant channels, while displaying abundant and clear signals in the ROX and CY5 wild-type channels, indicating good system specificity and no false positives. The detection results of positive plasmid samples ( Figure 2 The data shows that all four channels exhibit clearly clustered positive signal points, indicating that the system can effectively detect the target mutation. The original signal image after chip reading is shown below. Figure 3 Further, it was shown that the signals of each channel were evenly distributed in approximately 21,000 microchambers without cross-interference, demonstrating the excellent compatibility of this primer-probe set on the microfluidic chip platform.
[0032] Example 2: Establishment and Performance Verification of a Microfluidic Chip-Based Digital PCR Detection Method 2.1 Sample Pretreatment and DNA Extraction Clinical samples were peripheral blood from NSCLC patients. After collection, the plasma was centrifuged at 1600g at room temperature for 10 minutes, and the supernatant plasma was collected. It was then centrifuged again at 1600g at 4℃ for 10 minutes to completely remove cellular debris, yielding clear plasma, which was stored at -80℃. cfDNA was extracted from 2 mL of plasma using a beaver cell-free DNA extraction kit (magnetic bead method), and finally eluted with 50 μL of elution buffer. Tissue DNA was extracted using Qiagen's QIAamp DNA Blood Kits (column extraction method).
[0033] 2.2 Digital PCR reaction system and amplification procedure The total reaction volume is 30 μL. The specific preparation method is shown in the table below. All steps were performed in a PCR clean bench to prevent contamination.
[0034] Table 3. Preparation of Digital PCR Reaction System
[0035] The "BioDigital·Qing" fully automated digital PCR system was used. The reaction solution was added to a dedicated 8-tube container and placed together with an oil tank containing oil phases A and B in a microdroplet preparation instrument. The instrument automatically divided the reaction system into microfluidic array chips. The chips were then transferred to a PCR amplification instrument, and the following program was run: 50℃ for 10 min; 95℃ for 10 min; 45 cycles (95℃ for 20 s, 60℃ for 40 s); stored at 25℃.
[0036] 2.3 Fluorescence Detection and Result Interpretation Standards Immediately after amplification, fluorescence signals from four channels (FAM, HEX, ROX, and CY5) were acquired using a chip reader. The positive interpretation criteria established in this invention are as follows: For mutant channels (FAM, HEX): a channel is considered positive when the number of positive droplets is ≥ 3. This criterion balances sensitivity and specificity.
[0037] For wild-type internal control channels (ROX, CY5): when the number of positive droplets in a channel is ≥ 10, the channel is considered positive, indicating that the sample quality is qualified and the amplification is successful.
[0038] Final result determination: A positive result for a mutant channel at a given site is only considered valid if the wild-type channel at that site is positive. For example, if both the ROX and HEX channels are positive, then the result is considered a positive L858R mutation.
[0039] 2.4 Determination of Key Performance Parameters of the Method 2.4.1 Screening for Maximum Sample Loading Amount To determine the maximum sample size at which the chip does not produce signal suppression, approximately 3000 copies of the mutant template were fixed, and the amount of wild-type template was gradually increased (10). 1 Up to 10 7 (Copies / Reaction). Results are as follows: Figure 4 As shown, when the wild-type background reaches 10 5 During the copy / reaction phase, the signal-to-noise ratio begins to decrease significantly; reaching 10. 6 When the reaction occurs at a ratio of <3, the signal-to-noise ratio cannot distinguish between positive and negative results. Therefore, this invention determines the maximum recommended loading amount to be 10. 5 Copies / reaction (approximately 330 ng genomic DNA).
[0040] 2.4.2 Linear Range and Quantitative Accuracy The reference sample with a 50% mutation rate (10 4 copies / μL) and wild-type genomic DNA (10 copies / μL)4 Mix the samples (copies / μL) according to the proportions shown in Table 1 to prepare gradient samples with mutation rates ranging from 50% to 0.01%. Perform multiple digital PCR tests on each gradient sample.
[0041] Table 4. Linear evaluation sample gradient ratio
[0042] The results are as follows Figure 5 As shown, at different mutation rates, the number of positive signal points in the mutant channels (FAM, HEX) decreased systematically with decreasing mutation concentration, while the signal of the wild-type channels (CY5, ROX) remained stable. Linear regression analysis was performed on the theoretical mutation rate and the measured mutation rate by digital PCR, and the results are as follows: Figure 5 As shown in Figure EF, the linear correlation coefficient R between the two detection systems, 19del and L858R, is... 2 All are greater than 0.99 (y = 1.034665x, R). 2 = 0.999904; y = 1.002580x, R 2 = 0.999949). This indicates that the system of the present invention has a good linear relationship over a wide concentration range of 0.01% to 50%, and the quantitative results are accurate and reliable.
[0043] 2.4.3 Precision Assessment Experimental methods: Precision reference samples with mutation rates of 50%, 5%, and 0.5% were prepared. For the 5% mutation rate sample, intra-batch precision (24 repeated tests within the same batch) and inter-batch precision (72 tests across different devices and batches) were evaluated. Overall precision was evaluated for the three concentrations (two batches per day, twice per batch, for 10 consecutive days).
[0044] Table 5. Precision test results for samples with a 5% mutation rate
[0045] Table 6. Overall Precision Test Results
[0046] Experimental results: Table 5 shows the precision test results for samples with a 5% mutation rate. The specific results are as follows: the intra-assay imprecision of the 19del system was 9.35%, the inter-assay imprecision was 3.88%, and the total imprecision was 11.85%; the intra-assay imprecision of the L858R system was 10.09%, the inter-assay imprecision was 5.75%, and the total imprecision was 10.91%; the average intra-assay imprecision of the two systems was 9.72%, the inter-assay imprecision was 4.81%, and the total imprecision was 11.38%.
[0047] Table 6 shows the total imprecision test results for samples with mutation rates of 50%, 5%, and 0.5%. The total imprecision test results for the 19del mutation detection system are 4.66%, 11.85%, and 31.08%, respectively; the total imprecision test results for the L858R mutation detection system are 4.25%, 10.91%, and 33.49%, respectively.
[0048] The results are consistent with the characteristics of digital PCR technology, which is that imprecision increases as the mutation concentration decreases. However, within the clinically acceptable range of low-frequency mutation detection (e.g., above 0.5%), this system demonstrated good reproducibility.
[0049] 2.4.4 Accuracy Experimental methods: Human genomic DNA was extracted and serially diluted. The concentration was determined using a UV spectrophotometer (OD260) and the digital PCR detection system of this invention, respectively, and the consistency of the results of the two methods was compared.
[0050] Experimental results: such as Figure 5 As shown, there is an excellent linear correlation between the copy number measured by digital PCR and the DNA concentration measured by OD260 (R0). 2 >0.99). Furthermore, digital PCR quantification showed that 1 μL of human genomic DNA at a concentration of 1 ng / μL is approximately equal to 308.53 copies, with a relative deviation of only 1.85% from the theoretical value (1 ng / μL ≈ 302.94 copies / μL), fully demonstrating the accuracy of the absolute quantification of the system of this invention.
[0051] 2.4.5 Limit of Detection (LoD) Experimental methods: Under a background of 15 ng human genomic DNA (approximately 4500 copies / reaction), a series of samples with different mutation copy numbers were prepared by diluting 0.5% mutation rate reference. Each concentration was tested 21 times, and the detection rate under different positive judgment criteria (1, 2 or 3 positive signal points) was statistically analyzed.
[0052] Experimental results: See Tables 7 and 8 for detailed data.
[0053] Table 7. Detection Limit Test of 19del System
[0054] Table 8. Detection Limit Test of L858R System
[0055] Comprehensive analysis shows that when ≥3 positive signal points are used as the interpretation criterion, the limit of detection (LoD) of this system for 19del and L858R mutations is as low as 0.30% (approximately 16 copies / reaction), with a detection rate of ≥95%. If ≥1 positive signal point is used as the interpretation criterion, the sensitivity can be further improved to 0.10%, but to reduce the risk of false positives, it is recommended to use ≥3 positive signal points as the interpretation criterion in clinical applications. This sensitivity fully meets the requirements for detecting extremely low-frequency mutations in plasma cfDNA.
[0056] Example 3: Clinical Sample Testing Applications and Consistency Analysis 3.1 Clinical Sample Testing With ethical approval and informed consent from the patients, paired tissue (NGS) and plasma samples were collected from 25 NSCLC patients. Using the NGS results of the tissue samples as the gold standard, EGFR mutations were detected in plasma cfDNA using the primer-probe set and digital PCR system of this invention, and the consistency of the two methods was compared.
[0057] Experimental results: (1) Figure 6 This diagram illustrates the NGS testing results for a patient at the 19del & L858R and T790M & C797S loci. The sample showed numerous positive signal points in the L858R detection channel (HEX), with a mutation rate as high as 17%, and was therefore identified as L858R mutation positive; however, it was negative at the 19del, T790M, and C797S loci. This result is consistent with the NGS testing results.
[0058] (2) Table 9 shows that among the 25 patients with non-small cell lung cancer, digital PCR detected EGFR gene mutations in 7 patients, including 1 patient with double mutations, namely simultaneous mutations of T790M and L858R; NGS detected EGFR gene mutations in 4 patients, with no double mutations detected. All EGFR gene mutations detected by NGS could be detected by digital PCR. The Kappa value for EGFR gene mutations detected by digital PCR and NGS was 0.106, the positive concordance rate was 42.86%, the negative concordance rate was 85.71%, and the overall concordance rate was 84.00%.
[0059] Table 9. Results of EGFR gene mutation detection by digital PCR and NGS
[0060] (3) Table 10 shows the results of digital PCR detection of EGFR gene mutations. Mutation sites with a mutation abundance of ≥1% accounted for 37.50% (3 / 8) of the total mutation sites, and mutation sites with a mutation abundance of <1% accounted for 62.50% (5 / 8) of the total mutation sites.
[0061] Table 10. Results of EGFR gene mutation abundance detection by digital PCR
[0062] (4) Table 11 is a schematic diagram of the results of NGS detection of EGFR gene mutations. The mutation abundance of all (4 / 4) mutation sites is ≥1%.
[0063] Table 11. Results of EGFR gene mutation abundance detected by NGS
[0064] (5) For EGFR gene mutations with a mutation abundance of ≥1%, the Kappa value of digital PCR and NGS detection results was 0.834, the positive concordance rate was 75.00%, the negative concordance rate was 95.45%, and the total concordance rate was 96.00%; for EGFR gene mutations with a mutation abundance of <1%, the Kappa value of digital PCR and NGS detection results was 0.000, the positive concordance rate was 0.00%, the negative concordance rate was 80.00%, and the total concordance rate was 80.00%.
[0065] Conclusion: This invention fully discloses an innovative technical solution encompassing core reagents (primer and probe sets) and a complete methodology (digital PCR detection process based on microfluidic chips, key parameters, and interpretation criteria). Through systematic and detailed experimental data, the method has been fully validated for its superior performance, including a wide linear range, high precision, high accuracy, and excellent sensitivity (LoD of 0.30%). Clinical trials demonstrate that it effectively complements and even surpasses the capabilities of NGS in detecting low-frequency mutations, providing a powerful tool for the precision diagnosis and treatment of NSCLC.
[0066] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for detecting EGFR mutation based on microfluidic chip digital PCR, characterized in that, The method comprises the following steps: a. providing sample DNA: extracting genomic DNA or circulating free DNA from a sample to be tested; b. preparing a reaction system: mixing the sample DNA with a digital PCR reaction premix, which comprises specific primers and probes for detecting the 19th exon deletion mutation 19del and the 21st exon L858R point mutation of the EGFR gene; c. droplet preparation: adding the reaction system to a microfluidic array chip; d. PCR amplification: performing thermal cycling amplification on the microfluidic chip; e. fluorescence signal acquisition: detecting the signal of each microreaction unit under four fluorescence channels, which correspond to FAM, HEX, ROX and CY5 fluorescence groups respectively; f. result interpretation: determining the mutation status according to the fluorescence signal; wherein, when the number of positive microreaction units in the HEX channel corresponding to the L858R mutant probe is ≥ 3, and the number of positive microreaction units in the ROX channel corresponding to the L858R wild-type probe is ≥ 10, it is determined that the L858R mutation is positive; when the number of positive microreaction units in the FAM channel corresponding to the 19del mutant probe is ≥ 3, and the number of positive microreaction units in the CY5 channel corresponding to the 19del wild-type probe is ≥ 10, it is determined that the 19del mutation is positive.
2. The method of claim 1, wherein, The sequence of the primer pair for detecting 19del in step b is shown in SEQ ID NO: 3 and SEQ ID NO: 4; the sequence of the primer pair for detecting L858R is shown in SEQ ID NO: 1 and SEQ ID NO:
2.
3. The method of claim 2, wherein, The probe for detecting 19del in step b comprises: a wild-type probe with a sequence shown in SEQ ID NO: 7, modified with a CY5 fluorescence group at the 5' end and a BHQ2 quenching group at the 3' end; at least one mutant probe with a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, all modified with a FAM fluorescence group at the 5' end and a BHQ1 quenching group at the 3' end; The probe for detecting L858R comprises: a wild-type probe with a sequence shown in SEQ ID NO: 5, modified with a ROX fluorescence group at the 5' end and a BHQ2 quenching group at the 3' end; a mutant probe with a sequence shown in SEQ ID NO: 6, modified with a HEX fluorescence group at the 5' end and a MGB quenching group at the 3' end.
4. The method of claim 3, wherein, The mutant probe for detecting 19del comprises all five sequences shown in SEQ ID NO: 8 to SEQ ID NO:
12.
5. The method of claim 1, wherein, In step b, the sample DNA loading amount in the reaction system is not more than 1 x 10 5 copies / reaction in terms of wild-type genome equivalents.
6. The method of claim 1, wherein, In step d, the program of the thermal cycling amplification comprises: pre-denaturation at 95℃ for 10 minutes; then 45 cycles of denaturation at 95℃ for 20 seconds, annealing / elongation at 60℃ for 40 seconds.
7. The method of claim 1, wherein, The sample to be tested in step a is a body fluid sample, preferably a plasma or serum sample.
8. A kit for carrying out the method of any one of claims 1 to 7, characterized in that The kit comprises: Primers and probes for detecting EGFR gene 19del and L858R mutations Digital PCR reaction buffer and enzymes Components for building microfluidic array chips or finished chips.
9. Use of the method according to any one of claims 1 to 7 for the manufacture of a medical device or companion diagnostic agent for the diagnosis, prognosis assessment or guidance of targeted therapy with tyrosine kinase inhibitors of non-small cell lung cancer.