A reagent, kit, method for detecting EGFR gene mutation and application thereof

Abstract

This invention discloses a reagent, kit, method, and application for detecting EGFR gene mutations. The reagent includes a primer set comprising a first primer pair for detecting the EGFR gene mutation site 19del, a second primer pair for detecting the EGFR gene mutation site T790M, and / or a third primer pair for detecting the EGFR gene mutation site L858R. The primer set and probes in this invention for detecting EGFR gene mutations exhibit high sensitivity and specificity, and show no cross-reactivity with wild-type samples, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a reagent, kit, method, and application for detecting EGFR gene mutations. Background Technology

[0002] EGFR mutation ctDNA detection, as a core technology in liquid biopsy for lung cancer, has become a key basis for clinical targeted therapy selection, drug resistance monitoring, and recurrence early warning due to its advantages such as being non-invasive and allowing for dynamic monitoring. Its core process includes plasma sample pretreatment, EGFR mutation ctDNA enrichment, amplification, and quantitative analysis, while the stability of the probe immobilization on the chip surface is one of the core factors affecting target capture efficiency.

[0003] In clinical scenarios involving low-abundance EGFR mutation ctDNA detection (such as early-stage lung cancer and postoperative residual lesions, with mutation abundance ≤0.05%), existing technologies have significant limitations: (1) Insufficient stability of chip probe fixation: Traditional chip probes mostly adopt direct covalent bonding, which has weak bonding force. During the detection process, the probe detachment rate is >10%, resulting in low capture efficiency of low abundance targets (0.05% mutation abundance detection rate <55%). (2) Low abundance target enrichment efficiency and severe sample loss: Traditional detection methods require capturing EGFR mutant ctDNA with magnetic beads or chips, eluting it, and then transferring it to tubes for amplification. During the elution process, the loss rate of low abundance targets reaches 35%-55%; (3) High risk of non-specific interference and cross-contamination: Plasma samples have complex components, existing cleaning protocols are not targeted enough, and the residual rate of non-specific conjugates is >18%; batch plate cleaning is prone to cross-contamination of samples and has a high false positive rate. (4) The process is complex and has poor compatibility: the traditional method requires multiple steps such as “sample processing → capture → elution → pre-amplification → qPCR”, and the whole process takes more than 3.5 hours; the reagent compatibility of different steps is poor, which increases the experimental cost and operation difficulty. Summary of the Invention

[0004] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a reagent for detecting EGFR gene mutations.

[0005] The present invention also provides a kit for detecting EGFR gene mutations.

[0006] This invention also provides a method for detecting EGFR gene mutations.

[0007] The present invention also provides applications of the above-described reagents, kits, or methods.

[0008] According to a first aspect of the present invention, a reagent for detecting EGFR gene mutations includes a primer set comprising a first primer pair for detecting EGFR gene mutation site 19del, a second primer pair for detecting EGFR gene mutation site T790M, and / or a third primer pair for detecting EGFR gene mutation site L858R. The primer sequences of the first primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2; The primer sequences of the second primer pair are shown in SEQ ID NO:5 and SEQ ID NO:6; The primer sequences of the third primer pair are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0009] According to some embodiments of the present invention, the reagent further includes a nanoneedle chip on which probes for detecting EGFR gene mutation sites are immobilized; The probes include a first probe for detecting the EGFR gene mutation site 19del, a second probe for detecting the EGFR gene mutation site T790M, and / or a third probe for detecting the EGFR gene mutation site L858R. The sequence of the first probe is shown in SEQ ID NO:3; The sequence of the second probe is shown in SEQ ID NO:7; The sequence of the third probe is shown in SEQ ID NO:11.

[0010] According to some embodiments of the present invention, the probe is immobilized on the nanoneedle chip via biotin-streptavidin binding.

[0011] According to some embodiments of the present invention, the Tm value of the probe is 65-75°C.

[0012] According to some embodiments of the present invention, the GC content of the probe is 40%-60%.

[0013] According to some embodiments of the present invention, the nanoneedle chip is a biotin-modified nanoneedle chip.

[0014] According to some embodiments of the present invention, the nanoneedle chip is prepared by the following method: A silicon-based chip with amino-modified surface was reacted with NHS-biotin to obtain a chip in which amino groups on the surface of the silicon-based chip are covalently bound to NHS-biotin; the chip was reacted with streptavidin to construct a biotin-modified silicon-based chip; the biotin-modified silicon-based chip was reacted with a probe for detecting EGFR gene mutation sites to prepare a nanoneedle chip.

[0015] According to some embodiments of the present invention, the concentration of the NHS-biotin in the reaction system is 0.05-0.5 mg / mL. Optionally, the concentration is 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 mg / mL, or any two of these values, as a range between the endpoints.

[0016] According to some embodiments of the present invention, the reaction time of the surface-modified amino silicon-based chip with NHS-biotin is 30-90 min. Optionally, the time is 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 min or any two of these values ​​are used as a range between endpoint values.

[0017] According to some embodiments of the present invention, before reacting the chip with streptavidin, the step of cleaning the chip is further included, wherein the cleaning is performed using PBS buffer and the chip is cleaned 2-4 times.

[0018] According to some embodiments of the present invention, the concentration of the streptavidin is 0.25-0.3 mg / mL.

[0019] According to some embodiments of the present invention, the reaction time of the chip with streptavidin is 1.5-2.5 hours.

[0020] According to some embodiments of the present invention, the step of cleaning the biotin-modified silicon-based chip is further included, wherein the cleaning is performed using PBS buffer and is performed 2-4 times.

[0021] According to some embodiments of the present invention, the concentration of the probe for detecting EGFR gene mutation sites in the reaction system is 10-20 μM.

[0022] According to some embodiments of the present invention, the reaction time between the biotin-modified silicon-based chip and the probe used to detect EGFR gene mutation sites is 0.8-1.2 h.

[0023] According to some embodiments of the present invention, the method further includes a step of cleaning the nanoneedle chip, wherein the cleaning is performed using PBS buffer and is repeated 2-4 times.

[0024] According to some embodiments of the present invention, the reagents further include proteinase K, washing solution, hybridization buffer, nanoneedle chip in situ pre-amplification reagent, and real-time PCR reaction reagent.

[0025] According to some embodiments of the present invention, the cleaning solution comprises 1×SSC solution and Triton 20 at a concentration of 0.05%-0.12%.

[0026] According to some embodiments of the present invention, the cleaning solution is used for cleaning the chip after in-situ hybridization capture. The present invention proposes a dedicated formulation of "1× SSC + 0.1% Triton 20" and a matching independent EP tube gradient oscillation cleaning process (rather than batch plate cleaning), reducing the non-specific binding rate from the conventional >18% to 2.1%, while achieving a cross-contamination rate of 0%. This combination of cleaning strategy and formulation is designed for the specific needs of cleaning before in-situ chip amplification.

[0027] According to some embodiments of the present invention, the hybridization buffer comprises 2×SSC buffer.

[0028] According to some embodiments of the present invention, the nanoneedle chip in-situ pre-amplification reagent includes 2× PCR MasterMix.

[0029] According to some embodiments of the present invention, the real-time PCR reaction reagent includes 2× SYBR Green qPCRMasterMix.

[0030] A kit according to a second aspect of the present invention, the kit comprising the reagents described in the first aspect embodiment.

[0031] According to some embodiments of the present invention, the kit further includes a positive control and a negative control.

[0032] According to some embodiments of the present invention, the negative control comprises water.

[0033] According to some embodiments of the present invention, the positive control includes nucleic acid molecules containing EGFR 19del mutation, EGFR L858R mutation and / or EGFR T790M mutation; or a vector containing the nucleic acid molecule.

[0034] According to some embodiments of the present invention, the nucleic acid molecule includes EGFR 19del mutation, L858R mutation and / or T790M mutation cDNA.

[0035] According to a third aspect of the present invention, a method for detecting EGFR gene mutations includes the following steps: detecting the sample to be tested using the above-described reagents or kits.

[0036] According to some embodiments of the present invention, the types of samples to be tested include peripheral blood cells, peripheral blood plasma / serum, body fluids, exfoliated material from cavities, and lesion tissue.

[0037] According to some embodiments of the present invention, the method further includes a pretreatment step for the sample to be tested, the steps being as follows: performing solid-liquid separation on the sample to be tested and collecting the liquid phase; digesting the liquid phase with proteinase K and then performing high-temperature unwinding treatment.

[0038] According to some embodiments of the present invention, the solid-liquid separation method includes centrifugation, wherein the centrifugation conditions are: centrifugation at 10000-14000 rpm for 8-12 min.

[0039] According to some embodiments of the present invention, the final concentration of the proteinase K is 1-3 mg / mL. Optionally, the concentration is 1, 2, or 3 mg / mL.

[0040] According to some embodiments of the present invention, the conditions for proteinase K digestion are 60-70°C and 25-35 min.

[0041] According to some embodiments of the present invention, the conditions for proteinase K digestion are 62-68°C and 28-32 min.

[0042] According to some embodiments of the present invention, the digestion of proteinase K further includes a centrifugation step, wherein the centrifugation conditions are: centrifugation at 10000-14000 rpm for 8-12 min.

[0043] According to some embodiments of the present invention, the high-temperature unwinding conditions are 93-97°C for 4-6 minutes, followed by treatment at 0-4°C for 1-3 minutes.

[0044] According to some embodiments of the present invention, the method further includes a step of in situ hybridization capture of the sample obtained after high-temperature unwinding treatment using a nanoneedle chip, wherein the in situ hybridization capture conditions are: treatment at 130-170 rpm for 55-70 min. The present invention achieves stable capture in a complex body fluid environment through chip surface probes: traditional covalently immobilized probes are easily detached (>10%) during hybridization, washing, and amplification thermal cycling. This solution, through a precisely optimized three-step modification process of "NHS-Biotin → Streptavidin → Biotinylated probe" (including specific dilution factors, incubation time, and number of washing cycles), controls the probe detachment rate to ≤1.2%, thereby ensuring the capture efficiency of low-abundance targets at the source.

[0045] According to some embodiments of the present invention, the method further includes a step of cleaning the nanoneedle chip obtained by in situ hybridization capture, wherein the cleaning is performed 1-3 times with a cleaning solution comprising 1×SSC solution and 0.05%-0.12% Triton 20.

[0046] According to some embodiments of the present invention, the method further includes the step of adding the nanoneedle chip obtained by in situ hybridization capture to the in situ pre-amplification reaction system for in situ pre-amplification.

[0047] According to some embodiments of the present invention, the in situ pre-amplification reaction system includes: 0.3-0.7 μM upstream primer; 0.3-0.7 μM downstream primer; 1×PCR MasterMix.

[0048] According to some embodiments of the present invention, the amplification program for the in situ pre-amplification is as follows: pre-denaturation at 94-96℃ for 2-4 min; denaturation at 94-96℃ for 10-30 s, annealing at 50-60℃ for 10-20 s, extension at 65-75℃ for 15-25 s, 15-25 cycles; final extension at 70-74℃ for 4-6 min.

[0049] According to some embodiments of the present invention, the detection includes detection using quantitative real-time PCR, wherein the reaction system of the quantitative real-time PCR reaction includes: 0.3-0.7 μM upstream primer, 0.3-0.7 μM downstream primer, 1-3 μL of pre-amplified product, and 1×SYBR Green qPCR MasterMix.

[0050] According to some embodiments of the present invention, the amplification program of the real-time PCR reaction is as follows: pre-denaturation at 94-96℃ for 2-4 min; denaturation at 94-96℃ for 8-12 s, annealing at 58-62℃ for 20-40 s, 35-45 cycles, and collection of fluorescence data; melting curve analysis at 60℃-95℃, with a heating rate of 0.1℃ / s.

[0051] According to some embodiments of the present invention, the method further includes a step of calculating the mutation abundance of EGFR mutant ctDNA in the sample using a standard curve constructed by comparing the experimental group Ct value with a gradient dilution of the mutant plasmid.

[0052] The application of the above-described chip, kit, or method according to the fourth aspect of the present invention is in the preparation of reagents for detecting EGFR gene mutations.

[0053] According to some embodiments of the present invention, the EGFR gene mutation includes the EGFR gene mutation site 19del mutation, the EGFR gene mutation site L858R mutation, and / or the EGFR gene mutation site T790M mutation.

[0054] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The primer set and probe in the reagent for detecting EGFR gene mutations in this invention have high sensitivity and specificity, and no cross-reactivity with wild-type samples, showing great application potential.

[0055] This breakthrough solution combines highly stable immobilization with in situ amplification, not only avoiding elution loss but also revealing that the in situ pre-amplified product has extremely high specificity due to its extremely low probe shedding rate. Direct use in qPCR yields stable and reproducible results. Furthermore, through workflow integration and parameter optimization, the method reduces the overall process time from >3.5 hours in traditional methods to approximately 2 hours, while significantly improving detection sensitivity. This invention also solves the problem of capturing and preventing loss of low-abundance ctDNA. ctDNA abundance in plasma is extremely low (as low as 0.05%), and the traditional "capture-elution-transfer" process inevitably leads to sample loss (>35%). This innovative solution proposes an integrated design of "in situ hybridization capture + chip-based in situ pre-amplification," eliminating the elution step and reducing the target loss rate to <3%.

[0056] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0057] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a graph showing the detection results of the relationship between Ct value and biotin concentration in an embodiment of the present invention; Figure 2 This is a graph showing the detection results of the relationship between Ct value and incubation time in an embodiment of the present invention; Figure 3 This is a graph showing the detection results of the relationship between Ct value and enzyme digestion time in an embodiment of the present invention; Figure 4 This is a graph showing the detection results of the relationship between Ct value and unwinding time in an embodiment of the present invention. Detailed Implementation

[0058] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0059] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0060] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0061] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0062] Example 1 This example provides a method for detecting EGFR gene mutations based on nanoneedle chips (taking EGFR 19del mutation detection as an example). This embodiment uses the detection of EGFR 19del mutation as an example to describe the detection method of the present invention in detail. The detection methods for EGFR L858R and T790M mutations are exactly the same; only the specific probes and specific primers used in the following steps need to be replaced with sequences designed for L858R or T790M. The primers and probes used in this embodiment were designed through extensive screening, and the primer and probe sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0063] The method includes the following steps: 1. Fabrication of nanoneedle chips The specific method for fabricating nanoneedle chips is as follows: (1) Reagent preparation NHS-Biotin working solution: Dilute the stock solution 1000 times with PBS to a final concentration of 0.1-0.2 mg / mL. Prepare fresh before use (store protected from light).

[0064] Streptavidin working solution: Dilute the stock solution 400 times with PBS to a final concentration of 0.25-0.3 mg / mL, and store at 4℃ for ≤1 week.

[0065] EGFR19del mutation-specific probe working solution: Dilute the stock solution 100 times with PBS to a final concentration of 10-20 μM, and aliquot and store at -20℃.

[0066] Proteinase K working solution: ready-to-use liquid proteinase K (20 mg / mL), no dilution required, for direct use in plasma sample processing.

[0067] Hybridization buffer: 2×SSC buffer (pH 7.0) for hybridization reaction of EGFR mutant ctDNA with probe.

[0068] Cleaning solution: 1×SSC + 0.1% Triton 20 mixture (prepared with DEPC water) to remove non-specific conjugates; final rinse with enzyme-free water.

[0069] (2) Fabrication of biotin-modified silicon-based chips 1) NHS-Biotin modification: Take a silicon-based chip with amino-modified surface (commercially available) and place it in a light-protected reaction dish. Add NHS-Biotin working solution with a concentration of 0.1 mg / mL (the liquid level should cover the chip, about 80 μL). React at room temperature in the dark for 1 hour to allow the amino groups on the chip surface to covalently bind with NHS-Biotin.

[0070] 2) Cleaning: Discard the NHS-Biotin working solution and wash the chip three times with PBS buffer (pH 7.4) for 5 minutes each time (150 μL per wash) to remove unbound NHS-Biotin.

[0071] 3) Streptavidin binding: Add 0.25 mg / mL of Streptavidin working solution (the liquid level should cover the chip, about 80 μL) to the container containing the chip, and react at room temperature for 2 h to construct the biotin-streptavidin intermediate layer.

[0072] 4) Cleaning: Discard the Streptavidin working solution and wash the chip three times with PBS buffer for 5 minutes each time (150 μL each time) to remove unbound Streptavidin and obtain the biotin-modified silicon-based chip.

[0073] (3) Immobilization of EGFR mutation-specific probes: Add biotin-labeled EGFR mutation-specific probe working solution (the liquid level covers the chip, about 80 μL), incubate at room temperature in the dark for 1 hour, and stably fix the probe (sequence shown in SEQ ID NO:3) on the chip surface using the biotin-streptavidin system to prepare the nanoneedle chip.

[0074] (4) Chip storage: Discard the probe working solution and wash the chip three times with sterile PBS buffer for 5 minutes each time. After treatment, immerse the chip in PBS buffer and store at 4°C in the dark. It is effective for batch sample detection.

[0075] 2. Sample pretreatment Sample preparation: Take 5 mL of clinical plasma / serum sample, thaw at 4℃, centrifuge at 12000 rpm for 10 min to remove the precipitate (blood cell fragments), and take the sample supernatant for later use.

[0076] Proteinase K digestion: Take 200 μL of supernatant, add 20 μL of proteinase K working solution (20 mg / mL), incubate at 65 °C for 30 min to degrade the protein and release EGFR mutant ctDNA; then centrifuge at 12000 rpm for 10 min, and take the supernatant as the test sample for later use.

[0077] 3. High-temperature unwinding Add 90 μL of the above test sample (experimental group), 90 μL of EGFR 19del mutant cDNA working solution (positive control), and 90 μL of enzyme-free water (negative control) to PCR tubes respectively; incubate at 95℃ for 5 min (to unwind the ctDNA double strand into a single strand), and immediately incubate on ice for 2 min (to maintain the single-stranded state) to obtain the unwound samples of each group.

[0078] 4. In-situ hybridization capture on a chip: Take 80 μL of each unwound sample and add it to the corresponding well of a 96-well plate. Place a biotin-modified nanoneedle chip with EGFR probe immobilized in the plate (probe side up, completely immersed in the sample solution). Incubate at 150 rpm for 60 min at room temperature to allow the nanoneedle chip probe to specifically hybridize with the EGFR mutant single-stranded ctDNA.

[0079] 5. Independent chip cleaning (including Triton 20): Prepare four 1.5 mL enzyme-free EP tubes for each nanoneedle chip: add 1 mL of 1× SSC + 0.1% Triton 20 mixture to each of the first three tubes, and add 1 mL of enzyme-free water to the fourth tube.

[0080] Use enzyme-free tweezers to remove the nanoneedle chip from the well plate, and sequentially place it into the first 3 tubes for 3 min of agitation and washing (to remove non-specific conjugates), then place it into the 4th tube for 1 min of rinsing (to remove cleaning solution residue); after removal, hang it vertically in the air to air dry for 1 min (keeping the probe area moist).

[0081] 6. In-situ pre-amplification of the chip: Place the nanoneedle chip into a 0.2 mL PCR tube, add 50 μL of in situ pre-amplification system (covering the chip probe area, the system is shown in Table 1), and run the amplification program: 95℃ pre-denaturation for 3 min → (95℃ denaturation for 15 s → 58℃ annealing for 15 s → 72℃ extension for 20 s) × 20 cycles → 72℃ final extension for 5 min → store at 4℃ to obtain the pre-amplified product.

[0082] Table 1

[0083] Table 2

[0084] 7. Collection and dilution of pre-amplified products: After centrifuging the PCR tube containing the pre-amplified product, aspirate all the liquid (approximately 50 μL) into a new EP tube; take 2 μL of the product, add 18 μL of enzyme-free water, dilute 10-fold, and keep on ice for later use as a qPCR template.

[0085] 8. qPCR quantitative detection: Prepare 20 μL / well qPCR systems on ice (qPCR systems are shown in Table 3, with 3 technical replicates per group), using primer sequences as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0086] Table 3

[0087] Add the qPCR system to a 96-well qPCR plate and run the following program: 95℃ pre-denaturation for 3 min → (95℃ denaturation for 10 s → 60℃ annealing for 30 s, fluorescence acquisition) × 40 cycles → melting curve analysis (60℃ → 95℃, heating rate 0.1℃ / s).

[0088] 9. Result Interpretation: Acceptable criteria: Positive control: stable Ct value (≤28), single peak in melting curve; negative control: no fluorescence signal.

[0089] Quantitative analysis: To calculate the mutation abundance of EGFR mutant ctDNA in the sample, a standard curve can be constructed as follows: The synthesized EGFR19del mutant template is serially diluted 10-fold (10... 5 ~10 1 (Copies / μL) were detected according to the qPCR system and procedure in step 8. A standard curve was plotted with the logarithm of the standard copy number on the x-axis and the Ct value on the y-axis. Substituting the Ct value of the experimental group into the standard curve equation, the initial copy number of EGFR mutant ctDNA in the sample can be calculated, and then converted into mutation abundance.

[0090] Example 2: Screening of probes for nanoneedle chips To ensure high specificity and sensitivity of the detection, a strategy of "online bioinformatics screening → offline experimental verification and optimization" was adopted to design and determine the EGFR mutation-specific probes to be used in the final test.

[0091] Step 1: Online Bioinformatics Screening 1. Target for screening: High specificity: Ensures that the probe matches only the target mutant sequence and does not mismatch with wild-type or other mutant sequences.

[0092] Suitable hybridization characteristics: appropriate melting temperature (Tm value), avoidance of stable secondary structures and primer dimers.

[0093] 2. Screening tools and criteria: Sequence acquisition and alignment: Wild-type and mutant reference sequences of EGFR genes (including exon 19 deletion, L858R, and T790M sites) were obtained from databases such as NCBI, and accurate alignment was performed to determine the mutation region.

[0094] Probe design principles: For point mutations (L858R, T790M): the 3' end base of the probe must be a mutant base, using the "end-matching rule" to maximize specificity.

[0095] For deletion mutations (19del): the probe sequence must cross the deletion breakpoint region.

[0096] Feature analysis: Using tools such as OligoAnalyzer, candidate probes that meet the following criteria are evaluated and screened: Tm value: Controlled within the range of 65-75℃ to adapt to the hybridization conditions of this scheme (annealing at 58℃).

[0097] GC content: Maintain at 40%-60% to ensure hybridization stability.

[0098] Avoid hairpin structures: ensure probe stability and reduce non-specific binding.

[0099] 3. Output results: Through online screening, 2-3 candidate probe sequences were initially identified for each target mutation site (19del, L858R, T790M) for subsequent experimental verification.

[0100] Step 2: Offline experimental verification and final determination of the probe: All candidate probes 19del-P1 (i.e., 19del-Cap Probe in Table 2 of Example 1), 19del-P2 (sequence 5′-Biotin-CAACATCTCCGAAAGCCAAC (SEQ ID NO:13)), L858R-P1 (i.e., L858R-Cap Probe in Table 2 of Example 1), L858R-P2 (sequence 5′-Biotin-CATGTCAAGATCACAGATTT (SEQ ID NO:14)), and T790M-P1 (i.e., T790M-Cap Probe in Table 2 of Example 1) were immobilized on nanoneedle chips according to the "Biotin-modified linking process" in Example 1, thus preparing nanoprobe chips containing different candidate probes. Performance was tested in a complete "in situ hybridization-in situ amplification-qPCR" detection system. Specific testing methods are as shown in Example 1.

[0101] Validation metrics and results: The tests were conducted using simulated plasma samples containing specific mutations (0.05%-1.0% abundance) and wild-type samples. Key validation data are summarized below: Table 4 Comparison of Experimental Performance of Candidate Probes

[0102] The results are shown in Table 4. As can be seen from the table, the system using the method of this invention can effectively detect EGFR 19del, L858R, and T790M mutations. Specificity results show that the ΔCt values ​​with wild-type samples are all greater than 8, indicating almost no cross-reactivity and extremely high specificity. Sensitivity results show the lowest Ct value for low-abundance mutations (e.g., 0.05%), the highest capture and detection efficiency, good amplification specificity, and single-peak melting curves with no non-specific amplification. Furthermore, probes 19del-P1, L858R-P1, and T790M-P1 show better performance. Therefore, 19del-P1, L858R-P1, and T790M-P1 will be selected for the subsequent fabrication of nanoneedle chips for further detection.

[0103] Example 3: Screening of Method Parameters This embodiment filters the method parameters from Embodiment 1. The specific steps are as follows: 1. Optimization of biotin modification parameters for the chip Sample: A simulated plasma sample containing a specific 19del mutation (0.05% abundance).

[0104] The method used to optimize the biotin modification parameters of the chip differs from that of Example 1 only in the NHS-Biotin modification steps (concentration and time). The specific parameter settings for different groups are shown in Table 5. Meanwhile, the EGFR mutation-specific probe used in the nanoneedle chip is 19del-Cap Probe.

[0105] Table 5

[0106] The results are shown in Table 5 and Figure 1-2 As shown, the lowest Ct value was obtained when the NHS-Biotin working solution concentration was 0.1 mg / mL and the reaction time was 1 h. Therefore, the NHS-Biotin working solution concentration of 0.1 mg / mL and the reaction time of 1 h were selected for subsequent experiments.

[0107] 2. Proteinase K time screening This embodiment aims to screen the proteinase K time to fully degrade plasma proteins (especially nucleases and DNA-binding proteins) to release and protect ctDNA, while avoiding excessive digestion that could damage DNA or introduce PCR inhibitors.

[0108] The method used to screen proteinase K incubation time differs from that in Example 1 only in that the proteinase K incubation time is 15, 30, and 45 minutes. In addition, the EGFR mutation-specific probe used in the nanoneedle chip is 19del-Cap Probe.

[0109] Table 6

[0110] The results are shown in Table 6 and Figure 3 As shown, adding 10% volume (i.e., final concentration ~1.8 mg / mL) of the 20 mg / mL stock solution directly to the sample and digesting at 65°C for 30 minutes resulted in the highest recovery efficiency (>95%) of known concentrations of mutant ctDNA from simulated plasma samples, with no inhibition of downstream PCR amplification efficiency. Extending the digestion time or increasing the concentration did not further improve the recovery rate; instead, it may have increased background.

[0111] 3. Screening of high-temperature unwinding conditions This embodiment ensures the effective utilization of extremely low starting amounts of ctDNA by screening the high-temperature unwinding time.

[0112] The method used to screen the high-temperature unwinding time differed from that in Example 1 only in that the high-temperature unwinding conditions were incubation at 95°C for 3, 5, and 10 minutes. The effect of high-temperature unwinding time on hybridization efficiency was compared. Meanwhile, the EGFR mutation-specific probe used in the nanoneedle chip was the 19del-Cap Probe.

[0113] Table 7

[0114] The results are shown in Table 7 and Figure 4 As shown, 95℃ for 5 minutes is sufficient to fully unwind low-concentration ctDNA double strands into single strands, and an immediate ice bath can effectively maintain the single-stranded state for hybridization. Too short a time results in incomplete unwinding, while too long a time may lead to DNA damage.

[0115] Example 4: Detection of low-abundance EGFR 19del mutant ctDNA This embodiment uses the method in Example 1 to detect low abundance of EGFR 19del mutant ctDNA in clinical plasma samples.

[0116] Samples: 3 groups of 5 mL clinical plasma samples, containing EGFR 19del (0.05%), (0.5%), and (1.0%) mutant ctDNA (sequences shown in SEQ ID NO:4).

[0117] Experimental group: The method in Example 1 was used to detect low abundance of EGFR 19del mutant ctDNA in clinical plasma samples. At the same time, the EGFR 19del mutant specific probe used in the nanoneedle chip was 19del-Cap Probe.

[0118] Control group: The only difference from the method in Example 1 is that the nanoneedle chip was not modified with biotin.

[0119] Detection indicators: Ct value, coefficient of variation, detection rate.

[0120] Table 8

[0121] The conclusions are shown in Table 8. As can be seen from the table, the probe stability is significantly improved by the biotin-modified linking process of this invention. The detection rate of EGFR 19del mutation with a low abundance of 0.05% reaches 98.2%, which is 45.9 percentage points higher than that of traditional chips, and the process time is reduced by nearly 50%.

[0122] Example 5: Detection of different EGFR mutation types (mutation type suitability) This embodiment uses the method in Example 1 to detect different EGFR mutation types in clinical plasma samples. The EGFR mutation-specific probes used in the nanoneedle chip are 19del-P1, L858R-P1, and T790M-P1 probes.

[0123] Samples: 3 groups of 5 mL clinical plasma samples, containing EGFR 19del (0.05%), L858R (0.5%), and T790M (1.0%) mutant ctDNA respectively (the sequence of EGFR 19del mutant ctDNA is shown in SEQ ID NO:4, the sequence of L858R mutant ctDNA is shown in SEQ ID NO:8, and the sequence of T790M mutant ctDNA is shown in SEQ ID NO:12).

[0124] Detection indicators: Ct value, coefficient of variation, detection rate.

[0125] Table 9

[0126] The results are shown in Table 9. As can be seen from the table, the present invention has excellent compatibility with common clinical EGFR mutation types (19del, L858R, T790M), with a detection rate of ≥97.0% and a coefficient of variation of ≤1.5%, which can cover the core monitoring targets of lung cancer targeted therapy.

[0127] Example 6: Detection of different body fluid samples (sample type suitability) This embodiment uses the method in Example 1 (the chip used is a nanoneedle chip containing only EGFR 19del probes) to detect EGFR 19del mutations in different body fluid samples.

[0128] Samples: 5 mL plasma and 5 mL serum samples from the same donor (both containing EGFR 19del mutant ctDNA, abundance 0.05%); Detection indicators: Ct value, detection rate, and operational compatibility.

[0129] Table 10

[0130] The results are shown in Table 10. As can be seen from the table, the detection effect of the method of the present invention on plasma and serum samples is not significantly different. The operation process is completely consistent, the sample adaptability is wide, there is no need to adjust the parameters for different body fluid samples, the compatibility is strong, and it can meet the detection needs of different clinical samples.

[0131] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A reagent for detecting EGFR gene mutations, characterized in that, The reagent includes a primer set, which includes a first primer pair for detecting the EGFR gene mutation site 19del, a second primer pair for detecting the EGFR gene mutation site T790M, and / or a third primer pair for detecting the EGFR gene mutation site L858R. The primer sequences of the first primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2; The primer sequences of the second primer pair are shown in SEQ ID NO:5 and SEQ ID NO:6; The primer sequences of the third primer pair are shown in SEQ ID NO:9 and SEQ ID NO:

10.

2. The reagent according to claim 1, characterized in that, The reagent also includes a nanoneedle chip, on which probes for detecting EGFR gene mutation sites are immobilized; The probes include a first probe for detecting the EGFR gene mutation site 19del, a second probe for detecting the EGFR gene mutation site T790M, and / or a third probe for detecting the EGFR gene mutation site L858R. The sequence of the first probe is shown in SEQ ID NO:3; The sequence of the second probe is shown in SEQ ID NO:7; The sequence of the third probe is shown in SEQ ID NO:

11.

3. The reagent according to claim 2, characterized in that, The probe is immobilized on the nanoneedle chip via biotin-streptavidin binding.

4. The reagent according to claim 2, characterized in that, The nanoneedle chip was prepared by the following method: reacting an amino-modified silicon chip with NHS-biotin to obtain a chip in which amino groups on the surface of the silicon chip are covalently bound to NHS-biotin; reacting the chip with streptavidin to construct a biotin-modified silicon chip; and reacting the biotin-modified silicon chip with a probe for detecting EGFR gene mutation sites to prepare the nanoneedle chip. Preferably, the concentration of NHS-biotin in the reaction system is 0.05-0.5 mg / mL; Preferably, the reaction time of the surface-modified amino silicon-based chip with NHS-biotin is 30-90 min; Preferably, the concentration of streptavidin is 0.25-0.3 mg / mL; Preferably, the reaction time between the chip and streptavidin is 1.5-2.5 hours; Preferably, the concentration of the probe used to detect EGFR gene mutation sites in the reaction system is 10-20 μM; Preferably, the reaction time between the biotin-modified silicon-based chip and the probe used to detect EGFR gene mutation sites is 0.8-1.2 h.

5. The reagent according to claim 1, characterized in that, The reagents also include proteinase K, washing solution, hybridization buffer, nanoneedle chip in situ pre-amplification reagent, and real-time PCR reaction reagent; Preferably, the cleaning solution comprises 1×SSC solution and Triton 20 at a concentration of 0.05%-0.12%.

6. A reagent kit, characterized in that, The kit comprises the reagent as described in any one of claims 1-5; Preferably, the kit further includes a positive control and a negative control.

7. A method for detecting EGFR gene mutations, characterized in that, The method includes the following steps: detecting the sample to be tested using the reagent as described in any one of claims 1-5 or the kit as described in claim 6; Preferably, the types of samples to be tested include peripheral blood cells, peripheral blood plasma / serum, body fluids, exfoliated material from cavities, and lesion tissue.

8. The method according to claim 7, characterized in that, The method further includes a pretreatment step for the sample to be tested, the steps of which are as follows: the sample to be tested is subjected to solid-liquid separation, and the liquid phase is collected; the liquid phase is digested with proteinase K and then subjected to high-temperature unwinding treatment; Preferably, the final concentration of the proteinase K is 1-3 mg / mL; Preferably, the digestion conditions for proteinase K are 60-70°C for 25-35 minutes; Preferably, the high-temperature unwinding conditions are 93-97℃ for 4-6 minutes, followed by treatment at 0-4℃ for 1-3 minutes. Preferably, the method further includes a step of in situ hybridization capture of the sample obtained after high-temperature unwinding treatment using a nanoneedle chip, wherein the in situ hybridization capture conditions are: treatment at 130-170 rpm for 55-70 min; More preferably, the method further includes the step of adding the nanoneedle chip obtained by in situ hybridization to the in situ pre-amplification reaction system for in situ pre-amplification; More preferably, the in situ pre-amplification reaction system includes: 0.3-0.7 μM upstream primer; 0.3-0.7 μM downstream primer; 1×PCR MasterMix. More preferably, the amplification program for the in situ pre-amplification is as follows: pre-denaturation at 94-96℃ for 2-4 min; denaturation at 94-96℃ for 10-30 s, annealing at 50-60℃ for 10-20 s, extension at 65-75℃ for 15-25 s, for 15-25 cycles; and final extension at 70-74℃ for 4-6 min.

9. The method according to claim 8, characterized in that, The detection includes using quantitative real-time PCR, and the reaction system of the quantitative real-time PCR reaction includes: 0.3-0.7 μM upstream primer, 0.3-0.7 μM downstream primer, 1-3 μL of pre-amplified product, and 1× SYBR Green qPCR MasterMix; Preferably, the amplification program for the real-time PCR reaction is as follows: pre-denaturation at 94-96℃ for 2-4 min; denaturation at 94-96℃ for 8-12 s, annealing at 58-62℃ for 20-40 s, 35-45 cycles, during which fluorescence data are collected; melting curve analysis at 60℃-95℃, with a heating rate of 0.1℃ / s.

10. The use of the reagent according to any one of claims 1-5, the kit according to claim 6, or the method according to any one of claims 7-9 in the preparation of reagents for detecting EGFR gene mutations; Preferably, the EGFR gene mutation includes the EGFR gene mutation site 19del mutation, the EGFR gene mutation site L858R mutation, and / or the EGFR gene mutation site T790M mutation.

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