A sequence combination, kit, and method for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR.

Abstract

This invention discloses a sequence combination, kit, and method for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR. The method includes amplification primers and crRNA. The amplification primers include a universal primer pair, an upstream primer for amplifying the T790M mutation, and a first and second downstream primer for amplifying the two C797S mutation subtypes. The crRNA includes a first universal crRNA targeting T790M, a second universal crRNA targeting C797S, and a first and second specific crRNA targeting the two C797S mutation subtypes. This invention establishes a rapid, highly sensitive, signal amplification-based cis-trans typing detection method by combining ARMS-PCR, T7 transcription, and Cas13a signaling. This method can directly serve precision medicine for non-small cell lung cancer patients after drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a sequence combination, kit and method for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR. Background Technology

[0002] In targeted therapy for non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) gene mutations are a core molecular marker guiding the use of tyrosine kinase inhibitors (TKIs). Approximately 50%–60% of patients develop the T790M gatekeeper mutation after receiving first- or second-generation EGFR-TKI treatment; however, after receiving third-generation osimertinib treatment, secondary C797S mutations become the main resistance mechanism. Crucially, T790M and C797S have two genetic configurations: cis and trans. When both mutations are located on the same allele, it is a cis mutation, in which case the patient is insensitive to all current generations of TKIs, and standard treatment is primarily chemotherapy. When the two mutations are located on different alleles, it is a trans mutation, in which case the patient may benefit from the combination of first- and third-generation TKIs. Therefore, accurately distinguishing the cis and trans configurations of T790M / C797S is of paramount importance for guiding clinical medication decisions and avoiding indiscriminate treatment.

[0003] Currently, next-generation sequencing (NGS) is the gold standard method for detecting cis-trans gene relationships. NGS can directly determine whether two mutations are located on the same chromosome through phasing analysis of high-throughput sequencing reads. However, this technology suffers from drawbacks such as high detection costs, complex experimental procedures, long cycle times (usually 3-7 days), and strong dependence on equipment and bioinformatics analysis, making it difficult to meet the needs of rapid clinical decision-making. While digital PCR (ddPCR) can achieve absolute quantification, it can only indirectly infer mutations from the abundance ratio of two mutation sites, and cannot directly read mutation combinations on a single allele. Furthermore, it is costly in reagents and cumbersome to operate, making it unsuitable for routine clinical practice.

[0004] Allele-specific PCR (ARMS-PCR), due to its primer 3' end mismatch design, can distinguish single-base mutations by differences in DNA polymerase extension efficiency and is widely used in the clinical detection of EGFR hotspot mutations. However, ARMS-PCR has an inherent contradiction between sensitivity and specificity: in the context of extremely high wild-type (e.g., mutation frequency in plasma cfDNA below 0.1%), a high cycle number of 45-50 cycles is usually required to accumulate sufficient mutant products for detection. But as the cycle number increases, the "slippage extension" of the wild-type template at the primer mismatch site accumulates exponentially with the amplification, leading to saturation of non-specific products and a significant increase in the false positive rate. At this point, even with blocking probes or locked nucleic acids (LNAs), only non-specific amplification can be partially inhibited, and the detection sensitivity ceiling is usually only about 1%, which cannot meet the monitoring needs of low-frequency drug-resistant clones at the 0.01% level in cfDNA.

[0005] Existing technologies have attempted to use ARMS-PCR for cis-trans typing of EGFR T790M / C797S. For example, existing techniques use two tubes of qPCR reaction solution with TaqMan-MGB probes, indirectly inferring the cis-trans relationship by the presence or absence of fluorescence signal (see CN120775960A). Although this method can complete the detection within 2 hours, it is still based on the conventional TaqMan probe method, and its detection sensitivity is still limited by existing conventional PCR techniques. Other technologies have introduced locked nucleic acid (LNA) probes and highly specific DNA polymerases, employing a three-tube stepwise elimination strategy for cis-trans typing (see CN121249884A). While this improves the detection specificity to the 0.001% level, its sensitivity is configured based on wild-type plasmid templates and does not use real healthy human plasma cfDNA as a background for evaluation.

[0006] The CRISPR / Cas system, with its highly efficient signal amplification capability (trans-cleavage activity that non-specifically cleaves a large number of reporter molecules after target recognition) and sequence targeting characteristics, offers new possibilities for overcoming the aforementioned sensitivity bottlenecks. Existing technologies have disclosed methods for detecting EGFR mutations based on conventional PCR combined with CRISPR / Cas12a (see PMC12515118). This method utilizes Cas12a's target recognition trigger signal amplification, improving detection sensitivity. However, this technique uses conventional PCR instead of ARMS-PCR for pre-amplification, lacking the specific screening capability of primer ends, allowing wild-type background to directly flood into the CRISPR step. Simultaneously, Cas12a strictly relies on PAM (usually TTTV) when recognizing DNA targets, while natural PAM is scarce near the core regions of T790M and C797S mutations, often requiring artificial PAM sequences created through primer modification, altering the original sequence environment and increasing system complexity. More importantly, existing PCR-CRISPR protocols do not involve cis-trans typing detection logic, failing to meet clinical needs for interpreting the T790M / C797S phase relationship.

[0007] In summary, the core technical bottleneck of existing technologies lies in the fact that the high cycle number strategy (45-50 cycles) of ARMS-PCR leads to saturation of wild-type nonspecific amplification, making it impossible to simultaneously achieve both sensitivity and specificity; while simple CRISPR detection lacks an effective pre-screening mechanism and cannot cope with ultra-high wild-type background. There is an urgent need in this field for an EGFR T790M / C797S cis-trans typing detection scheme that can cascade and synergistically combine the terminal specificity of ARMS-PCR with the signal amplification effect of CRISPR to achieve ultra-high sensitivity while fundamentally suppressing wild-type nonspecific amplification. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a sequence combination, kit, and method for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR. It establishes a rapid, ultra-sensitive, signal amplification-based cis-trans typing detection method, effectively solving the technical problems of ARMS non-specific amplification saturation, cis-trans interpretation dependence on indirect inference, and long detection cycle in existing technologies. This method can directly serve the precision medicine treatment of non-small cell lung cancer patients after drug resistance.

[0009] The technical solution provided by this invention is as follows:

[0010] This invention provides a sequence combination for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR, including amplification primers and crRNA. The amplification primers include a universal primer pair, an upstream primer for amplifying the T790M mutation, and a first and second downstream primers for amplifying the two C797S mutation subtypes. The crRNA includes a first universal crRNA targeting T790M, a second universal crRNA targeting C797S, and a first and second specific crRNA targeting the two C797S mutation subtypes. The sequences of the upstream and downstream primers of the universal primer pair are shown in SEQ ID NO:7 and SEQ ID NO:8; the sequence of the upstream primer used to amplify the T790M mutation is shown in SEQ ID NO:4; the sequences of the first downstream primer and the second downstream primer are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; and the sequences of the first universal crRNA, the second universal crRNA, the first specific crRNA, and the second specific crRNA are shown in SEQ ID NO:12, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

[0011] The present invention also provides the application of the sequence combination as described above in the preparation of a kit for detecting EGFR T790M / C797S cis-trans mutations.

[0012] The present invention also provides a kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR, comprising the sequence combination described above.

[0013] Furthermore, its ARMS-PCR reaction system is 20 μL, including the following components: 10 μL of AceQ® Universal U+Probe Master Mix V2, 0.5 μL each of 10 μM upstream and downstream primers, 5 μL of template DNA, and nuclease-free water to a final volume of 20 μL.

[0014] Furthermore, the CRISPR detection reaction system is 20 μL, including the following components: 1 μL of 1 M HEPES buffer, 1 μL of 1 M MgCl2, 2 μL of 25 mM NTP, 1 μL of T7 RNA polymerase, 0.5 μL of 1 μM LwCas13a protein, 0.4 μL of 10 μM crRNA, 1 μL of 10 μM RNA fluorescent reporter probe, 5 μL of ARMS-PCR reaction product, and nuclease-free water to a final volume of 20 μL.

[0015] Furthermore, the ARMS-PCR amplification conditions were: 37℃ for 2 min; 95℃ for 5 min; 95℃ for 20 s, 60℃ for 40 s, for a total of 33 cycles; 40℃ for 30 s; and the CRISPR detection reaction conditions were: incubation at 37℃ for 15-20 min.

[0016] Furthermore, the RNA fluorescent reporter probe is an oligonucleotide molecule with fluorescent groups and quenching groups labeled at both ends, and the RNA fluorescent reporter probe is FAM-ssRNA-BHQ1.

[0017] Furthermore, the kit comprises six detection systems, namely: Group 1: Internal control system, using the primer pairs shown in SEQ ID NO:7 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:12; The second group: the T790M mutation screening system, using the primer pairs shown in SEQ ID NO:4 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:12; The third group: C797S-1 mutation screening system, using the primer pairs shown in SEQ ID NO:5 and SEQ ID NO:7, and the crRNA shown in SEQ ID NO:9; Group 4: C797S-2 mutation screening system, using the primer pairs shown in SEQ ID NO:6 and SEQ ID NO:7, and the crRNA shown in SEQ ID NO:9; Group 5: Cis-trans assay of the C797S-1 system, using the primer pairs shown in SEQ ID NO:4 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:10; Group 6: Cis-trans assay of the C797S-2 system, using the primer pairs shown in SEQ ID NO:4 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:11.

[0018] Furthermore, the interpretation criteria for the kit are as follows: First, if there is a signal in the first group, the sample is valid; if there is no signal, it is invalid. Second, the T790M, C797S-1, or C797S-2 single mutation is determined based on the signals from the second to the fourth groups. Finally, when there is a signal in the second group, cis-trans typing is performed based on the signals from the fifth and sixth groups: if there is a signal in the fifth or sixth group, it is a cis double mutation; if there is no signal in either group, it is a trans double mutation.

[0019] This invention also provides a method for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR, the method comprising the following steps: Extract DNA from the sample; Using the extracted DNA as a template, ARMS-PCR amplification and CRISPR detection reactions were performed using the reagents in the kit described above; Based on the fluorescence signal generated by the CRISPR detection reaction, the mutation status and cis-trans type of EGFR T790M and C797S are comprehensively determined.

[0020] Beneficial effects

[0021] This invention provides a systematically designed and validated ARMS-PCR primer set that can specifically recognize the T790M and C797S drug resistance mutation sites of the EGFR gene and generate short fragment amplification products suitable for downstream CRISPR signal detection. Simultaneously, corresponding crRNA sequences were designed for the T7 transcription of the amplification products, including a universal crRNA for confirming successful ARMS amplification, and C797S-targeting crRNA and T790M-targeting crRNA for detecting the second mutation sequence region in the same amplification product, thereby achieving joint interpretation of cis-trans mutation status based on signal amplification.

[0022] This invention optimizes ARMS-PCR pre-amplification from the conventional 45-50 cycles to 33 cycles, stopping amplification before the wild-type template reaches exponential saturation due to non-specific amplification. Subsequently, the trans-cleavage signal amplification effect of Cas13a enables highly sensitive detection of the relatively limited product, significantly enhancing the mutant signal. This cascade strategy fundamentally overcomes the inherent bottleneck of ARMS—"high cycle number leads to wild-type saturation"—maintaining high specificity while maintaining a detection sensitivity of 0.1%.

[0023] This invention provides a two-tube orthogonal cascade system based on T790M-ARMS and C797S-ARMS. Each tube of PCR product is transcribed via T7 and then detected with corresponding universal crRNA and target crRNA using Cas13a. After the ARMS primers specifically capture alleles containing the target mutation, Cas13a recognizes a specific sequence region in the amplified product via the target crRNA and amplifies the signal by trans-cleaving a large number of fluorescent reporter probes. This allows for the detection of successful amplification and the presence of a second mutation sequence in the amplified product, achieving precise differentiation between cis and trans amplification.

[0024] This invention utilizes the synergistic effect of ARMS-PCR pre-amplification and Cas13a signal amplification to stably detect drug-resistant clones with a mutant allele frequency of 0.1% against a background of 10 ng wild-type cfDNA, improving sensitivity by one order of magnitude compared to conventional qPCR methods. The entire detection process can be completed in approximately 70 minutes, significantly shorter than NGS (3-7 days).

[0025] This invention's kit employs a UDG-dUTP time-series anti-contamination system. Pre-treatment at 37°C before ARMS-PCR pre-amplification degrades historical aerosol contamination, and the amplification products are incorporated with dUTP, making them less susceptible to re-amplification by conventional PCR even in the event of aerosol leakage. The detection process does not require high-end sequencing equipment, and results can be interpreted using a portable fluorescence spectrometer.

[0026] The cis-trans typing results of this invention are directly linked to clinical treatment plans: the detection of cis double mutations indicates that the patient is insensitive to all current generations of EGFR-TKIs, and the clinical approach can be promptly adjusted to chemotherapy or recommended for enrollment in a fourth-generation TKI clinical trial; the detection of trans double mutations indicates that the patient may benefit from a combination therapy of first-generation TKIs (such as gefitinib) and third-generation TKIs (such as osimertinib). This test provides a clear molecular stratification basis for individualized medication decisions after drug resistance, avoiding treatment delays and economic burdens caused by blind drug trials.

[0027] In summary, this invention establishes a rapid, ultra-sensitive, signal amplification-based cis-trans typing detection method by combining ARMS-PCR, T7 transcription, and Cas13a signaling. This method effectively solves the technical problems of ARMS non-specific amplification saturation, cis-trans interpretation dependence on indirect inference, and long detection cycle in existing technologies. It can directly serve the precision medicine for non-small cell lung cancer patients after drug resistance. Attached Figure Description

[0028] Figure 1 This is a flowchart of the detection process of the present invention. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0030] Example 1: Construction of the Detection System

[0031] 1. Experimental Materials

[0032] (1) Positive control: mutant plasmid and wild-type plasmid (Sangon Biotech (Shanghai) Co., Ltd.)

[0033] (2) Negative control: Nuclease-free water (Solepro)

[0034] (3) Nucleic acid extraction: Qiagen AllPrep DNA / RNA Mini Kit (Qiagen)

[0035] (4) PCR reagent: AceQ® Universal U + Probe Master Mix V2 (Novizan)

[0036] (5) CRISPR reagents: LwCas13a (Genscript), RNase inhibitor (Novizan), fluorescent reporter probe FAM-ssRNA-BHQ1 (5′FAM-rUrUrUrUrU-BHQ1-3′ (Qingke Biotechnology))

[0037] (6) Instrument: Fluorescence detector (Roche)

[0038] 2. Target sequence acquisition and conserved region screening

[0039] Based on the EGFR exon 20 sequence published in the COSMIC database, EGFR T790M and C797S mutation detection sites were identified, and the samples were sent to Sangon Biotech for plasmid synthesis. Details are shown in Table 1.

[0040] Table 1 Plasmid templates

[0041]

[0042] 3. Primer and crRNA sequence design: Referring to the EGFR gene exon 20 sequence in the COSMIC database, conventional ARMS-PCR control primers, the ARMS-T7 fusion primers of this invention, and the Cas13a crRNA sequence were designed. Specific sequences are shown in Table 2.

[0043] Table 2 Primer and crRNA Sequences

[0044]

[0045] 4. Testing process

[0046] (1) Conventional ARMS-PCR amplification system

[0047] Prepare a PCR reaction solution with a total volume of 20 μL, containing 10 μL of AceQ Universal U+Probe Master Mix V2, 0.5 μL each of forward and reverse primers (10 μM), 5 μL of template DNA, and make up the remainder with nuclease-free water.

[0048] The reaction procedure was as follows: first, UDG enzyme digestion was performed at 37℃ for 2 min; then, pre-denaturation was performed at 95℃ for 5 min; followed by an amplification phase of 45 cycles (denaturation at 95℃ for 20 s, annealing and extension at 60℃ for 40 s, during which FAM fluorescence signals were collected); and finally, cooling was performed at 40℃ for 30 s.

[0049] (2) CRISPR detection process

[0050] The Cas13a detection process includes T7 transcription and Cas13a signal amplification detection. A 20 μL reaction system was prepared, containing 1 μL of 1 M HEPES buffer, 1 μL of 1 M MgCl2, 2 μL of 25 mM NTP, 1 μL of T7 RNA polymerase, 0.5 μL of 1 μM Cas13a protein, 0.4 μL of 10 μM targeting crRNA, 1 μL of 10 μM RNA fluorescent reporter probe, 5 μL of ARMS-PCR reaction product, and the remainder was made up with nuclease-free water.

[0051] The reaction procedure was as follows: incubation at 37℃ for 15-20 min to complete T7 transcription and CRISPR cleavage, and collection of fluorescence signals.

[0052] 5. Detection combination method

[0053] Based on the designed ARMS-PCR primer pairs and crRNA sequences, six detection systems were constructed according to functional localization: the first group was an internal control system, the second to fourth groups were single mutation screening systems, and the fifth and sixth groups were cis-trans typing systems. The fifth and sixth groups shared the same ARMS primer pairs as the second group, and the scanning for the second mutation on the T790M positive allele was achieved by changing the target crRNA. The correspondence between primers, crRNAs, and detection targets for each group is shown in Table 3.

[0054] Table 3. Correspondence between primers, crRNA and detection system.

[0055]

[0056] 6. Interpretation criteria

[0057] Based on the above six detection systems, the analysis is performed step by step according to the following logic:

[0058] (1) First step (quality control interpretation): If there is a signal in No. 1 (internal reference system), the sample is considered valid; if there is no signal, the result is considered invalid and needs to be retested.

[0059] (2) Second step (mutation screening): Based on the signals numbered 2 to 4, determine whether there are T790M, C797S-1 or C797S-2 mutations in the sample.

[0060] (3) Third step (cis-trans typing): When there is a signal in number 2 (T790M screening), further test numbers 5 and 6. If there is a signal in number 5 or number 6, it indicates that T790M and the corresponding C797S mutation are located in the same allele, and it is determined to be a cis double mutation; if there is no signal in number 5 and number 6, it indicates that T790M and C797S are located in different alleles, and it is determined to be a trans double mutation.

[0061] The complete six-state interpretation criteria are shown in Table 4.

[0062] Table 4. Criteria for interpreting six mutation states

[0063]

[0064] Example 2: Optimization of ARMS-PCR cycle number

[0065] 1. Preliminary confirmation of ARMS-PCR cycle number

[0066] 1.1 Experimental Design

[0067] To screen for the optimal number of cycles for ARMS-PCR pre-amplification, a cycle number gradient was set, and the following templates were tested respectively:

[0068] (1) Positive plasmid dilutions (100 copies / μL and 10 copies / μL) were used to evaluate the ability to detect mutants.

[0069] (2) Cell-free DNA (cfDNA) from healthy human plasma was used as a negative control to assess nonspecific amplification.

[0070] (3) The primers used for this test are: T790M-ARMS-T7-F / COM-R; the crRNA to be detected is: T790M-C-crRNA; the template to be detected is: mutant template 1-T790M.

[0071] 1.2 Experimental Results

[0072] ARMS-PCR pre-amplification and subsequent Cas13a signal detection were performed under each cycle number condition. The signal intensity was compared with the wild-type background signal to preliminarily optimize the optimal cycle number.

[0073] Table 5 Optimization Results of Loop Count - 1

[0074]

[0075] 1.3 Experimental Conclusions

[0076] The results showed that the detection results with a cycle number of 30 and 35 met the requirements. If the cycle number was too low (≤25), the positive plasmid could not be detected accurately. If the cycle number was too high (≥40), the negative samples would show false positives.

[0077] 2. Repeatedly verify the optimal number of cycles for ARMS-PCR

[0078] 2.1 Experimental Design

[0079] To repeatedly confirm the optimal number of cycles for ARMS-PCR pre-amplification, a cycle number gradient was set, and the following templates were tested respectively:

[0080] (1) Positive plasmid dilutions (100 copies / μL and 10 copies / μL) were repeated three times to assess the ability to detect stable mutants.

[0081] (2) Cell-free DNA (cfDNA) from the plasma of 10 healthy individuals was used as a negative control to assess nonspecific amplification.

[0082] (3) The primers used for this test are: T790M-ARMS-T7-F / COM-R; the crRNA to be detected is: T790M-C-crRNA; the template to be detected is: mutant template 1-T790M.

[0083] 2.2 Experimental Results

[0084] ARMS-PCR pre-amplification and subsequent Cas13a signal detection were performed under each cycle number condition. The signal intensity was compared with the wild-type background signal, and the detection results were used to repeatedly verify the optimal cycle number conditions.

[0085] Table 6 Optimization Results of Loop Count - 2

[0086]

[0087] 2.3 Experimental Conclusions

[0088] The results showed that the detection results were best when the cycle number was 32 and 34. If the cycle number was too low (<32), the positive plasmid of 10 copies / μL could not be detected accurately and stably. If the cycle number was too high (>34), false positives occurred in negative samples.

[0089] 3. Determine the optimal number of cycles for ARMS-PCR

[0090] 3.1 Experimental Design

[0091] Based on the initial screening of the T790M ARMS-PCR system by cycle number, further simultaneous validation was performed on other ARMS-PCR systems and their corresponding CRISPR detection systems.

[0092] (1) Positive plasmid dilutions (10,000 copies / μL and 10 copies / μL) were repeated five times to assess the ability to detect stable mutants and to examine whether there is cross-contamination between different types at high concentrations.

[0093] (2) Cell-free DNA (cfDNA) from the plasma of 10 healthy individuals was used as a negative control to assess nonspecific amplification.

[0094] (3) The primers and crRNA combinations used in this test are shown in Table 2.

[0095] 3.2 Experimental Results

[0096] ARMS-PCR pre-amplification and subsequent Cas13a signal detection were performed under each cycle number condition. The signal intensity was compared with the wild-type background signal, and the detection results were used to repeatedly verify the optimal cycle number conditions.

[0097] Table 7 Optimization Results of Loop Count - 3

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] 3.3 Experimental Conclusions

[0108] Under 32 cycles, C797S (2389T>A) showed a false negative in detecting mutant template 4 (mutant template 4-T790M-C797S-A); under 34 cycles, both high and low concentrations of template could be stably detected with positive signals, but false positives occurred in healthy human samples; and C797S (2390G>C) showed cross-false positives in detecting high concentrations of template containing the C797S (2389T>A) mutation.

[0109] Positive signals were stably detected under both high and low template concentrations for 33 cycles, and no cross-reactivity was observed in the negative control sample of healthy human plasma cfDNA. This number of cycles ensured that the mutant product was sufficient to activate Cas13a signal amplification while wild-type nonspecific amplification had not yet reached exponential saturation, achieving an optimal balance between sensitivity and specificity (the detection procedure of this invention is as follows). Figure 1 ).

[0110] Example 3: Comparison of the sensitivity and specificity of the detection system with conventional ARMS-PCR

[0111] 1. Experimental Design

[0112] To evaluate the sensitivity of the ARMS-CRISPR combined detection system of this invention, healthy human plasma cfDNA was used as wild-type background. Synthetic T790M and C797S mutant positive plasmids were diluted to equivalent copy numbers to prepare a series of simulated samples with different mutation frequencies. The final formulation consisted of reference samples with a concentration of 2 ng / μL and mutation frequency gradients of 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.02%. cfDNA was extracted from plasma samples from 10 healthy individuals as an independent wild-type negative control group.

[0113] The detection method confirmed in Example 2 was used to detect the above-mentioned simulated samples and negative controls, and a simultaneous comparative analysis was performed with the conventional ARMS-PCR methods (PCR-1~PCR-4). This example aims to confirm the detection capability at each mutation frequency and does not involve cis-trans configuration determination. Therefore, only four systems were used: CRISPR-1 (internal control), CRISPR-2 (T790M screening), CRISPR-3 (C797S-1 screening), and CRISPR-4 (C797S-2 screening). The specific combinations and corresponding relationships are shown in Table 8.

[0114] Table 8. Correspondence between the conventional ARMS-PCR control system and the cascade detection system of this invention.

[0115]

[0116] 2. Experimental Results

[0117] Table 9. Statistical Table of CRISPR / PCR Detection Sensitivity Results

[0118]

[0119] 3. Experimental Conclusions

[0120] The above results demonstrate that the ARMS-T7-Cas13a cascade detection system of this invention achieves an order-of-magnitude improvement in detection sensitivity compared to the conventional ARMS-PCR control system. With the same input amount of 10 ng of DNA, the conventional ARMS-PCR method can only stably detect positive signals when the mutation frequency is ≥1% (≥5% for some sites); while this invention, through 33 cycles of restriction amplification combined with Cas13a signal amplification, can stably detect mutant allele frequencies as low as 0.05%, with a sensitivity 20 to 100 times higher than the conventional ARMS-PCR method.

[0121] Example 4: Verification of Detection Sensitivity

[0122] 1. Experimental Design

[0123] The plasmids (mutant template 4 / mutant template 5) of known concentration were diluted to 10-fold serial dilutions. 3 10 2 10 1 and 10 0 Different concentrations of copies / μL were used as amplification templates to evaluate amplification sensitivity. At 10... 5 10 copies / μL of wild type 3 10 2 10 1 and 10 0 The mutant plasmid product was prepared into reference samples at concentrations of 1%, 0.1%, 0.01%, and 0.001% using copies / μL and then analyzed.

[0124] 2. Experimental Results

[0125] Table 10 Detection Results of Plasmid Reference Standards - 1

[0126]

[0127] Table 11 Detection Results of Plasmid Reference Standards - 2

[0128]

[0129] 3. Experimental Conclusions

[0130] The above results demonstrate that the kit of the present invention achieves a detection sensitivity of 0.001% for the detection of serially diluted positive plasmid standards, which is consistent with the sensitivity level of the reported patented method (CN121249884A).

[0131] Example 5: Clinical Sample Test Results

[0132] 1. Experimental Design

[0133] Several clinical lung cancer samples validated by NGS were collected and tested using the detection method confirmed in Example 2. The consistency between the results and the NGS results was statistically analyzed.

[0134] 2. Experimental Results

[0135] Table 12 Clinical Sample Test Results

[0136]

[0137] 3. Experimental Conclusions

[0138] The above results show that the kit of this invention has 100% consistency with NGS in the detection of EGFR T790M / C797S mutations and cis-trans typing, proving that the accuracy and reliability of the detection system of this invention reach the gold standard level. At the same time, compared with the NGS method, which usually takes 3-7 days and relies on high-throughput sequencers and professional bioinformatics analysis, the entire process of this invention can be completed in about 90 minutes, the cost of reagents and consumables is only about one-tenth of that of NGS, and the detection can be carried out on a conventional real-time PCR platform. It does not require expensive sequencing equipment or professional personnel, and only a small amount of plasma cfDNA (10 ng) is needed to complete the detection of fragmented samples. Therefore, it can better meet the needs of rapid clinical decision-making, dynamic monitoring of drug resistance, and routine implementation in medical institutions at all levels.

[0139] This invention achieves high sensitivity and specificity for the detection of EGFR T790M / C797S drug resistance mutations through optimized ARMS primer design, a 33-cycle restriction amplification strategy, and a T7 transcription-Cas13a cascade signal amplification system. Experimental data show that the detection method of this invention achieves a minimum stable detection limit of 0.05% for low-abundance mutant alleles, significantly better than the sensitivity ceiling of conventional ARMS-PCR methods (1%-5%). Furthermore, in a head-to-head comparison with the NGS gold standard, this invention shows 100% consistency in mutation detection and cis-trans typing, and no false positives due to non-specific amplification occurred even in a background of extremely high wild-type (99.95%), indicating its excellent detection specificity and reliability.

[0140] It should be noted that the embodiments and descriptions described herein are intended to illustrate the technical principles and implementation methods of the invention and do not constitute a limitation on the scope of protection of the invention. Those skilled in the art should understand that various adjustments, substitutions, or improvements can be made to the invention without departing from its design principles and technical purpose. For example, adjusting the mismatch positions of ARMS primers, changing the target region or fluorescent reporter group of crRNA, and optimizing T7 transcription and Cas13a reaction times are all within the scope of protection covered by the claims and their equivalents of this invention.

Claims

1. A sequence combination for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR, characterized in that, It includes amplification primers and crRNA. The amplification primers include universal primer pairs, an upstream primer for amplifying the T790M mutation, and a first and second downstream primer for amplifying the two C797S mutation subtypes. The crRNA includes a first universal crRNA targeting T790M, a second universal crRNA targeting C797S, and a first and second specific crRNA targeting the two C797S mutation subtypes. The sequences of the upstream and downstream primers of the universal primer pair are shown in SEQ ID NO:7 and SEQ ID NO:8; the sequence of the upstream primer used to amplify the T790M mutation is shown in SEQ ID NO:4; the sequences of the first downstream primer and the second downstream primer are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; and the sequences of the first universal crRNA, the second universal crRNA, the first specific crRNA, and the second specific crRNA are shown in SEQ ID NO:12, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

2. The application of the sequence combination as described in claim 1 in the preparation of a kit for detecting EGFR T790M / C797S cis-trans mutations.

3. A kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR, characterized in that, Includes the sequence combination described in claim 1.

4. The kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR according to claim 3, characterized in that, The ARMS-PCR reaction system is 20 μL, including the following components: 10 μL of AceQ® Universal U+ProbeMaster Mix V2, 0.5 μL each of 10 μM upstream and downstream primers, 5 μL of template DNA, and nuclease-free water to a final volume of 20 μL.

5. The kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR according to claim 4, characterized in that, The CRISPR detection reaction system is 20 μL, including the following components: 1 μL of 1 M HEPES buffer, 1 μL of 1 M MgCl2, 2 μL of 25 mM NTP, 1 μL of T7 RNA polymerase, 0.5 μL of 1 μM LwCas13a protein, 0.4 μL of 10 μM crRNA, 1 μL of 10 μM RNA fluorescent reporter probe, 5 μL of ARMS-PCR reaction product, and nuclease-free water to a final volume of 20 μL.

6. The kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR according to claim 5, characterized in that, The ARMS-PCR amplification conditions were: 37℃ for 2 min; 95℃ for 5 min; 95℃ for 20 s, 60℃ for 40 s, for a total of 33 cycles; 40℃ for 30 s. The CRISPR detection reaction conditions were: incubation at 37℃ for 15-20 min.

7. The kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR according to claim 5, characterized in that, The RNA fluorescent reporter probe is an oligonucleotide molecule with fluorescent and quenching groups labeled at both ends, and the RNA fluorescent reporter probe is FAM-ssRNA-BHQ1.

8. The kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR according to claim 3, characterized in that, The kit contains six detection systems, namely: Group 1: Internal control system, using the primer pairs shown in SEQ ID NO:7 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:12; The second group: the T790M mutation screening system, using the primer pairs shown in SEQ ID NO:4 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:12; The third group: C797S-1 mutation screening system, using the primer pairs shown in SEQ ID NO:5 and SEQ ID NO:7, and the crRNA shown in SEQ ID NO:9; Group 4: C797S-2 mutation screening system, using the primer pairs shown in SEQ ID NO:6 and SEQ ID NO:7, and the crRNA shown in SEQ ID NO:9; Group 5: Cis-trans assay of the C797S-1 system, using the primer pairs shown in SEQ ID NO:4 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:10; Group 6: Cis-trans assay of the C797S-2 system, using the primer pairs shown in SEQ ID NO:4 and SEQ ID NO:8, and the crRNA shown in SEQ ID NO:

11.

9. The kit for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR according to claim 8, characterized in that, The interpretation criteria for the kit are as follows: First, if there is a signal in the first group, the sample is valid; if there is no signal, it is invalid. Second, determine the single mutation of T790M, C797S-1, or C797S-2 based on the signals from the second to the fourth groups. Finally, when there is a signal in the second group, perform cis-trans typing based on the signals from the fifth and sixth groups: if there is a signal in the fifth or sixth group, it is a cis double mutation; if there is no signal in either group, it is a trans double mutation.

10. A method for detecting EGFR T790M / C797S cis-trans mutations based on ARMS-CRISPR, characterized in that, The method includes the following steps: Extract DNA from the sample; Using the extracted DNA as a template, ARMS-PCR amplification and CRISPR detection reactions were performed using the reagents in the kit described in any one of claims 3-8; Based on the fluorescence signal generated by the CRISPR detection reaction, the mutation status and cis-trans type of EGFR T790M and C797S are comprehensively determined.

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