Pre-amplification detection method of low-abundance fusion gene

Abstract

The invention relates to the technical field of nucleic acid molecule detection, and discloses a pre-amplification detection method of a low-abundance fusion gene. According to the invention, a high-sensitivity SNT-MRD platform technology and the specificity of a qPCR primer probe system are combined, and a two-stage PCR amplification strategy and an optimized primer probe design are adopted, so that effective enrichment and detection of trace fusion genes in plasma ctDNA are realized. Compared with the existing qPCR detection method, the method disclosed by the invention has the advantages that the target sequence is firstly enriched, and then specific detection is performed, so that the problem of non-specific amplification caused by a large number of cycles in single PCR is avoided; compared with a method purely depending on NGS deep sequencing, the method has the advantages that the dependence on sequencing depth and complex biological information analysis is reduced, sensitive detection can be realized on a conventional qPCR instrument, the cost is lower, and the speed is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid molecule detection, and in particular to a pre-amplification detection method for low-abundance fusion genes. Background Art

[0002] A fusion gene refers to a process in which partial or complete sequences of two or more genes combine to form a new chimeric gene. Fusion genes can lead to abnormalities in protein expression levels, function, and sites of action, which can cause abnormal cell proliferation and promote the occurrence or development of tumors. It is important to note that the presence of trace amounts of fusion gene sequences in blood does not indicate that the blood sample originated from a cancer patient, nor can it provide a diagnostic result or health status. The sampled blood is processed or tested to determine the fusion gene content, which serves as intermediate information to determine whether further testing is needed to detect abnormal cell proliferation.

[0003] For example, thyroid cancer, particularly papillary thyroid carcinoma, often harbors fusion gene abnormalities such as RET / PTC. RET / PTC refers to the RET proto-oncogene rearrangement in papillary thyroid carcinoma (PTC). In patients with early-stage thyroid cancer or minimal residual disease after surgery, the amount of ctDNA released into the blood by the tumor is extremely low, and fusion gene sequences often constitute only a tiny fraction of peripheral blood cfDNA, typically less than 1%. Furthermore, ctDNA fragments are short (approximately 150-180 bp) and vary widely between individuals. This makes direct detection of these low-abundance fusion genes using traditional PCR or NGS techniques challenging with insufficient sensitivity and high false-negative rates.

[0004] In current technology, for the detection of low-content fusion genes, the Chinese patent with authorization announcement number CN107885972B provides a fusion gene detection method based on single-end sequencing and its application. The fusion gene detection method includes: (1) designing a capture probe for the first gene of the fusion gene and performing single-end sequencing; (2) analyzing the single-end sequencing results to obtain the sequencing depth of the second gene strong promoter; (3) comparing the obtained strong promoter sequencing depth with the sequencing depth baseline. If the sequencing depth of the strong promoter of the sample to be tested is significantly greater than the sequencing depth baseline, it is judged that the sample to be tested has a gene fusion mutation, otherwise it is judged that no gene fusion has occurred; the sequencing depth baseline is the sequencing depth baseline of the second gene strong promoter of the fusion gene negative sample. The fusion gene detection method provided by this patent improves the sensitivity of detecting fusion genes through single-end detection. However, the following problems still exist: 1. Insufficient sensitivity (failure in ultra-low abundance scenarios) Relying on direct sequencing for deep comparison, it lacks a mechanism for pre-enrichment of target sequences. When the target fusion gene abundance is ≤0.1% (e.g., in plasma ctDNA), the detection limit is only 0.1% due to high sequencing noise (nonspecific capture interference). This patented detection method cannot meet the detection requirements of ultra-low abundances, such as 0.05%.

[0005] 2. Lack of specificity design (high risk of false positives) Positive determination was determined solely by statistical thresholds (U test P>0.95), which did not address issues of primer cross-hybridization or nonspecific amplification. Window division (100-150 bp) diluted local signals, resulting in insensitive threshold determination. Probes were not used to block non-target binding, resulting in a low signal-to-noise ratio (background noise S / N<5:1) during multiplex detection.

[0006] 3. Pollution control mechanism gap The dUTP / UNG enzyme anti-contamination system is not integrated, and aerosol contamination of pre-amplification products can easily lead to false positives.

[0007] 4. Inefficient operation A large number of negative sample baselines (≥37 cases) need to be established in advance, and statistical analysis needs to be repeated when new detection targets are added, which is inflexible and time-consuming. Summary of the Invention

[0008] Low-abundance fusion genes, such as RET / PTC fusion genes in plasma ctDNA, suffer from insufficient sensitivity and low signal-to-noise ratio. To address these issues, the present invention provides a pre-amplification detection method for low-abundance fusion genes. This method combines the highly sensitive SNT-MRD platform technology with the specificity of a qPCR primer-probe system, employing a two-stage PCR amplification strategy and optimized primer-probe design to effectively enrich and detect trace fusion genes in plasma ctDNA.

[0009] The specific technical solutions of the present invention are: The present invention provides a method for pre-amplification detection of low-abundance fusion genes, which comprises the following steps: Step (S.1): Design a specific PCR primer pair and a TaqMan® MGB fluorescent probe for the target fusion gene, wherein the two primers in the specific PCR primer pair are respectively targeted to the sequences on both sides of the fusion breakpoint, and the TaqMan® MGB fluorescent probe is designed to span the fusion breakpoint; Step (S.2), using the specific PCR primer pair to perform PCR amplification on the extracted free DNA sample, the number of cycles is 8-15, a high-fidelity DNA polymerase is added to the amplification system, and the annealing temperature is controlled to be above 60°C to inhibit nonspecific amplification, thereby obtaining a pre-amplified product; In step (S.3), a qPCR system (quantitative PCR system) with an embedded TaqMan® MGB probe is used to perform real-time qPCR detection using the preamplification product as a template. At least one primer is a semi-nested primer located within the preamplification product, and the presence of the target fusion gene is determined by fluorescence signal.

[0010] The method of the present invention combines the high-sensitivity SNT-MRD platform technology with the specificity of the qPCR primer-probe system, and adopts a two-stage PCR amplification strategy and optimized primer-probe design to achieve effective enrichment and detection of trace fusion genes in plasma ctDNA. The present invention greatly improves the detection sensitivity and signal-to-noise ratio of low-abundance fusion genes through the "double PCR amplification + MGB probe detection" scheme. Compared with the existing qPCR detection method, this method first enriches the target sequence and then specifically detects it, avoiding the problem of non-specific amplification caused by a large number of cycles in a single PCR; compared with the method that simply relies on NGS deep sequencing, the present invention reduces the dependence on sequencing depth and complex bioinformatics analysis, and can achieve sensitive detection on conventional qPCR instruments, with lower cost and faster speed.

[0011] As a preferred embodiment of the above method, in step (S.1), the primers in the specific PCR primer pair are 20-25 bases in length, have a Tm value of 60-62°C, and have no complementary sequence at the 3' end of the specific PCR primer pair.

[0012] As a preferred embodiment of the above method, in step (S.2), the number of cycles of the pre-amplification is 10.

[0013] As a preferred embodiment of the above method, in step (S.2), the amplification system comprises dNTPs, wherein dUTP accounts for 40%-60% of the total dTTP concentration.

[0014] As a preferred embodiment of the above method, in step (S.2), the annealing temperature is 62°C.

[0015] As a preferred embodiment of the above method, step (S.3) includes treating the pre-amplification product with UNG enzyme before qPCR detection, and the treatment conditions are reaction at 50°C for 2 minutes.

[0016] As a preferred embodiment of the above method, in step (S.3), the TaqMan® MGB probe has a length of 16 to 20 bases, a Tm value 1 to 5°C higher than the Tm value of the specific PCR primer, and the 5' end of the probe is labeled with a reporter fluorescent group and the 3' end is labeled with an MGB quencher group.

[0017] As a preferred embodiment of the above method, the free DNA sample is plasma circulating tumor DNA (ctDNA), and the fragment length is 150-180 bp.

[0018] As a preferred embodiment of the above method, the target fusion gene is a fusion gene selected from RET / PTC, NTRK or ALK.

[0019] As a preferred embodiment of the above method, the detection limit of the method is 0.05% to 0.1% variant allele frequency.

[0020] Compared with the prior art, the present invention has the following technical effects: The method of the present invention combines the high-sensitivity SNT-MRD platform technology with the specificity of the qPCR primer-probe system, and adopts a two-stage PCR amplification strategy and optimized primer-probe design to achieve effective enrichment and detection of trace fusion genes in plasma ctDNA. The present invention greatly improves the detection sensitivity and signal-to-noise ratio of low-abundance fusion genes through the "double PCR amplification + MGB probe detection" scheme. Compared with the existing qPCR detection method, this method first enriches the target sequence and then specifically detects it, avoiding the problem of non-specific amplification caused by a large number of cycles in a single PCR; compared with the method that simply relies on NGS deep sequencing, the present invention reduces the dependence on sequencing depth and complex bioinformatics analysis, and can achieve sensitive detection on conventional qPCR instruments, with lower cost and faster speed. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the following embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0022] Example 1 In one embodiment, the present invention provides a method for pre-amplification detection of low-abundance fusion genes, comprising the following steps: Step (S.1): Design a specific PCR primer pair and a TaqMan® MGB fluorescent probe for the target fusion gene, wherein the two primers in the specific PCR primer pair are respectively targeted to the sequences on both sides of the fusion breakpoint, and the TaqMan® MGB fluorescent probe is designed to span the fusion breakpoint; Step (S.2), using the specific PCR primer pair to perform PCR amplification on the extracted free DNA sample, the number of cycles is 8-15, a high-fidelity DNA polymerase is added to the amplification system, and the annealing temperature is controlled to be above 60°C to inhibit nonspecific amplification, thereby obtaining a pre-amplified product; In step (S.3), a qPCR system (quantitative PCR system) with an embedded TaqMan® MGB probe is used to perform real-time qPCR detection using the preamplification product as a template. At least one primer is a semi-nested primer located within the preamplification product, and the presence of the target fusion gene is determined by fluorescence signal.

[0023] The method of this embodiment combines the high-sensitivity SNT-MRD platform technology with the specificity of the qPCR primer probe system, and adopts a two-stage PCR amplification strategy and optimized primer probe design to achieve effective enrichment and detection of trace fusion genes in plasma ctDNA. This method greatly improves the detection sensitivity and signal-to-noise ratio of low-abundance fusion genes through the "double PCR amplification + MGB probe detection" scheme. Compared with the existing qPCR detection method, this method first enriches the target sequence and then specifically detects it, avoiding the problem of non-specific amplification caused by a large number of cycles in a single PCR; compared with the method that relies solely on NGS deep sequencing, the present invention reduces the dependence on sequencing depth and complex bioinformatics analysis, and can achieve sensitive detection on conventional qPCR instruments, which is lower cost and faster.

[0024] In this example, the two primers in the specific PCR primer pair target sequences at either end of the fusion gene (one at each end of the two fused genes), and the probes are designed to span the fusion breakpoint. Primers were designed to avoid complementary sequences at the 3' end, thereby reducing primer-dimer formation. The TaqMan® MGB fluorescent probe utilizes MGB technology, allowing for shorter lengths to enhance discrimination and reduce background noise.

[0025] TaqMan® MGB fluorescent probe is a probe used for real-time fluorescence quantitative PCR (qPCR). Its structure includes a reporter group, a quencher group, and an MGB modification. Specifically: Reporter group: The 5' end of the probe is labeled with a fluorescent reporter group, such as FAM, VIC, etc. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group.

[0026] Quencher: The 3' end is labeled with a quencher. Traditional quenchers have a certain fluorescence background at the fluorescence emission wavelength, while the TaqMan® MGB probe uses a non-fluorescent quencher (NFQ) to effectively reduce the background signal.

[0027] MGB modification: A minor groove binder (MGB) is attached to the 3' end of the probe. MGB is a molecule that binds to the minor groove of the DNA double helix. It increases the affinity and stability of the probe with the target DNA, enabling the probe to hybridize with the target sequence at lower temperatures.

[0028] Therefore, this example uses a qPCR system with an embedded TaqMan® MGB probe to specifically detect the target fusion gene. The amplification curve is monitored in real time using the probe's fluorescence signal to determine the presence of the fusion gene and quantify its initial abundance. The PCR utilizes semi-nested primers, meaning at least one primer is located within the pre-amplification product, which enhances detection specificity.

[0029] The use of TaqMan® MGB fluorescent probe combined with double PCR amplification to detect fusion genes can greatly improve the sensitivity of detection.

[0030] Preferably, in step (S.1), the primers in the specific PCR primer pair are 20-25 bases in length, have a Tm value of 60-62°C, and have no complementary sequence at the 3' end of the specific PCR primer pair.

[0031] Preferably, in step (S.2), the number of cycles of the pre-amplification is 10.

[0032] A preliminary PCR amplification of the target fusion gene region is performed using the specific PCR primers. If the target fusion gene is present in the pre-amplification product, its copy number will be enriched and amplified. This pre-amplification stage is preferably performed for a minimum of 8-15 cycles, with 10 cycles being preferred. Adding a high-fidelity enzyme to the amplification system and increasing the annealing temperature can effectively inhibit nonspecific amplification and primer cross-talk.

[0033] Preferably, in step (S.2), the amplification system comprises dNTPs, wherein dUTP accounts for 40%-60% of the total dTTP concentration.

[0034] In order to prevent aerosol contamination of pre-amplification products from affecting the accuracy of qPCR, this method can also introduce a dUTP / UNG enzyme system into the pre-amplification reaction system, and degrade possible contaminating products through UNG enzyme during the qPCR stage, thereby further improving the detection reliability.

[0035] Preferably, in step (S.2), the annealing temperature is 62°C.

[0036] Preferably, in step (S.3), the pre-amplification product is treated with UNG enzyme before qPCR detection, and the treatment conditions are 50° C. for 2 minutes.

[0037] Preferably, in step (S.3), the TaqMan® MGB probe has a length of 16 to 20 bases, a Tm value 1 to 5°C higher than the Tm value of the specific PCR primer, and the 5' end of the probe is labeled with a reporter fluorescent group and the 3' end is labeled with an MGB quencher group.

[0038] Preferably, the cell-free DNA sample is plasma circulating tumor DNA (ctDNA), and the fragment length is 150-180 bp.

[0039] Preferably, the target fusion gene is a fusion gene selected from RET / PTC, NTRK or ALK.

[0040] Preferably, the detection limit of the method is 0.05% to 0.1% variant allele frequency.

[0041] Example 2 Based on the method provided in Example 1, this example further describes the present invention using the detection of the RET / PTC1 fusion gene as an example. This example includes the following steps: (1) Primer and probe design: Take the RET / PTC1 fusion gene as an example (fusion of the RET gene and the CCDC6 gene). The upstream primer is designed in the upstream region of the RET gene fusion breakpoint, and the downstream primer is designed in the downstream region of the CCDC6 gene fusion breakpoint, so that only templates with RET / PTC1 fusion can be amplified by both primers at the same time. The probe is designed across the RET / PTC1 fusion point, with one end targeting the RET sequence and the other end targeting the CCDC6 sequence, with an MGB group in the middle to shorten the probe length and improve specificity. The probe is labeled with a reporter fluorescent group at the 5′ end and a quencher group at the 3′ end (MGB-NFQ). The designed primers are approximately 20–25 bases long, with a Tm value of 62°C and no obvious complementarity with each other; the probe is approximately 18 bases long, with a Tm value slightly higher than the primer, approximately 65°C.

[0042] (2) Pre-amplification reaction: cfDNA was extracted from the subject's plasma, and ctDNA was obtained using a commercial kit. 50 µL of ctDNA solution (equivalent to extraction from approximately 5-10 mL of plasma) was added to the pre-amplification PCR system (total volume 50 µL), which contained the above-mentioned specific primer pairs (final concentration 0.2 µM each), high-fidelity DNA polymerase (with proofreading function), dNTPs (dUTP partially replaced dTTP, accounting for 50% of the total dTTP concentration), and an appropriate amount of Mg²⁺ buffer system. The PCR amplification program used a hot start method (pre-denaturation at 95°C for 2 minutes), followed by pre-amplification cycles: denaturation at 95°C for 15 seconds, annealing and extension at 62°C for 1 minute, for a total of 10 cycles. A higher annealing temperature helps reduce non-target binding. After pre-amplification, the product can be optionally purified and diluted 10-fold to reduce background interference.

[0043] (3) Real-time PCR detection: Take 1 µL of the above pre-amplification product as a template and add it to the qPCR reaction mixture containing the inner primer pair and TaqMan MGB probe (total volume 20 µL). The inner primer design is slightly different from the pre-amplification primer. One of the inner primers is a nested primer located inside the fusion fragment to ensure that even if a small amount of non-specific products remain in the pre-amplification product, these non-specific fragments will not be amplified in the second round of amplification. The probe concentration in the qPCR system is 0.1 µM and the primer concentration is 0.3 µM each. After treating the pre-amplification template with UNG enzyme (50°C for 2 minutes), PCR amplification is performed: 95°C for 3 minutes, followed by 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, repeated for 40 cycles, and FAM fluorescence signal is collected during the 60°C annealing extension stage. If the RET / PTC1 fusion gene is present in the sample, the fluorescence signal of the second round of amplification will increase exponentially and cross the threshold line at a specific cycle number (Ct value); otherwise, if there is no target, there will only be a baseline noise level signal.

[0044] To validate the sensitivity of the present method, a plasmid containing the RET / PTC1 fusion fragment and the corresponding wild-type fragment were constructed in this example. The two were mixed at a molar ratio of 1:1000 (mutant abundance 0.1%) and ultrasonically fragmented into approximately 170 bp fragments to simulate ctDNA fragments. This simulated low-abundance sample served as a template for analysis using both traditional single-round qPCR and the present method's two-step preamplification followed by qPCR. The traditional single-round qPCR method involves direct, single-cycle amplification.

[0045] The results showed that while conventional qPCR failed to detect a specific signal after 40 cycles of amplification, this method, after 10 cycles of pre-amplification, exhibited a clear exponential amplification curve at cycle 32, with a significantly earlier Ct value, successfully detecting a fusion gene at a 0.1% abundance. Further reducing the mutation abundance to 0.01% (i.e., 1:10,000, exceeding the pre-amplification detection limit), this method still reproducibly obtained a weak positive signal, while the conventional method detected no signal at all. This demonstrates that this method significantly improves the sensitivity of fusion gene detection.

[0046] Compared with the prior art, the method of this embodiment has the following beneficial effects: 1. Significantly Improved Detection Sensitivity: By pre-amplifying and enriching the target sequence, the sensitivity of fusion gene detection is increased by more than an order of magnitude. Experiments have demonstrated that this method has a detection limit of 0.05%–0.1% variant allele frequency for fusion genes, significantly superior to the approximately 1% detection limit of conventional qPCR. This method can reliably detect fusion gene signals, particularly in early-stage patient samples with extremely low ctDNA levels, increasing the likelihood of early diagnosis.

[0047] 2. Improved signal-to-noise ratio and specificity: Utilizing TaqMan® MGB probe technology and a two-step PCR strategy, false positive signals are effectively reduced. The short length and low background fluorescence of the MGB probes provide a clearer detection signal. Pre-amplification using specific primers and optimized conditions avoids cross-amplification and primer-dimer formation between different target primers, significantly improving signal specificity and accuracy.

[0048] 3. Fast detection and easy operation: This method does not rely on complex and expensive second-generation sequencers. It only requires a conventional PCR amplification instrument and a real-time quantitative PCR instrument to complete the test. The entire process is completed within 4 hours, making it suitable for rapid clinical screening. Compared to high-depth NGS, this method simplifies data processing, eliminates the need for complex bioinformatics analysis, and provides intuitive result interpretation.

[0049] 4. Broad Applicability: This method is flexible in design and can be expanded to detect low-abundance fusion genes or mutated genes in other cancers, beyond RET / PTC fusion genes. For example, the detection of other RET fusion types and NTRK fusions in thyroid cancer, as well as low-abundance genetic abnormalities in liquid biopsies of leukemia and lung cancer, can be accomplished simply by replacing the corresponding specific primer and probe combinations. This method is also applicable to low-concentration nucleic acid samples (such as cerebrospinal fluid ctDNA), demonstrating broad application prospects.

[0050] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for pre-amplification detection of low-abundance fusion genes, characterized by: The following steps are involved: Step (S.1): Design a specific PCR primer pair and a TaqMan® MGB fluorescent probe for the target fusion gene, wherein the two primers in the specific PCR primer pair are respectively targeted to the sequences on both sides of the fusion breakpoint, and the TaqMan® MGB fluorescent probe is designed to span the fusion breakpoint; Step (S.2), using the specific PCR primer pair to perform PCR amplification on the extracted free DNA sample, the number of cycles is 8-15, a high-fidelity DNA polymerase is added to the amplification system, and the annealing temperature is controlled to be above 60°C to inhibit nonspecific amplification, thereby obtaining a pre-amplified product; In step (S.3), a qPCR system with an embedded TaqMan® MGB probe is used to perform real-time qPCR detection using the preamplification product as a template. At least one primer is a semi-nested primer located within the preamplification product, and the presence of the target fusion gene is determined by fluorescence signal.

2. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: In step (S.1), the primers in the specific PCR primer pair are 20-25 bases in length, have a Tm value of 60-62°C, and have no complementary sequence at the 3' end of the specific PCR primer pair.

3. A method according to claim 1, characterized in that: In step (S.2), the number of cycles of the pre-amplification is 10.

4. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: In step (S.2), the amplification system comprises dNTPs, wherein dUTP accounts for 40%-60% of the total dNTP concentration.

5. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: In step (S.2), the annealing temperature is 62°C.

6. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: In step (S.3), the pre-amplification product is treated with UNG enzyme before qPCR detection, and the treatment condition is 50°C for 2 minutes.

7. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: In step (S.3), the TaqMan® MGB probe has a length of 16 to 20 bases, a Tm value 1 to 5°C higher than the Tm value of the specific PCR primer, and a reporter fluorescent group is labeled at the 5' end of the probe and an MGB quencher group is labeled at the 3' end.

8. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: The cell-free DNA sample is plasma circulating tumor DNA, and the fragment length is 150~180 bp.

9. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: The target fusion gene is a fusion gene selected from RET / PTC, NTRK or ALK.

10. The method for pre-amplification detection of a low-abundance fusion gene according to claim 1, wherein: The detection limit of the method is 0.05%~0.1% variant allele frequency.