Fusion gene detection system based on multiple fluorescent quantitative PCR (Polymerase Chain Reaction)

Abstract

The invention relates to the technical field of nucleic acid molecule detection, and discloses a fusion gene detection system based on multiple fluorescent quantitative PCR. The TaqMan-MGB probe group, the specific primer pair and the PCR buffer system are optimally designed, so that the binding specificity of the probe group and the primer pair is effectively improved, the binding efficiency is improved, and the sensitivity of fusion gene detection is improved. Specifically, the probe is arranged across the fusion connection area, so that the specificity is ensured to the maximum extent. The primer crosses two sides of a fusion breakpoint to ensure that only fused templates can be amplified at the same time. The multiple fluorescent quantitative PCR detection system provided by the invention can simultaneously amplify and monitor the existence condition of various thyroid cancer fusion genes in one tube reaction.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid molecule detection, and in particular to a fusion gene detection system based on multiplex fluorescence quantitative PCR. 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 site of action, which can cause abnormal cell proliferation and promote the development or progression of tumors. For example, thyroid cancer, particularly papillary thyroid cancer, may harbor RET-CCDC6, RET-NCOA4, PAX8-PPARG, NTRK1 / 3 fusions, and the rare ALK fusion. Accurate detection of these fusion genes is crucial for selecting treatment options for thyroid cancer. Detecting the presence of certain fusion genes can aid in selecting treatment options for patients.

[0003] However, if traditional single-target detection methods are used, for example, single-plex PCR detection for each fusion is performed separately, the process is not only cumbersome and time-consuming, but also requires a large amount of precious samples, making it difficult to obtain comprehensive molecular information in a timely manner.

[0004] Multiplex fluorescence quantitative PCR technology is suitable for detection panels containing multiple genes. However, in practical applications, the design of primer probes and system optimization for multiplex PCR are very challenging. The main challenges of applying multiplex fluorescence quantitative PCR technology to thyroid cancer fusion genes are: (1) The simultaneous presence of multiple sets of primers can easily induce primer dimers or non-specific amplification, and the primers may interfere with each other, resulting in a decrease in the amplification efficiency of certain targets or the occurrence of false positive signals; (2) The fluorescence signals of different target probes may overlap or have cross-color spectral overlap, and the fluorescent reporter groups need to be carefully selected to ensure that the signals of each channel can be clearly distinguished; (3) When the length of each amplicon and the primer efficiency are inconsistent, the strongly amplified product may consume too much reagent and inhibit the amplification of other products.

[0005] Therefore, how to optimize the design of primer-probe combinations to avoid cross-interference, balance amplification efficiency, and rationally configure fluorescence channels is a key technical issue in the development of multiplex qPCR detection systems. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems in applying the multiplex fluorescence quantitative PCR technology to the detection of thyroid cancer fusion genes, the present invention provides a fusion gene detection system based on multiplex fluorescence quantitative PCR.

[0007] The specific technical solutions of the present invention are: In one aspect, the present invention provides a fusion gene detection system based on multiplex fluorescence quantitative PCR, which comprises: a specific primer pair for the fusion gene and a TaqMan-MGB probe set; In the fusion gene detection system, the 5' end of the probe in the TaqMan-MGB probe group designed for different fusion genes is labeled with different fluorescent reporter groups, and the 3' end of the probe is connected to MGB and a quencher group; The probes are designed to span the fusion breakpoints; The fusion genes include RET-CCDC6, RET-NCOA4, PAX8-PPARG, NTRK1 fusion, NTRK3 fusion and ALK fusion; The PCR buffer system of the detection system includes: hot start DNA polymerase, 1.5~3.0mM Mg 2+ , 0.1~0.5mM dNTPs, 5~10wt% glycerol.

[0008] To reduce primer cross-interference and address low sensitivity in multiplex fluorescence quantitative PCR, the present invention optimizes the design of TaqMan-MGB probe sets, specific primer pairs, and a PCR buffer system. This effectively enhances the binding specificity and efficiency of the probe sets and primer pairs, thereby improving the sensitivity of fusion gene detection. The multiplex fluorescence quantitative PCR detection system described above can simultaneously amplify and monitor the presence of multiple thyroid cancer fusion genes in a single reaction tube.

[0009] Specifically, the probes are set to span the fusion junction region to maximize specificity. The primers span both sides of the fusion breakpoint to ensure that only the fusion template is amplified simultaneously. It has been experimentally verified that the probes are set to span the fusion junction region to maximize specificity. 2+ In the PCR buffer system of "0.1~0.5mM dNTPs, 5~10wt% glycerol", the amplification efficiency and sensitivity are high.

[0010] As a preferred embodiment of the above detection system, the fusion gene is a combination of RET-CCDC6, PAX8-PPARG and ETV6-NTRK3.

[0011] The detection system is set as a quantitative PCR of the combination of RET-CCDC6, PAX8-PPARG and ETV6-NTRK3. In the above PCR buffer system, the efficiency of synchronous detection can be improved and primer cross-interference can be avoided.

[0012] As a preferred embodiment of the above detection system, the probe is 18 to 30 bp in length and contains at least 5 base sequences at the fusion breakpoint.

[0013] More preferably, the Tm value of the probe is 3-5° C. higher than the Tm value of the corresponding specific primer.

[0014] By making the Tm value of the probe 3-5°C higher than the Tm value of the corresponding specific primer, the probe is preferentially bound, avoiding primer-dominated non-specific amplification.

[0015] As a preferred embodiment of the above detection system, the fluorescent reporter groups include at least three of FAM, VIC, ROX and Cy5, and the spectral overlap rate of each fluorescent channel is less than 5%.

[0016] As a preferred embodiment of the above detection system, an internal reference gene primer probe set is further included, and the internal reference gene is selected from GAPDH, β-actin, and ACTB.

[0017] As a preferred embodiment of the above detection system, the two primers in the specific PCR primer pair are respectively targeted at sequences on both sides of the fusion breakpoint.

[0018] As a preferred embodiment of the above detection system, the specific primer is a primer sequence with a homology of <70% with the human genome, and the probe has ≥2 base mismatches in the wild-type sequence.

[0019] By utilizing the high sensitivity of MGB to mismatches, nonspecific signals were reduced by more than 10 times.

[0020] As a preferred embodiment of the above detection system, in the PCR of the detection system, the primer concentration is adjusted to 0.1-0.5 μM and the probe concentration is adjusted to 0.05-0.2 μM, so that the Ct value of each target in the multiplex PCR differs from that of the singlex reaction by ≤1 cycle.

[0021] On the other hand, the present invention provides a thyroid cancer fusion gene detection kit, which comprises the above-mentioned fusion gene detection system.

[0022] Compared with the prior art, the present invention has the following technical effects: (1) The present invention optimizes the design of TaqMan-MGB probe sets and specific primer pairs, as well as the PCR buffer system, to effectively improve the binding specificity of the probe sets and primer pairs, and to improve the binding efficiency, thereby providing sensitivity for fusion gene detection. Specifically, the above-mentioned probes are set to span the fusion junction region to maximize specificity. The primers span both sides of the fusion breakpoint to ensure that only templates with fusion can be amplified simultaneously. It has been experimentally verified that in the presence of "hot start DNA polymerase, 1.5~3.0mM Mg 2+ In the PCR buffer system of "0.1~0.5mM dNTPs, 5~10wt% glycerol", the amplification efficiency and sensitivity are high.

[0023] (2) The multiplex fluorescence quantitative PCR detection system provided by the present invention can simultaneously amplify and monitor the presence of multiple thyroid cancer fusion genes in a single tube reaction. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1 This embodiment provides a fusion gene detection system based on multiplex fluorescence quantitative PCR, which includes: a specific primer pair for the fusion gene and a TaqMan-MGB probe set; In the fusion gene detection system, the 5' end of the probe in the TaqMan-MGB probe group designed for different fusion genes is labeled with different fluorescent reporter groups, and the 3' end of the probe is connected to MGB and a quencher group; The probes are designed to span the fusion breakpoints; The fusion genes include RET-CCDC6, RET-NCOA4, PAX8-PPARG, NTRK1 fusion, NTRK3 fusion and ALK fusion; The PCR buffer system of the detection system includes: hot start DNA polymerase, 1.5~3.0mM Mg 2+ , 0.1~0.5mM dNTPs, 5~10wt% glycerol.

[0026] To reduce primer cross-interference in multiplex fluorescence quantitative PCR and address low sensitivity, this detection system optimizes the design of the TaqMan-MGB probe set, specific primer pairs, and PCR buffer system to effectively enhance the binding specificity and efficiency of the probe set and primer pairs, thereby increasing the sensitivity of fusion gene detection. The multiplex fluorescence quantitative PCR detection system provided in this example can simultaneously amplify and monitor the presence of multiple thyroid cancer fusion genes in a single reaction tube.

[0027] Specifically, the probes are set to span the fusion junction region to maximize specificity. The primers span both sides of the fusion breakpoint to ensure that only the fusion template is amplified simultaneously. It has been experimentally verified that the probes are set to span the fusion junction region to maximize specificity. 2+In the PCR buffer system of "0.1~0.5mM dNTPs, 5~10wt% glycerol", the amplification efficiency and sensitivity are high.

[0028] More specifically, the specific primer pairs and probes of this embodiment are illustrated using RET-CCDC6, PAX8-PPARG, and ETV6-NTRK3 fusion genes. The specific design of the primer probe set for each fusion gene is as follows: RET-CCDC6 fusion: The forward primer is selected to target a region approximately 100 bp upstream of the RET gene fusion breakpoint (the end of the RET gene exon), and the backward primer is selected to target a region approximately 100 bp downstream of the CCDC6 gene breakpoint (the corresponding exon of the CCDC6 gene). The amplified product is approximately 120 bp in length. A TaqMan MGB probe is designed to span the RET-CCDC6 fusion site. The probe is 20 bp long and includes the fusion junction sequence. An "MGB" moiety is inserted in the middle to shorten the probe and improve specificity. The probe is labeled with a FAM fluorophore at the 5' end and a BHQ1 quencher at the 3' end. This probe only binds perfectly when the RET and CCDC6 sequences are connected. If the fusion is not present (RET or CCDC6 alone), the probe will not fully bind due to spanning the breakpoint, resulting in no fluorescent signal.

[0029] PAX8-PPARG fusion: The forward primer is selected at the end of exon 7 of the PAX8 gene, and the reverse primer is selected at the start of exon 1 of the PPARG gene (designed to address the most common breakpoint types). The amplified product is approximately 90 bp in length, with shorter products accommodating the length of RNA transcripts or free DNA fragments. The TaqMan probe, covering the fusion junction between PAX8 and PPARG, is 18 bp long and labeled with a VIC fluorophore at the 5' end and an MGB and NFQ quencher at the 3' end. The use of the MGB destabilizes probe binding in the presence of single-base mismatches (e.g., the normal sequence in the absence of the fusion), ensuring that fluorescent signal is generated only when the fusion sequence is correct. This design is expected to avoid amplifying wild-type sequences of PAX8 and PPARG, providing extremely high specificity.

[0030] ETV6-NTRK3 fusion: Although uncommon in thyroid cancer, this fusion has clinical significance as a broad-spectrum tumor fusion marker. The forward primer is located at the end of a specific exon of the ETV6 gene (e.g., the end of exon 5), and the reverse primer is located in the corresponding exon of the NTRK3 gene. The amplified product is approximately 150 bp. The probe is designed to span the ETV6-NTRK3 fusion site and is approximately 22 bp in length. It is labeled with a ROX fluorophore at the 5' end and quenched with MGB and BHQ2 at the 3' end. ROX was chosen because its emission spectrum (approximately 608 nm) is distinct from that of FAM and VIC, allowing it to serve as a third channel. The probe sequence was also optimized to avoid nonspecific binding to normal ETV6 or NTRK3 sequences.

[0031] Therefore, for the combined detection of RET-CCDC6, PAX8-PPARG, and ETV6-NTRK3 fusion genes, the three specific primer pairs and TaqMan-MGB probe set designed above constitute the core components of the fusion gene detection system based on multiplex fluorescence quantitative PCR provided in this example. In addition, the internal reference gene ACTB (β-Actin) was introduced as an internal control. The ACTB primer pair amplifies a fragment of approximately 100 bp, and the probe is labeled with Cy5 fluorescence. This allows the system to occupy a total of four fluorescence channels (FAM: RET-CCDC6, VIC: PAX8-PPARG, ROX: ETV6-NTRK3, Cy5: internal control gene), making it compatible with most four-channel real-time PCR instruments.

[0032] As a preference in this embodiment, the fusion gene is a combination of RET-CCDC6, PAX8-PPARG and ETV6-NTRK3.

[0033] The detection system is set as a quantitative PCR of the combination of RET-CCDC6, PAX8-PPARG and ETV6-NTRK3. In the above PCR buffer system, the efficiency of synchronous detection can be improved and primer cross-interference can be avoided.

[0034] As a preference in this embodiment, the probe is 18 to 30 bp in length and contains at least 5 base sequences at the fusion breakpoint.

[0035] More preferably, the Tm value of the probe is 3-5° C. higher than the Tm value of the corresponding specific primer.

[0036] By making the Tm value of the probe 3-5°C higher than the Tm value of the corresponding specific primer, the probe is preferentially bound, avoiding primer-dominated non-specific amplification.

[0037] Preferably, the fluorescent reporter groups include at least three of FAM, VIC, ROX and Cy5, and the spectral overlap rate of each fluorescent channel is less than 5%.

[0038] As a preference of this embodiment, an internal reference gene primer probe set is also included, and the internal reference gene is selected from GAPDH, β-actin, and ACTB.

[0039] As a preference in this embodiment, the two primers in the specific PCR primer pair are respectively targeted at sequences on both sides of the fusion breakpoint.

[0040] As a preference in this embodiment, the specific primer is a primer sequence having a homology of <70% with the human genome, and the probe has ≥2 base mismatches in the wild-type sequence.

[0041] By utilizing the high sensitivity of MGB to mismatches, nonspecific signals were reduced by more than 10 times.

[0042] As a preference of this embodiment, in the PCR of the detection system, the primer concentration is adjusted to 0.1-0.5 μM and the probe concentration is adjusted to 0.05-0.2 μM, so that the Ct value of each target in the multiplex PCR differs from that of the singlex reaction by ≤1 cycle.

[0043] Compared with the prior art, this embodiment has the following technical effects: (1) This embodiment optimizes the design of TaqMan-MGB probe set and specific primer pair, as well as the PCR buffer system, to effectively improve the binding specificity of the probe set and primer pair, improve the binding efficiency, and thus provide sensitivity for fusion gene detection. Specifically, the above-mentioned probe is set to span the fusion junction region to maximize specificity. The primers span both sides of the fusion breakpoint to ensure that only templates with fusion can be amplified simultaneously. It has been experimentally verified that in the presence of "hot start DNA polymerase, 1.5~3.0mM Mg 2+ In the PCR buffer system of "0.1~0.5mM dNTPs, 5~10wt% glycerol", the amplification efficiency and sensitivity are high.

[0044] (2) The multiplex fluorescence quantitative PCR detection system provided in this embodiment can simultaneously amplify and monitor the presence of multiple thyroid cancer fusion genes in a single tube reaction.

[0045] 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.

[0046] 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 fusion gene detection system based on multiplex fluorescence quantitative PCR, characterized by: Includes specific primer pairs and TaqMan-MGB probe sets targeting fusion genes; In the fusion gene detection system, the 5' end of the probe in the TaqMan-MGB probe group designed for different fusion genes is labeled with different fluorescent reporter groups, and the 3' end of the probe is connected to MGB and a quencher group; The probes are designed to span the fusion breakpoints; The fusion genes include RET-CCDC6, RET-NCOA4, PAX8-PPARG, NTRK1 fusion, NTRK3 fusion and ALK fusion; The PCR buffer system of the detection system includes: hot start DNA polymerase, 1.5~3.0mM Mg 2+ , 0.1~0.5mM dNTPs, 5~10wt% glycerol.

2. The fusion gene detection system according to claim 1, wherein: The fusion gene is a combination of RET-CCDC6, PAX8-PPARG and ETV6-NTRK3.

3. The fusion gene detection system according to claim 1, wherein: The probe is 18 to 30 bp in length and contains at least 5 base sequences at the fusion breakpoint.

4. The fusion gene detection system according to claim 3, wherein: The Tm value of the probe is 3-5° C. higher than the Tm value of the corresponding specific primer.

5. The fusion gene detection system according to claim 1, wherein: The fluorescent reporter groups include at least three of FAM, VIC, ROX and Cy5, and the spectral overlap rate of each fluorescent channel is less than 5%.

6. The fusion gene detection system according to claim 1, wherein: Also included is an internal reference gene primer probe set, wherein the internal reference gene is selected from GAPDH, β-actin, and ACTB.

7. The fusion gene detection system according to claim 1, wherein: The two primers in the specific PCR primer pair are respectively directed to sequences on both sides of the fusion breakpoint.

8. The fusion gene detection system according to claim 1, wherein: The specific primer is a primer sequence with a homology of <70% with the human genome, and the probe has ≥2 base mismatches in the wild-type sequence.

9. The fusion gene detection system according to claim 1, wherein: In the PCR of the detection system, the primer concentration is adjusted to 0.1-0.5 μM and the probe concentration is adjusted to 0.05-0.2 μM, so that the Ct value of each target in the multiplex PCR differs from that of the singlex reaction by ≤1 cycle.

10. A thyroid cancer fusion gene detection kit, characterized by: Comprising the detection system according to any one of claims 1 to 9.