Multi-gene locus joint detection method based on target enrichment

Abstract

The invention relates to a multi-gene locus joint detection method based on target enrichment, and belongs to the technical field of gene detection. In order to solve the problems of low specificity and poor low-abundance site enrichment effect of the existing multi-gene target site detection method, the invention provides a multi-gene site joint detection method based on target enrichment, which comprises the steps of primary amplification reaction, specific ligation reaction, label hybridization enrichment and detection of a label signal. Qualitative detection and quantitative analysis of target sites of different genes are realized through existence and intensity of signals and corresponding tag sequences. The triple guarantee mechanism can effectively eliminate non-specific amplification product interference, greatly improve the specificity and sensitivity of multi-gene target site detection, realize accurate detection of single base difference, simplify the operation process and shorten the detection period, has the advantages of simple operation, high detection efficiency and accurate and reliable result, and is suitable for popularization and application. And the defects in the prior art are overcome.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and in particular relates to a multi-gene locus joint detection method based on target enrichment. Background Technology

[0002] In the field of gene testing, single-gene locus detection is no longer sufficient to meet the needs of accurate testing. The characteristic expression and locus distribution of genes are not determined by a single gene locus, but are the result of the synergistic effect of multiple gene loci. By jointly detecting multiple gene target loci, specific information about multiple gene loci can be obtained simultaneously, providing a more comprehensive and accurate basis for gene locus analysis, genotyping research, and gene characteristic judgment, significantly improving the scientificity and effectiveness of gene testing. Therefore, the application demand for multi-gene target locus detection is becoming increasingly urgent.

[0003] Currently, commonly used methods for multi-gene target site detection include microarray detection, digital PCR, and next-generation sequencing, but all of these methods have significant drawbacks. While microarray detection can achieve high-throughput detection, its specificity is poor, and it is susceptible to interference from non-specific amplification products and cross-hybridization, leading to false positives and false negatives, thus affecting detection accuracy. Furthermore, it has low capture efficiency for low-abundance target sites, making it unsuitable for micro-sample detection scenarios. Digital PCR has better specificity than microarray detection, but its operation is complex, sample pretreatment steps are cumbersome, and the number of gene sites covered in a single detection is limited, resulting in a long detection cycle. This fails to meet the demand for rapid results in the field of gene detection, limiting its application in high-efficiency gene detection scenarios.

[0004] Second-generation sequencing has significant advantages in high throughput, enabling the simultaneous detection of a large number of gene loci. However, it also suffers from problems such as complex operation, long detection cycle, poor enrichment effect on low-abundance target loci, consumption of a large amount of sample nucleic acid, insufficient accuracy in the detection of trace samples, and high detection cost, which hinders its widespread adoption.

[0005] In summary, existing multi-gene target site detection methods generally suffer from problems such as low specificity, poor enrichment of low-abundance sites, complex operation, and long detection cycle, making it difficult to balance detection accuracy, efficiency, and sample adaptability. Summary of the Invention

[0006] To address the problems of low specificity, poor enrichment of low-abundance sites, complex operation, and long detection cycle in existing multi-gene target site detection methods, this invention provides a non-diagnostic target-based multi-gene site joint detection method.

[0007] The technical solution of the present invention: A multi-gene locus joint detection method based on target enrichment includes the following steps: Step 1: Design specific amplification primer pairs for each target site of multi-gene detection. Using the nucleic acid of the sample to be tested as a template, add all specific amplification primer pairs simultaneously to perform a single amplification reaction to obtain a single amplification product containing multiple gene target sites. Step 2: Design specific ligation primer pairs for each target site amplification product in the amplification product obtained in Step 1. The upstream primer of the specific ligation primer pair has a tag sequence at the 5' end, the downstream primer has a label at the 5' end, and the 3' end has phosphorylation modification. The amplification product obtained in step one, all specific ligation primer pairs, and ligase are mixed. The amplification product is pre-denatured to unwind into single strands, exposing each target site and its upstream and downstream specific sequence regions. Then, under primer annealing conditions, the specific ligation primer pairs are initiated to specifically bind to the single strands of the amplification product, forming a continuous aligned structure of upstream specific ligation primer-target site-downstream specific ligation primer. The ligase catalyzes the covalent ligation of the upstream and downstream specific ligation primers to form a complete ligation product. Step 3: Prepare a vector coupled with an inverse complementary anchor tag sequence. Mix the ligation product obtained in Step 2 with the vector to carry out a hybridization reaction. The ligation product is adsorbed onto the surface of the vector by specifically binding its own tag sequence to the anchor tag sequence on the vector, thereby achieving efficient enrichment of the target ligation product. Step 4: Detect the label signal of the ligation product adsorbed on the carrier. By detecting the presence, intensity and corresponding tag sequence of the signal, qualitative detection and quantitative analysis of different gene target sites can be achieved. By combining the correspondence between different tag sequences and target sites, the presence status and content of multiple gene target sites in the sample can be determined simultaneously, and finally, the joint detection results of multiple gene target sites can be obtained.

[0008] Furthermore, the sample to be tested in step one is one or more of the following: blood sample, plasma sample, FFPE sample, tissue sample, fecal sample, or urine sample, which are processed in vitro for non-diagnostic purposes and used for gene locus analysis.

[0009] Furthermore, in step two, the upstream primers of the specific ligation primer pairs corresponding to different target sites have different tag sequences, and the tag sequences have no homology with the sample nucleic acid.

[0010] Furthermore, the 5' end of the tag sequence described in step two is modified with a functional group, which is an amino, carboxyl, mercapto, aldehyde, or azide group modification.

[0011] Furthermore, the functional group modification is directly or indirectly connected to the 5' end of the tag sequence. The direct connection is that the functional group is covalently connected to the nucleotide at the 5' end of the tag sequence, and the indirect connection is that the functional group is indirectly connected to the 5' end of the tag sequence through a short linker arm.

[0012] Furthermore, the labeling described in step two enables the linker product to directly develop color when irradiated with excitation light of the corresponding wavelength, or to develop color immediately upon addition of the matching reaction substrate or affinity ligand.

[0013] Furthermore, the labeling described in step two is fluorescent labeling and / or biotin.

[0014] Furthermore, the carrier mentioned in step three is a solid-phase carrier and / or a liquid-phase carrier. The solid-phase carrier is a magnetic bead, a microporous plate, or a glass sheet; the liquid-phase carrier is a nanoparticle dispersion, a polymer solution, or a biomacromolecule solution.

[0015] Furthermore, the carrier surface described in step three has active modifying groups, which are carboxyl, amino, or N-hydroxy-succinimide groups; the carrier and the anchoring tag sequence are coupled through covalent bonds mediated by the active modifying groups, which are amide bonds, ester bonds, or disulfide bonds.

[0016] Furthermore, the multi-gene target site is specifically distinguished by at least one of the following two methods: (1) The types of marker signals carried by the ligation products corresponding to each target site are different; (2) The types of carriers that carry the ligation products corresponding to each target site are different.

[0017] The beneficial effects of this invention are: This invention proposes a multi-stage combined detection method consisting of "one-time specific amplification + dual-primer specific ligation + tag hybridization enrichment." This method achieves a significant improvement in detection performance through a three-stage synergistic strategy: primary amplification completes initial enrichment of the target site; upstream primers with tag sequences, labeled downstream primers, and ligase work synergistically to achieve secondary specific screening of the amplification products; and specific hybridization of the tag sequence with the vector's reverse complementary sequence completes a tertiary enrichment. This triple-protection mechanism effectively eliminates interference from non-specific amplification products, significantly improving the specificity and sensitivity of multi-gene target site detection. It enables accurate detection of single-base differences while simplifying the operation process and shortening the detection cycle. It combines the advantages of ease of operation, high detection efficiency, and accurate and reliable results, overcoming the shortcomings of existing technologies.

[0018] This method possesses broad applicability and expansion value, enabling multiplex detection and simultaneous detection of multiple samples and / or multiple target loci. It exhibits low primer bias and clear result interpretation. Applicable sample ranges include, but are not limited to, blood, plasma, FFPE samples, tissues, feces, urine samples, as well as plant and microbial samples. It can be widely applied to multi-site DNA detection scenarios across various fields, including but not limited to SNP, mutation, deletion, methylation detection, and microbial identification. This invention aims to provide a highly efficient detection scheme for enriching multiple gene target loci, effectively adapting to the needs of micro-volume DNA sample detection, improving the sensitivity, accuracy, and throughput of nucleic acid detection, and providing strong technical support for the field of gene detection. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the three-level enrichment process of a single gene target site in the multi-gene target site joint detection method based on target enrichment of the present invention, which consists of "one-time specific amplification + dual primer specific ligation + tag hybridization enrichment". Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0021] Example 1

[0022] This embodiment provides a set of specific amplification primer pairs, specific ligation primer pairs, and vectors used in a multi-gene target site joint detection method for target enrichment.

[0023] The methylation sites of protein phosphatase 1 regulatory subunit 16B (PPP1R16B) are mostly located in its gene promoter region, making them important sites for epigenetic modification. Its methylation status can serve as an important indicator for analyzing the epigenetic characteristics of this gene, enabling precise assessment of gene methylation features. Branched-chain amino acid transaminase 1 (BCAT1) is a key enzyme gene involved in branched-chain amino acid metabolism. Its methylation status is an important epigenetic feature related to the functional regulation of this gene and can be used for the analysis and study of its methylation level. Icarus family zinc finger protein 1 (IKZF1) is a transcriptional regulation-related gene. Methylation of its promoter region is a typical feature of its epigenetic modification and can serve as a target for epigenetic research related to transcriptional regulation. It should be noted that the epigenetic feature detection of the above gene targets is not for disease diagnosis purposes, but only for target research related to multi-gene joint detection.

[0024] I. This embodiment uses the methylation sites of PPP1R16B, BCAT1, and IKZF1 as target sites and provides specific amplification primer pairs designed for the methylation sites of PPP1R16B, BCAT1, and IKZF1.

[0025] The nucleotide sequence of the upstream primer for PPP1R16B methylation site-specific amplification is shown in SEQ ID No:1, specifically 5'-CGTTTATTTTCGGCGTTC-3', and the nucleotide sequence of the downstream primer for PPP1R16B methylation site-specific amplification is shown in SEQ ID No:2, specifically 5'-CCCTCTAAACGACTCCGAC-3'.

[0026] The nucleotide sequence of the upstream primer for BCAT1 methylation site-specific amplification is shown in SEQ ID No:3, specifically 5'-GTTGATGTAATTCGTTAGGTCG-3', and the nucleotide sequence of the downstream primer for BCAT1 methylation site-specific amplification is shown in SEQ ID No:4, specifically 5'-ATACCCGAAACGACGACG-3'.

[0027] The nucleotide sequence of the upstream primer for IKZF1 methylation site-specific amplification is shown in SEQ ID No:5, specifically 5'-GGACGACGTATTTTTTTCG-3', and the nucleotide sequence of the downstream primer for IKZF1 methylation site-specific amplification is shown in SEQ ID No:6, specifically 5'-CACCTCTCGACCGCCT-3'.

[0028] The three sets of specific amplification upstream primer pairs in this embodiment have primer lengths of 18-25 bp and GC content of 40-60%. There is no complementary pairing between the primers, and they do not form hairpin structures. All primers were synthesized by Genewiz Biotechnology Co., Ltd. Each synthesized primer was prepared into a 100 pmol / mL stock solution using TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0029] 2. Design specific ligation primer pairs based on the target methylation site sequence, the terminal sequence of the primary amplification product, the tag sequence, and the labeling requirements.

[0030] The nucleotide sequence of the specific upstream primer corresponding to the PPP1R16B methylation site is shown in SEQ ID No:7. The 5' end to the 3' end are the tag sequence and the upstream primer sequence, respectively. The nucleotides from position 1 to position 24 at the 5' end are the tag sequence, and the nucleotides from position 25 to the 3' end are the upstream primer sequence, specifically 5'-CTTTCTCATACTTTCAACTAATTTCGGGCGTTTTTTTGGGTC-3'.

[0031] The nucleotide sequence of the specific downstream primer corresponding to the PPP1R16B methylation site is shown in SEQ ID No:8. It has a biotin label at the 5' end and a phosphorylation modification at the 3' end, specifically 5'-biotin-ACTCCGACTCATCGATTCC-P-3'.

[0032] The specific nucleotide sequence of the upstream primer corresponding to the BCAT1 methylation site is shown in SEQ ID No:9. The 5' end to the 3' end are the tag sequence and the upstream primer sequence, respectively. The nucleotides from position 1 to position 24 at the 5' end are the tag sequence, and the nucleotides from position 25 to the 3' end are the upstream primer sequence, specifically 5'-ACTACTTATTCTCAAACTCTAATACGTTAGGTCGCGAGTTTTC-3'.

[0033] The nucleotide sequence of the specific downstream primer corresponding to the BCAT1 methylation site is shown in SEQ ID No:10. The 5' end is marked with biotin and the 3' end is phosphorylated, specifically 5'-biotin-CCGACCCTCTCGCGAC--P3'.

[0034] The nucleotide sequence of the specific upstream primer corresponding to the IKZF1 methylation site is shown in SEQ ID No:11. The 5' end to the 3' end are the tag sequence and the upstream primer sequence, respectively. The nucleotides from position 1 to position 24 at the 5' end are the tag sequence, and the nucleotides from position 25 to the 3' end are the upstream primer sequence, specifically 5'-CATCTTCATATCAATTCTCTTATTTCGTGTTTCGTTTTGCG-3'.

[0035] The nucleotide sequence of the specific downstream primer corresponding to the IKZF1 methylation site is shown in SEQ ID No:12. It has a biotin label at the 5' end and a phosphorylation modification at the 3' end, specifically 5'-biotin-CGAAACGCGCAAAAAAA-P-3'.

[0036] All primers were synthesized by Genewiz Biotechnology Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL using TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0037] 3. Based on the tag sequences of the upstream primers that are specifically linked, design reverse complementary anchoring tag sequences and couple them to the carrier magnetic beads.

[0038] This embodiment uses three types of MagPlex magnetic beads, all surface-carboxyl-modified from Luminex Corporation. These are suitable for all Luminex liquid-phase suspension chip systems, such as the MAGPIX or Luminex 200 liquid-phase suspension chip systems. MagPlex beads are 6.5-micron ultramagnetic polystyrene microspheres doped with three red / infrared fluorescent dyes. By precisely controlling the concentration ratio of each dye, different fluorescent coding combinations are formed, each corresponding to a unique "region number." This embodiment uses beads numbered 20, 33, and 35. During detection, the Luminex instrument uses a red laser to excite the fluorescent dyes inside the magnetic beads. The combination of fluorescence signal intensity is used to identify the number, thus distinguishing the detection target points corresponding to different magnetic beads.

[0039] In this embodiment, the vector corresponding to the PPP1R16B methylation site uses magnetic beads of number 20. The nucleotide sequence of the anchor tag sequence coupled on the magnetic beads is shown in SEQ ID No:13, specifically 5'-AAATTAGTTGAAAGTATGAGAAAG-3'.

[0040] The vector corresponding to the BCAT1 methylation site uses magnetic beads number 33. The nucleotide sequence of the anchor tag sequence coupled to the magnetic beads is shown in SEQ ID No:14, specifically 5'-TATTAGAGTTTGAGAATAAGTAGT-3'.

[0041] The vector corresponding to the IKZF1 methylation site uses magnetic beads of number 35. The nucleotide sequence of the anchor tag sequence coupled to the magnetic beads is shown in SEQ ID No:15, specifically 5'-AATAAGAGAATTGATATGAAGATG-3'.

[0042] All anchor tag sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized anchor tag sequences were prepared into a 0.1 nM stock solution using nuclease-free water for later use.

[0043] The method for coupling the anchor tag sequence to the vector is as follows: Take 5×10 6 MagPlex magnetic beads with uncoupled surface carboxyl groups were magnetically separated, the supernatant was discarded, and the beads were resuspended in 45 μL of 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 4.5).

[0044] Add 2 μL of the corresponding anchoring tag sequence (0.1 nM) to MagPlex magnetic beads of different types with carboxyl groups modified on the surface. Add 2.5 μL of freshly prepared 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, purchased from Thermo Fisher Scientific) solution to the magnetic bead suspension, mix well, and react at 25°C in the dark for 30 minutes. Add 2.5 μL of freshly prepared 10 mg / mL EDC solution to the magnetic bead suspension again, mix well, and react at 25°C in the dark for 30 minutes. Magnetic separation of the magnetic beads and discarding the supernatant, wash once with 1 mL of 0.02% Tween 20, magnetic separation again and discarding the supernatant. Resuspend the precipitate with 0.1% SDS for 40 seconds, magnetic separation again and discarding the supernatant. Resuspend in 80 μL of LTE (tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid) buffer and store at 2–8°C in the dark.

[0045] Example 2

[0046] This embodiment provides a multi-gene target site joint detection method for methylation sites of PPP1R16B, BCAT1 and IKZF1 genes, based on the specific amplification primer pairs, specific ligation primer pairs and vector provided in Example 1, and its specificity verification.

[0047] I. Experimental Materials 1. Standard quality granules Positive standard plasmid PC3, containing the target region after bisulfite transformation of the methylation sites of the PPP1R16B, BCAT1, and IKZF1 genes, and negative standard plasmid NC3, which does not contain the target region after transformation of the methylation sites of the above three genes, were both synthesized by Genewiz Biotechnology Co., Ltd.

[0048] 2. Main reagents High-specificity Taq Pro multiplex DNA polymerase: purchased from Nanjing Novizan Biotechnology Co., Ltd., product number Vazyme-PM202-01; T4 DNA ligase: purchased from NEB, catalog number M0202S; Tris-HCl (hydroxymethyl)aminomethane hydrochloride: purchased from Beijing Solarbio Technology Co., Ltd., product number Solarbio T8230; Polyethylene glycol p-isooctylphenyl ether (Triton X-100): purchased from Beijing Solarbio Technology Co., Ltd., product number Solarbio T8200; Sodium chloride (NaCl): purchased from Tianjin University Chemical Reagent Center; ExoSAP-IT™ PCR Product Purification Kit: Purchased from ABI, USA.

[0049] Hybrid magnetic bead mixture (Microspheres): purchased from Luminex; Enzyme-free water and TE buffer (pH 8.0): both were prepared in the laboratory according to standard procedures and met the standards for molecular biology experiments.

[0050] 3. Preparation of dedicated buffer solution The formulation of 2×Tm buffer is shown in Table 1.

[0051] Table 1

[0052]

[0053] The preparation method for 2×Tm buffer is as follows: Add approximately 200 mL of distilled water to a 500 mL beaker. Accurately weigh the NaCl and Tris-HCl using an electronic balance. Transfer Triton X-100 vial and add it to the distilled water. Place the beaker on a magnetic stirrer and stir at room temperature until all components are completely dissolved. Transfer the solution to a 250 mL volumetric flask and bring the volume to 250 mL with distilled water. Invert the flask to mix thoroughly, then pour the solution back into the beaker and adjust the pH to 8.0 using concentrated hydrochloric acid. Filter the solution and store at 4 °C.

[0054] II. Experimental Methods 1. Dilution of positive and negative standards for the target region Take the PC3 and NC3 plasmid dry powders, dissolve them separately in an appropriate amount of enzyme-free water, and then dilute the dissolved plasmid stock solution to 10. 15 copies / mL, followed by 10-fold serial dilutions, for a total of 10 dilutions.5 The final concentration was obtained by multiplying the concentration by 10. 10 Positive standard 5PC3 and negative standard 5NC3 (copies / mL) were used as experimental templates. 5PC3 and 5NC3 were used to verify the specificity of the magnetic bead-tag sequence conjugates and primers for each gene.

[0055] 2. The first amplification reaction of the sample to be tested. (1) Preparation of PCR primer working solution For each target gene site, 10 μL each of the upstream primer stock solution and the corresponding downstream primer stock solution for the methylation site-specific amplification of PPP1R16B, BCAT1, and IKZF1 provided in Example 1 were added to a 1.5 mL microcentrifuge tube, followed by 140 μL of TE buffer (pH 8.0). The mixture was vortexed for 30 seconds and then centrifuged briefly (3000 rpm, 1 minute) to allow the liquid on the tube wall to settle to the bottom. This yielded PCR primer working solution with a final concentration of 5 pmol / mL for each primer. The solution was then stored at 4°C for later use.

[0056] (2) Preparation of PCR reaction system Dilute to 10 10 Use 5PC3 positive standard or 5NC3 negative standard per copy / mL as the template to be tested, and prepare the PCR reaction solution according to the system shown in Table 2.

[0057] Table 2

[0058]

[0059] (3) PCR amplification program Vortex the prepared PCR reaction solution, centrifuge briefly (3000 rpm, 1 minute), and place it in a PCR instrument. Perform the amplification reaction according to the following program: pre-denaturate at 95℃ for 2 minutes; then proceed to the cycling stage, each cycle consisting of: denaturation at 95℃ for 30 seconds, annealing at 57℃ for 30 seconds, and extension at 72℃ for 30 seconds, for a total of 30 cycles; after the cycling is completed, perform a final extension at 72℃ for 5 minutes; finally, incubate the reaction system at 12℃ to complete the PCR amplification process, obtaining PCR products with 5PC3 template and PCR products with 5NC3 template, respectively.

[0060] (4) Purification of PCR products Add 5 μL of PCR amplification product to a new 1.5 mL microcentrifuge tube, then add 2 μL of ExoSAP-IT reagent. Vortex for 10 seconds, then briefly centrifuge to bring the liquid to a boil. Place the tube in a PCR instrument and incubate under the following conditions: 37°C for 15 minutes to degrade residual primers and dNTPs, then 80°C for 15 minutes to inactivate excess ExoSAP-IT reagent. The purified PCR product can be used directly for subsequent ligation reactions.

[0061] 3. Specific linker reaction: (1) Preparation of working solution for ligation primers Take 10 μL each of the specific upstream primer and downstream primer stock solutions corresponding to the methylation sites of PPP1R16B, BCAT1, and IKZF1, and add a total of 60 μL to a 1.5 mL microcentrifuge tube. Add 140 μL of TE buffer (pH 8.0), vortex for 30 seconds, and centrifuge briefly (3000 rpm, 1 minute) to obtain the ligation reaction primer working solution with a final concentration of 5 pmol / mL for each primer. Store at 4℃ for later use.

[0062] (2) Preparation of the connecting reaction system Using purified PCR products with 5PC3 or 5NC3 as templates, prepare ligation reaction systems according to the systems shown in Table 3.

[0063] Table 3

[0064]

[0065] (3) Connecting the reaction program After vortexing the ligation reaction mixture to mix thoroughly, perform a brief centrifugation (3000 rpm, 1 minute), and then place it in a PCR instrument to execute the following program: First, the reaction system was pre-denatured at 96℃ for 2 minutes; then, the cyclic amplification stage was entered, with each cycle consisting of denaturation at 94℃ for 15 seconds and annealing at 37℃ for 1 minute. This cycle was repeated 30 times. After the cycle, the reaction system was kept at 12℃ to complete the ligation reaction, yielding ligation products with 5PC3 as a template and ligation products with 5NC3 as a template, respectively.

[0066] 4. Tag-based hybridization enrichment: The three types of carrier magnetic beads corresponding to the methylation sites of PPP1R16B, BCAT1 and IKZF1 obtained in Example 1 were mixed to make the total number of the three types of magnetic beads in the resulting hybridization magnetic bead mixture reach 2500, ensuring that the number of each type of magnetic bead is relatively balanced.

[0067] The ligation product obtained using 5PC3 or 5NC3 as a template was hybridized with a mixture of hybridization magnetic beads. A 200 μL centrifuge tube was filled with the following components: 5 μL of the ligation product, 35 μL of 2×Tm buffer, 4 μL of the hybridization magnetic bead mixture, and 16 μL of enzyme-free water. The mixture was vortexed for 30 seconds and then briefly centrifuged (3000 rpm, 1 minute). The centrifuge tube was then placed in a PCR instrument and hybridization was performed under the following conditions: denaturation at 96℃ for 90 seconds, followed by isothermal hybridization at 37℃ for 20 minutes. This allowed the ligation product to specifically bind to the anchoring tag sequence on the vector via its own tag sequence, thus adsorbing onto the vector surface and achieving efficient enrichment of the target ligation product.

[0068] 5. Detect the marker signal: After the hybridization reaction was complete, remove the centrifuge tube, gently invert it five times to mix, and immediately place it on the Luminex 200 liquid chromatography-chip platform for detection. The detection procedures and instrument parameter settings were strictly performed in accordance with the Luminex 200 instrument operating manual.

[0069] The test results are shown in Table 4.

[0070] Table 4

[0071]

[0072] As can be seen from the data in Table 4, the primers and magnetic bead-anchored tag sequences of the three genes in the detection method of this embodiment can specifically distinguish them, indicating that the method has good specificity.

[0073] The fluorescence intensity (MFI) values ​​of the ligation products using 5PC3 as a template were compared with those of the ligation products using 5NC3 as a template. Combined with experimental repeatability verification and data statistical analysis, a unified and rigorous result judgment standard was set: if the MFI value of a certain gene locus in the sample to be tested is ≥100, it indicates that there is a specific hybridization reaction at that locus, and the gene methylation site is judged to be positive; if the MFI value of a certain gene locus in the sample to be tested is <100, no specific hybridization signal is generated, and the gene methylation site is judged to be negative.

[0074] Example 3

[0075] This embodiment verifies that the multi-gene target site joint detection method based on target enrichment of the present invention can effectively distinguish between positive and negative samples. Using methylation sites of PPP1R16B, BCAT1, and IKZF1 genes as detection targets, and using the specific amplification primer pairs, specific ligation primer pairs, and vector provided in Example 1, the joint detection of multiple gene target sites is performed on positive standard samples containing target gene methylation sites and negative standard samples without target gene methylation sites.

[0076] I. Sample DNA Extraction Five positive standard samples containing methylation sites of the PPP1R16B, BCAT1, and IKZF1 genes were numbered C1-C5, and five negative standard samples not containing methylation sites of the above target genes were numbered H1-H5. All samples were standard control samples commonly used in the field of gene testing.

[0077] Genomic DNA was extracted from 5 positive standard samples and 5 negative standard samples using the paraffin-embedded tissue DNA extraction kit (catalog number: DP331) produced by Tiangen Biotech (Beijing) Co., Ltd., following the instructions in the kit's manual. The resulting DNA samples were then processed.

[0078] II. DNA bisulfite conversion Using the DNA bisulfite conversion kit (catalog number: D5005) manufactured by Zymo Research, the extracted DNA samples were subjected to bisulfite conversion according to the kit instructions to achieve the conversion of unmethylated cytosine to uracil while retaining the base characteristics of methylated cytosine.

[0079] III. First PCR Amplification Using the transformed DNA sample as a template, and the PCR primer working solution containing the upstream primer and the corresponding downstream primer for methylation site specific amplification of PPP1R16B, BCAT1 and IKZF1 prepared in Example 2 as amplification primers, positive standard 5PC3 and negative standard 5NC3 were set as controls to construct the PCR amplification system as shown in Table 5.

[0080] Table 5

[0081]

[0082] After mixing the above components and shaking them thoroughly, the mixture was briefly centrifuged and then placed in a PCR instrument to perform the following amplification program: pre-denaturation at 95°C for 2 minutes; followed by a cycling phase, each cycle consisting of: denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles; after the cycling phase, a final extension was performed at 72°C for 5 minutes; finally, the reaction system was incubated at 12°C to complete the PCR amplification process, yielding PCR amplification products using each test sample as a template.

[0083] Take 5 μL of each PCR amplification product, add 2 μL of ExoSAP-IT reagent, and incubate at 37℃ for 15 minutes to remove residual primers and dNTPs, then incubate at 80℃ for 15 minutes to inactivate ExoSAP-IT reagent to obtain purified PCR products of each sample to be tested, which can be used directly for subsequent reactions.

[0084] IV. Specific Linkage Reaction Using the PCR products of each sample to be tested as templates, the ligation primer working solution containing PPP1R16B, BCAT1 and IKZF1 methylation site-specific upstream primers and corresponding downstream primers prepared in Example 2 was used as ligation primers to construct the ligation reaction system shown in Table 6.

[0085] Table 6

[0086]

[0087] After mixing the above components and vortexing them thoroughly, perform a brief centrifugation and then place them in a PCR instrument to execute the following procedure: First, the reaction system was pre-denatured at 96℃ for 2 minutes; then, the cyclic amplification stage was entered, with each cycle consisting of denaturation at 94℃ for 15 seconds and annealing at 37℃ for 1 minute. This cycle was repeated 30 times. After the cycle, the reaction system was kept at 12℃ to complete the ligation reaction, and ligation products were obtained using each test sample as a template.

[0088] V. Tag-based hybridization enrichment and detection Take 5 μL of each ligation product, add 35 μL of 2×Tm buffer, then add 4 μL of the mixed hybridization magnetic bead solution prepared in Example 2 containing the three vector magnetic beads corresponding to the methylation sites of PPP1R16B, BCAT1, and IKZF1, and finally add 16 μL of ddH2O. After mixing and shaking evenly, place in a PCR instrument and perform the following hybridization program: denaturation at 96℃ for 90 seconds, and isothermal hybridization at 37℃ for 20 minutes. This allows the ligation product to specifically bind to the anchor tag sequence on the vector through its own tag sequence and adsorb onto the vector surface, thus achieving efficient enrichment of the target ligation product.

[0089] The hybridization products of each sample were placed on a Luminex 200 liquid crystal chip platform. The detection parameters were set according to the Luminex 200 operating instructions, and the average fluorescence intensity (MFI) of the corresponding gene loci for each sample was obtained. The detection results are shown in Table 7.

[0090] Table 7

[0091]

[0092] Using the MFI value of the negative standard 5NC3 as a reference, and combining experimental repeatability verification and data statistical analysis, a unified and rigorous result judgment standard was set: if the MFI value of a certain gene locus in the sample to be tested is ≥100, it indicates that there is a specific hybridization reaction at that locus, and the gene methylation site is judged to be positive; if the MFI value of a certain gene locus in the sample to be tested is <100, no specific hybridization signal is generated, and the gene methylation site is judged to be negative.

[0093] As shown in Table 7, the detection data indicates that in the five positive standard samples (C1~C5), each sample had at least two target gene loci with MFI values ​​≥100, meeting the positive criteria and demonstrating good consistency in positive detection. Among them, samples C1 and C3 had three positive loci, while samples C2, C4, and C5 each had two positive loci, fully demonstrating the high enrichment of methylation sites of the PPP1R16B, BCAT1, and IKZF1 genes in the positive standard samples, and verifying the efficient capture capability of this method for target methylation sites.

[0094] All target gene loci in the five negative standard samples (H1~H5) and negative standard 5NC3 had MFI values ​​<100, and were therefore judged as negative, with no false positive results. The MFI values ​​of the three target gene loci in positive standard 5PC3 were all significantly higher than 100, with stable and strong positive signals, further verifying the reliability and accuracy of the detection system. This indicates that the method can effectively distinguish between positive and negative samples of target gene methylation, with good specificity and sensitivity.

[0095] The above results fully demonstrate that the efficient multi-gene target site enrichment joint detection method constructed in this invention can effectively distinguish between target gene methylation positive and negative samples, has the advantages of high specificity and high sensitivity, can accurately capture target gene methylation signals, and provides a scientific and reliable multi-gene site joint detection technology solution for the field of gene detection, solving the technical problems of insufficient specificity and difficulty in achieving multiple simultaneous detection in traditional detection methods.

Claims

1. A multi-gene locus joint detection method based on target enrichment, characterized in that, Includes the following steps: Step 1: Design specific amplification primer pairs for each target site of multi-gene detection. Using the nucleic acid of the sample to be tested as a template, add all specific amplification primer pairs simultaneously to perform a single amplification reaction to obtain a single amplification product containing multiple gene target sites. Step 2: Design specific ligation primer pairs for each target site amplification product, with the upstream primer having a tag sequence at the 5' end and the downstream primer having a tag at the 5' end and a phosphorylation modification at the 3' end; The amplification product, all specific ligation primer pairs, and ligase are mixed. The single strands obtained by pre-denaturing and de-stranding the amplification product are annealed with the specific ligation primer pairs to form a continuous aligned structure. The ligase catalyzes the covalent ligation of the upstream and downstream primers of the specific ligation primer pairs to form a complete ligation product. Step 3: Use a vector coupled with an inversely complementary anchor tag sequence to enable the ligation product to specifically bind to the vector through the tag sequence, thereby achieving enrichment of the target ligation product. Step 4: Detect the label signal of the ligation product on the vector. By analyzing the presence, intensity, and corresponding tag sequence of the signal, qualitative and quantitative analysis of multiple gene target sites can be achieved.

2. The multi-gene locus joint detection method based on target enrichment according to claim 1, characterized in that, The sample to be tested in step one is one or more of the following: blood sample, plasma sample, FFPE sample, tissue sample, fecal sample, or urine sample, which are processed in vitro for non-diagnostic purposes and used for gene locus analysis.

3. The multi-gene locus joint detection method based on target enrichment according to claim 2, characterized in that, In step two, the upstream primers of the specific ligation primer pairs corresponding to different target sites have different tag sequences, and the tag sequences have no homology with the sample nucleic acid.

4. The multi-gene locus joint detection method based on target enrichment according to claim 3, characterized in that, The 5' end of the tag sequence described in step two is modified with a functional group, which is an amino, carboxyl, thiol, aldehyde, or azide group.

5. The multi-gene locus joint detection method based on target enrichment according to claim 4, characterized in that, The functional group modification is directly or indirectly connected to the 5' end of the tag sequence. The direct connection is that the functional group is covalently connected to the nucleotide at the 5' end of the tag sequence, and the indirect connection is that the functional group is indirectly connected to the 5' end of the tag sequence through a short linker arm.

6. A multi-gene locus joint detection method based on target enrichment according to any one of claims 1-5, characterized in that, The labeling described in step two enables the ligation product to develop color directly upon irradiation with excitation light of the corresponding wavelength, or to develop color immediately upon addition of the matching reaction substrate or affinity ligand.

7. The multi-gene locus joint detection method based on target enrichment according to claim 6, characterized in that, The labeling described in step two is fluorescent labeling and / or biotin.

8. A multi-gene locus joint detection method based on target enrichment according to any one of claims 1-5, characterized in that, The carrier described in step three is a solid-phase carrier and / or a liquid-phase carrier. The solid-phase carrier is a magnetic bead, a microporous plate, or a glass slide; the liquid-phase carrier is a nanoparticle dispersion, a polymer solution, or a biomacromolecule solution.

9. The multi-gene locus joint detection method based on target enrichment according to claim 8, characterized in that, Step 3 describes a carrier surface with active modifying groups, which are carboxyl, amino, or N-hydroxy-succinimide groups; the carrier and the anchoring tag sequence are coupled through covalent bonds mediated by the active modifying groups, which are amide bonds, ester bonds, or disulfide bonds.

10. A multi-gene locus joint detection method based on target enrichment according to any one of claims 1-5, characterized in that, The multi-gene target sites are specifically distinguished by at least one of the following two methods: (1) The types of marker signals carried by the ligation products corresponding to each target site are different; (2) The types of carriers that carry the ligation products corresponding to each target site are different.

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