A multi-gene target site joint detection method

Abstract

The present application relates to a kind of multi-gene purpose site combined detection method, belong to gene detection technical field.To solve the problems of insufficient specificity, high background value, complicated operation and low detection efficiency of existing multi-gene parallel detection technology, the present application provides a kind of multi-gene purpose site combined detection method, steps include single system connection reaction, single system amplification reaction, hybridization enrichment and signal detection.The present application realizes the significant improvement of detection efficiency and the synchronous reduction of cost through innovative single system multi-gene parallel detection design.By designing multiple specific primer combinations, different targets can complete the connection amplification in the same reaction system, realize the specific separation and fixation of different target nucleic acid purpose sites, completely solve the cross interference problem in multi-gene purpose site detection, with the advantages of high specificity, low background value, high efficiency and low cost, can be widely used in gene detection related fields.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and in particular relates to a method for joint detection of multiple gene target sites and its application. Background Technology

[0002] In the field of gene testing, samples often exhibit the coexistence of multiple target nucleic acid sequences. Parallel specific detection of multiple targets is not only necessary to ensure the comprehensiveness of results but also crucial for improving testing efficiency and controlling costs. In gene testing, simultaneous screening of multiple sequences and parallel analysis of multiple gene loci require the simultaneous acquisition of information on multiple targets to avoid missed detections. Food and environmental testing also require the simultaneous screening of nucleic acid sequences related to multiple pollutants or indicator organisms. Single-target detection is insufficient to meet these needs; parallel detection can complete multi-target analysis within a single system, significantly optimizing testing efficiency.

[0003] Nucleic acid amplification and hybridization-based detection methods are mainstream technologies, achieving detection by amplifying the target sequence with primers and binding it to a labeled signal. However, this type of technology has significant drawbacks in parallel detection of multiple genes: First, insufficient amplification specificity, as co-amplification with multiple primer pairs easily leads to primer dimers or non-specific products, interfering with detection results; second, high background signal values, as non-specific binding in traditional labeling methods causes signal superposition, reducing discriminative power; and third, cumbersome operation, requiring the design of independent systems for different targets, resulting in low efficiency and high cost. Furthermore, traditional hybridization exhibits low specificity in the binding of vectors to amplified products, and cross-hybridization further reduces detection accuracy.

[0004] In summary, existing technologies are insufficient to simultaneously achieve the high specificity, low background value, and high efficiency required for parallel detection of multiple genes, and cannot meet the practical application needs of various fields. There is an urgent need to develop a method for joint detection of multiple gene target sites that can be performed in parallel using a single system to solve the above-mentioned technical problems. Summary of the Invention

[0005] To address the problems of insufficient specificity, high background values, cumbersome operation, and low detection efficiency in existing multi-gene parallel detection technologies, this invention provides a non-diagnostic multi-gene target site joint detection method and its application.

[0006] The technical solution of the present invention:

[0007] A method for joint detection of multiple gene target sites includes the following steps:

[0008] Step 1, Single-system linkage reaction:

[0009] Based on various target nucleic acid sites, multiple sets of specific ligation primer pairs were designed. Each primer pair consisted of a specific upstream ligation primer and a specific downstream ligation primer, and the 5' end of the specific downstream ligation primer was connected to a non-specific universal primer sequence.

[0010] Using the nucleic acid of the sample to be tested as a template, all specific ligation primer pairs, ligase and ligation reaction buffer are added. The reaction system is heated to the pre-denaturation temperature to unwind the double-stranded nucleic acid in the mixed sample template into a single-stranded state, exposing the target sites of each target nucleic acid and their upstream and downstream specific sequence regions, providing identifiable target sites for primer binding. Then the temperature of the reaction system is lowered to the primer annealing temperature to initiate the specific binding of multiple sets of specific ligation primer pairs to the single-stranded template.

[0011] The multiple sets of specific ligation primer pairs correspond one-to-one with various target nucleic acid sites in the template. Each primer pair consists of a specific upstream ligation primer and a specific downstream ligation primer. The nucleotide sequence of the specific upstream ligation primer is completely complementary to the upstream specific region of the corresponding target nucleic acid site, and can accurately identify and bind to that region. The 3' end nucleotide sequence of the specific downstream ligation primer is completely complementary to the downstream specific region of the corresponding target nucleic acid site, and can also accurately bind to that region. Its 5' end is pre-linked with a non-specific universal primer sequence that is not complementary to any segment of the template sequence. Only when a set of specific ligation primer pairs simultaneously and accurately bind to the upstream and downstream regions of the same target nucleic acid site can a continuous aligned structure of "specific upstream ligation primer - target nucleic acid site - specific downstream ligation primer" be formed on the single-stranded template.

[0012] The temperature of the ligation reaction system is then adjusted to the optimal reaction temperature of the ligase. The ligase specifically recognizes the phosphodiester bond gap at the junction of the primer and the target nucleic acid, catalyzing the formation of stable covalent bonds between the nucleotides on both sides of the gap. This covalently links the upstream specific ligation primer, the target nucleic acid fragment, and the downstream specific ligation primer to form a complete ligation product. After the ligation reaction, a mixture of ligation products is formed in the system. Each ligation product in this mixture is a single-stranded nucleic acid fragment with specific sequences at both ends and containing the target nucleic acid site in the middle, and it is still attached to the template, providing a precise and stable template substrate for subsequent amplification reactions. Miscellaneous nucleic acids that do not bind primer pairs or fragments that only bind a single primer cannot form a complete ligation structure and cannot be used as effective templates for subsequent amplification, thus improving the specificity of the entire detection process from the source.

[0013] Step 2: Single-system amplification reaction:

[0014] Based on the product structure of the specific ligation reaction in step one and the nucleotide sequences of multiple sets of specific ligation primer pairs, multiple sets of specific amplification upstream primers and one set of universal amplification downstream primers are designed. The 5' ends of the multiple sets of specific amplification upstream primers are linked with tag sequences, and the universal amplification downstream primers carry detectable labeling groups.

[0015] Using the ligation product mixture obtained in step one as a template, multiple sets of specific amplification upstream primers and one set of universal amplification downstream primers are added. Specific amplification is performed in the PCR reaction system. The multiple sets of specific amplification upstream primers only recognize and bind to the corresponding upstream primer sequence region of the ligation product obtained in step one. The universal amplification downstream primer is completely complementary to the non-specific universal primer sequence connected to the 5' end of the downstream primer of the ligation product obtained in step one. Only when the specific amplification upstream primer and the universal amplification downstream primer bind to both ends of the same ligation product can an effective amplification initiation complex be formed, and an amplification product mixture is obtained through the amplification reaction.

[0016] Step 3: Hybridization enrichment reaction:

[0017] The amplification product mixture obtained in step two is hybridized with a vector coupled with multiple reverse complementary anchoring tag sequences. The amplification products corresponding to different target nucleic acid sites in the amplification product mixture bind complementaryly to the corresponding reverse complementary anchoring tag sequences through their respective tag sequences, and are respectively fixed on the surface of the vector to form hybridization enrichment.

[0018] Step 4: Signal Detection

[0019] The vector after hybridization in step three is tested. By identifying the presence, intensity, and corresponding tag sequence of the marker signal, the simultaneous qualitative or quantitative detection of multiple target nucleic acid sites can be achieved.

[0020] Furthermore, in step one, the nucleotide sequences of the upstream primers of the multiple sets of specific connecting primer pairs are completely complementary to the upstream specific regions of the corresponding target nucleic acid target sites; the 3' end nucleotide sequences of the downstream primers are completely complementary to the downstream specific regions of the corresponding target nucleic acid target sites.

[0021] Furthermore, the 5' tag sequence of the multiple sets of specific amplification upstream primers in step two is a specific nucleotide sequence of 15-30 bp in length, and this tag sequence does not have complementary binding ability with the target nucleic acid sequence or the universal primer sequence.

[0022] Furthermore, the detectable label carried by the universal amplification downstream primer in step two is one of a fluorescent label, a radioactive label, an enzyme label, or a biotin label.

[0023] Furthermore, the carrier mentioned in step three is a solid-phase carrier, specifically including nitrocellulose membrane, nylon membrane, supermagnetic polystyrene microspheres, or glass sheet.

[0024] 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.

[0025] Furthermore, in step four, the corresponding detection method is selected according to the type of label. For fluorescent labels, the fluorescence signal intensity is detected using a fluorescence detector; for radioactive labels, the radioactivity intensity is detected using autoradiography or a liquid scintillation counter; for enzyme labels, the corresponding substrate is added, and signal detection is achieved by detecting the absorbance or fluorescence intensity of the substrate reaction product; for biotin labels, streptavidin is added, and signal detection is achieved by detecting the signal of the streptavidin-coupled group using a fluorescence detector or the absorbance or fluorescence intensity of the substrate reaction product.

[0026] The beneficial effects of this invention are:

[0027] This invention achieves a significant improvement in detection efficiency and a simultaneous reduction in cost through an innovative single-system, multi-gene parallel detection design. By designing multiple sets of specific primer combinations, different targets can be ligated and amplified in the same reaction system, eliminating the need to design separate detection systems for different gene target sites, simplifying the operation process while reducing sample and reagent consumption.

[0028] This invention significantly improves the specificity of multi-gene target site detection through a multi-specific screening design. In the first ligation reaction, multiple sets of specific primer pairs bind specifically to the corresponding target nucleic acid sequences and target sites, achieving preliminary specific screening of multiple target nucleic acid target sites. In the second amplification reaction, multiple sets of upstream primers with specific tag sequences bind specifically to the corresponding target sequences and target sites again, while the shared universal marker primer binds only to the universal sequence of the downstream primer, avoiding cross-binding and dimer formation between primers. In the third hybridization reaction, different amplification products bind to the corresponding reverse complementary anchoring tag sequence on the vector through a unique tag sequence, achieving specific separation and immobilization of different target nucleic acid target sites, completely solving the problem of cross-interference in multi-gene target site detection.

[0029] This invention links the labeling group to a universal primer rather than a specific primer. This avoids the influence of the labeling group on the binding efficiency of the specific primer to the target nucleic acid sequence, thus improving the efficiency of the amplification reaction. Furthermore, since all amplification products share the same labeling group, there is no need to label multiple sets of specific primers separately, reducing labeling costs. At the same time, it avoids background signal superposition caused by multiple labels, thus improving the clarity of signal detection.

[0030] The detection method of the present invention has clear operation steps, mild reaction conditions, and can flexibly adjust the number of target gene loci according to detection needs, adapting to different multi-gene target locus detection scenarios, and has broad application prospects. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] This embodiment provides a set of specific ligation primer pairs, a set of specific amplification upstream primers, a set of universal amplification downstream primers, and a vector for a multi-gene target site joint detection method.

[0034] The methylation sites of open reading frame 50 (C9orf50) on chromosome 9 are mostly located in the CpG islands and 5'UTR region of the promoter region, making them epigenetic targets. The core methylation site of Septin9 (SEPT9) is concentrated in CpG island 3 (CGI3) of the V2 spliceosome promoter region, making it a highly specific methylation target. Icarus family zinc finger protein 1 (IKZF1) is a transcriptional regulation-related gene, and its methylation sites are mainly distributed in the promoter region, making it an epigenetic target suitable for multi-gene joint detection. It should be noted that the epigenetic characteristic detection of the above gene targets is not for disease diagnosis purposes, but only for research purposes related to multi-gene joint detection targets.

[0035] I. This embodiment uses the methylation sites of C9orf50, SEPT9 and IKZF1 genes as target sites and provides multiple sets of specific linker pairs designed for the methylation sites of C9orf50, SEPT9 and IKZF1.

[0036] The nucleotide sequence of the upstream primer specifically linked to the C9orf50 methylation site is shown in SEQ ID No:1, specifically 5'-GTTTGGGCGTCGATTTTC-3'. The nucleotide sequence of the downstream primer specifically linked to the C9orf50 methylation site is shown in SEQ ID No:2. The 5' end to the 3' end are the non-specific universal primer sequence and the specific downstream primer sequence, respectively. The nucleotides from position 1 to position 20 at the 5' end are the non-specific universal primer sequence, and the nucleotides from position 21 to the 3' end are the specific downstream primer sequence. The 3' end is phosphorylated, specifically 5'-TCGGTGGTCGCCGTATCATTCGACCGACGCGTCCC-P-3'.

[0037] The nucleotide sequence of the SEPT9 methylation site-specific upstream primer is shown in SEQ ID No:3, specifically 5'-TATTAGTTATTATGTCGGATTTCG-3'. The nucleotide sequence of the SEPT9 methylation site-specific downstream primer is shown in SEQ ID No:4. The 5' end to the 3' end are the non-specific universal primer sequence and the specific downstream primer sequence, respectively. The nucleotides from position 1 to position 20 at the 5' end are the non-specific universal primer sequence, and the nucleotides from position 21 to the 3' end are the specific downstream primer sequence. The 3' end is phosphorylated, specifically 5'-TCGGTGGTCGCCGTATCATTCCAACTACGCGTTAACCG-P-3'.

[0038] The nucleotide sequence of the upstream primer specifically linked to the IKZF1 methylation site is shown in SEQ ID No:5, specifically 5'-TCGTGTTTCGTTTTGCG-3'. The nucleotide sequence of the downstream primer specifically linked to the IKZF1 methylation site is shown in SEQ ID No:6. The 5' end to the 3' end are the non-specific universal primer sequence and the specific downstream primer sequence, respectively. The nucleotides from position 1 to position 20 at the 5' end are the non-specific universal primer sequence, and the nucleotides from position 21 to the 3' end are the specific downstream primer sequence. The 3' end is phosphorylated, specifically 5'-TCGGTGGTCGCCGTATCATTCGAAACGCGCAAAAAAA-P-3'.

[0039] The three sets of specific ligation primer pairs in this embodiment have phosphorylation modification at the 3' end of the downstream primer. All primers were synthesized by Genewiz Biotechnology Co., Ltd., and 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).

[0040] 2. Based on the product structure of the specific ligation reaction in step one and the nucleotide sequences of multiple sets of specific ligation primer pairs, design multiple sets of specific amplification upstream primers and one set of universal amplification downstream primers.

[0041] The specific amplification upstream primer corresponding to the C9orf50 methylation site consists of the tag sequence -C9orf50, iSpC18 (18-atom-spaced phosphoramidite linker), and the upstream primer sequence -C9orf50 from 5' to 3'. The nucleotide sequence of the tag sequence -C9orf50 is shown in SEQ ID No:7, specifically 5'-CTTTCTCATACTTTCAACTAATTT-3', and the nucleotide sequence of the upstream primer sequence -C9orf50 is shown in SEQ ID No:8, specifically 5'-GTTTGGGCGTCGATTTTC-3'.

[0042] The specific amplification upstream primer corresponding to the SEPT9 methylation site consists of the tag sequence -SEPT9, iSpC18, and upstream primer sequence -SEPT9 at the 5' to 3' ends, respectively. The nucleotide sequence of the tag sequence -SEPT9 is shown in SEQ ID No:9, specifically 5'-ACTACTTATTCTCAAACTCTAATA-3', and the nucleotide sequence of the upstream primer sequence -SEPT9 is shown in SEQ ID No:10, specifically 5'-TATTAGTTATTATGTCGGATTTCG-3'.

[0043] The specific amplification upstream primer corresponding to the IKZF1 methylation site consists of the tag sequence -IKZF1, iSpC18, and upstream primer sequence -IKZF1 from 5' to 3'. The nucleotide sequence of the tag sequence -IKZF1 is shown in SEQ ID No:11, specifically 5'-CATCTTCATATCAATTCTCTTATT-3', and the nucleotide sequence of the upstream primer sequence -IKZF1 is shown in SEQ ID No:12, specifically 5'-TCGTGTTTCGTTTTGCG-3'.

[0044] A set of universal amplification downstream primers has the following nucleotide sequence as shown in SEQ ID No:13, with a biotinylated label at the 5' end, specifically 5'-biotin-AATGATACGGCGACCACCGA-3'.

[0045] In this embodiment, the upstream primer and the universal downstream primer were 18-25 bp in length, with a GC content of 40%-60%. There was no complementary pairing between the primers, and they did 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).

[0046] 3. Design reverse complementary tag sequences based on the tag sequences of the upstream primers for specific amplification, and couple them with the vector magnetic beads.

[0047] 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.

[0048] In this embodiment, the vector corresponding to the C9orf50 methylation site uses magnetic beads of number 20. The nucleotide sequence of the reverse complementary anchor tag sequence coupled on the magnetic beads is shown in SEQ ID No:14, specifically 5'-AAATTAGTTGAAAGTATGAGAAAG-3'.

[0049] The vector corresponding to the SEPT9 methylation site uses magnetic beads number 33. The nucleotide sequence of the reverse complementary anchor tag sequence coupled to the magnetic beads is shown in SEQ ID No:15, specifically 5'-TATTAGAGTTTGAGAATAAGTAGT-3'.

[0050] The vector corresponding to the IKZF1 methylation site uses magnetic beads of number 35. The nucleotide sequence of the reverse complementary anchor tag sequence coupled to the magnetic beads is shown in SEQ ID No:16, specifically 5'-AATAAGAGAATTGATATGAAGATG-3'.

[0051] All anchor tag sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized tag sequences were prepared into a 0.1 nM stock solution using nuclease-free water for later use.

[0052] The method for coupling the anchor tag sequence to the vector is as follows:

[0053] 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).

[0054] 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 LEDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, purchased from Thermo) 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.

[0055] Example 2

[0056] This embodiment provides a method for joint detection of multiple gene target sites targeting methylation sites of C9orf50, SEPT9 and IKZF1 genes, based on multiple sets of specific ligation primer pairs, multiple sets of specific amplification upstream primers, a set of universal amplification downstream primers and vectors provided in Example 1, and its specificity verification.

[0057] I. Experimental Materials

[0058] 1. Standard quality granules

[0059] Positive standard plasmid PC3, containing the target region after bisulfite transformation of the methylation sites of C9orf50, SEPT9 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.

[0060] 2. Main reagents

[0061] High-specificity Taq Pro multiplex DNA polymerase: purchased from Nanjing Novizan Biotechnology Co., Ltd., product number Vazyme-PM202-01;

[0062] T4 DNA ligase: purchased from NEB, catalog number M0202S;

[0063] Tris-HCl (hydroxymethyl)aminomethane hydrochloride: purchased from Beijing Solarbio Technology Co., Ltd., product number Solarbio T8230;

[0064] Polyethylene glycol p-isooctylphenyl ether (Triton X-100): purchased from Beijing Solarbio Technology Co., Ltd., product number Solarbio T8200;

[0065] Sodium chloride (NaCl): purchased from Tianjin University Chemical Reagent Center;

[0066] ExoSAP-IT™ PCR Product Purification Kit: Purchased from ABI, USA.

[0067] Hybrid magnetic bead mixture (Microspheres): purchased from Luminex;

[0068] 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.

[0069] 3. Preparation of dedicated buffer solution

[0070] The formulation of 2×Tm buffer is shown in Table 1.

[0071] Table 1

[0072]

[0073] The preparation method for 2×Tm buffer is as follows:

[0074] 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.

[0075] II. Experimental Methods

[0076] 1. Dilution of positive and negative standards for the target region

[0077] 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.

[0078] 2. Single-system linkage reaction

[0079] (1) Preparation of working solution for ligation primers

[0080] Take 10 μL each of the specific upstream primer and downstream primer stock solutions corresponding to the methylation sites of C9orf50, SEPT9, and IKZF1, and add a total of 60 μL to a 1.5 mL microcentrifuge tube. Add 140 μL of LTE 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.

[0081] (2) Preparation of the connecting reaction system

[0082] Dilute to 10 10 Use 5PC3 positive standard or 5NC3 negative standard per copy / mL as the template to be tested, and prepare the ligation reaction system according to the system shown in Table 2.

[0083] Table 2

[0084]

[0085] (3) Connecting the reaction program

[0086] 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:

[0087] 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 mixed ligation products with 5PC3 as a template and mixed ligation products with 5NC3 as a template, respectively.

[0088] 3. Single-system amplification reaction

[0089] (1) Preparation of PCR primer working solution

[0090] For each target gene site, take 10 μL each of the specific upstream primers and 30 μL of the universal downstream primers provided in Example 1 for amplification of the C9orf50, SEPT9, and IKZF1 methylation sites, and add them to a 1.5 mL microcentrifuge tube. Then add 140 μL of TE buffer (pH 8.0), vortex for 30 seconds, and centrifuge briefly (3000 rpm, 1 minute) to allow the liquid on the tube wall to settle to the bottom. This yields PCR primer working solutions with a final concentration of 5 pmol / mL for each primer. Store at 4°C for later use.

[0091] (2) Preparation of PCR reaction system

[0092] Using the mixed ligation product of 5PC3 or 5NC3 as a template, PCR reaction solution was prepared according to the system shown in Table 3.

[0093] Table 3

[0094]

[0095] (3) PCR amplification program

[0096] 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 amplification products with 5PC3 as template and 5NC3 as template, respectively.

[0097] (4) Purification of PCR products

[0098] 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 amplification product can be used directly for subsequent hybridization reactions.

[0099] 4. Tag-based hybridization enrichment:

[0100] The three types of carrier magnetic beads corresponding to the methylation sites of C9orf50, SEPT9 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.

[0101] The amplification products using 5PC3 or 5NC3 as templates were hybridized with a mixture of magnetic hybridization beads. A 200 μL centrifuge tube was filled with the following components: 5 μL of the ligation reaction product, 35 μL of 2×Tm buffer, 4 μL of the magnetic hybridization 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 amplified product to specifically bind to the anchoring tag sequence on the vector and adsorb onto the vector surface, achieving efficient enrichment of the target amplification.

[0102] 5. Detect the marker signal:

[0103] 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.

[0104] The test results are shown in Table 4.

[0105] Table 4

[0106]

[0107] 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.

[0108] The fluorescence intensity (MFI) values ​​obtained from amplification using 5PC3 as a template were compared with those obtained from amplification 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.

[0109] Example 3

[0110] This embodiment verifies that the multi-gene target site joint detection method of the present invention can effectively distinguish between positive and negative samples. The methylation sites of C9orf50, SEPT9 and IKZF1 genes are used as detection targets. The multiple sets of specific ligation primer pairs, multiple sets of specific amplification upstream primers, a set of universal amplification downstream primers and vectors provided in Example 1 are used to perform joint detection of multiple gene target sites on positive standard samples containing target gene methylation sites and negative standard samples without target gene methylation sites.

[0111] I. Sample DNA Extraction

[0112] Five positive standard samples containing methylation sites of the C9orf50, SEPT9, 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.

[0113] 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.

[0114] II. DNA bisulfite conversion

[0115] 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.

[0116] III. Single-system linkage reaction

[0117] The transformed DNA sample was used as the template to be tested. The ligation primer working solution containing the upstream primer and the corresponding downstream primer of C9orf50, SEPT9 and IKZF1 methylation site specific ligation prepared in Example 2 was used as the amplification primer. At the same time, positive standard 5PC3 and negative standard 5NC3 were set as controls. The ligation reaction system was prepared according to the system shown in Table 5.

[0118] Table 5

[0119]

[0120] (3) Connecting the reaction program

[0121] 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:

[0122] 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 each test sample and the mixed ligation product with 5PC3 or 5NC3 as a template were obtained.

[0123] IV. Single-system amplification reaction

[0124] Using the mixed ligation products of each test sample and 5PC3 or 5NC3 as templates, and the amplification primer working solution containing C9orf50, SEPT9 and IKZF1 methylation site-specific upstream primers and universal amplification downstream primers prepared in Example 2 as amplification primers, PCR reaction solutions were prepared according to the system shown in Table 6.

[0125] Table 6

[0126]

[0127] Vortex the prepared PCR reaction solution, briefly centrifuge (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 amplification products with mixed ligation products as templates.

[0128] 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 amplification product can be used directly for subsequent hybridization reactions.

[0129] V. Tag-based hybridization enrichment:

[0130] The purified amplification product was hybridized with a hybridization magnetic bead mixture. A 200 μL centrifuge tube was filled with the following components: 5 μL of the amplification product, 35 μL of 2×Tm buffer, 4 μL of the hybridization magnetic bead mixture containing the three carrier magnetic beads corresponding to the methylation sites of C9orf50, SEPT9, and IKZF1 prepared in Example 2, 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 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 amplification 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 amplification product.

[0131] 5. Detect the marker signal:

[0132] 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.

[0133] The test results are shown in Table 7.

[0134] Table 7

[0135]

[0136] 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.

[0137] As shown in Table 7, the detection data shows that in the five positive standard samples (C1~C5), each sample had at least two target gene loci with MFI values ​​≥100, which met the positive judgment criteria and showed good consistency in positive detection. Among them, samples C2, C4 and C5 were positive at all three loci, while samples C1 and C3 were positive at two loci each, which fully reflects the high enrichment characteristics of C9orf50, SEPT9 and IKZF1 gene methylation sites in the positive standard samples.

[0138] 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, and the positive signal was stable and strong, further verifying the reliability and accuracy of this detection system.

[0139] The above results fully demonstrate that the multi-gene target site joint detection method constructed in this invention can effectively distinguish between positive and negative samples, and has the advantages of high specificity and high sensitivity. It can accurately capture characteristic gene methylation signals in positive samples, providing a scientific and reliable molecular detection basis for gene detection, and solving the problems of insufficient specificity and difficulty in achieving multiple simultaneous detection in traditional detection methods.

Claims

1. A method for joint detection of multiple gene target sites, characterized in that, Includes the following steps: Step 1: Design multiple sets of specific ligation primer pairs based on various target nucleic acid sites. The downstream primer of each primer pair is connected to a non-specific universal primer sequence at its 5' end. Using the nucleic acid of the sample to be tested as a template, add all the specific ligation primer pairs and ligase. The template is unwound by pre-denaturation. After annealing, the primers bind to the single-stranded template to form a continuous aligned structure, and a mixture of complete ligation products is formed under the action of ligase. Step 2: Design multiple sets of specific amplification upstream primers with tagged sequences at the 5' end and a set of universal amplification downstream primers carrying detectable markers. Use the ligation product mixture as a template for PCR specific amplification. An effective amplification complex is formed only when the upstream and downstream amplification primers bind to the corresponding ligation products at the same time, and an amplification product mixture is obtained. Step 3: Hybridize the amplified product mixture with a vector coupled with a reverse complementary anchoring tag, and fix different target amplified products on the surface of the vector through complementary binding of tag sequences; Step 4: Detect the hybridized vector. By identifying the presence, intensity, and corresponding tag sequence of the labeled signal, simultaneous qualitative or quantitative detection of multiple target nucleic acid sites can be achieved.

2. The method for joint detection of multiple gene target sites according to claim 1, characterized in that, In step one, the nucleotide sequences of the upstream primers of the multiple sets of specific ligation primer pairs are completely complementary to the upstream specific regions of the corresponding target nucleic acid target sites; the 3' end nucleotide sequences of the downstream primers are completely complementary to the downstream specific regions of the corresponding target nucleic acid target sites.

3. The method for joint detection of multiple gene target sites according to claim 1 or 2, characterized in that, The tag sequence described in step two is a specific nucleotide sequence of 15-30 bp in length, and the tag sequence does not have complementary binding ability with the target nucleic acid sequence or the universal primer sequence.

4. The method for joint detection of multiple gene target sites according to claim 3, characterized in that, The detectable label carried by the universal amplification downstream primer in step two is one of fluorescent label, radioactive label, enzyme label, or biotin label.

5. The method for joint detection of multiple gene target sites according to claim 4, characterized in that, The carrier mentioned in step three is a solid-phase carrier, specifically including nitrocellulose membrane, nylon membrane, supermagnetic polystyrene microspheres, or glass sheet.

6. The method for joint detection of multiple gene target sites according to claim 5, 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.

7. The method for joint detection of multiple gene target sites according to claim 6, characterized in that, Step four involves selecting the appropriate detection method based on the type of label. For fluorescent labels, a fluorescence detector is used to detect the fluorescence signal intensity; for radioactive labels, autoradiography or a liquid scintillation counter is used to detect the radioactivity intensity; for enzyme labels, the corresponding substrate is added, and signal detection is achieved by detecting the absorbance or fluorescence intensity of the substrate reaction product; for biotin labels, streptavidin is added, and signal detection is achieved by detecting the signal of the streptavidin-coupled group using a fluorescence detector or the absorbance or fluorescence intensity of the substrate reaction product.

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