A capture probe, magnetic bead extraction reagent and nucleic acid extraction method

Abstract

The application belongs to the technical field of nucleic acid target capture, and particularly relates to a capture probe, a magnetic bead extraction reagent and a nucleic acid extraction method. The magnetic bead extraction reagent for extracting nucleic acid based on the magnetic bead method is based on a traditional magnetic bead method reagent system, and a DNA capture probe with a universal double-grabbing structure is designed. The magnetic bead for grabbing nucleic acid does not inhibit a PCR reaction, can be put into a PCR reaction system to participate in the PCR reaction, nucleic acid on the magnetic bead does not need to be eluted, and automatic operation is easier.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid targeted capture technology, specifically relating to a capture probe, magnetic bead extraction reagent, and nucleic acid extraction method. Background Technology

[0002] Nucleic acids are the carriers of genetic information and the most important biological information molecules, making them a primary focus of molecular biology research. Therefore, nucleic acid extraction is the most important and fundamental operation in molecular biology experimental techniques. With the rapid development of molecular biology techniques, nucleic acid research and analysis are increasingly being promoted and applied in fields such as clinical diagnosis, environmental monitoring, and food safety testing, playing a significant role. Currently, magnetic bead-based nucleic acid extraction has unparalleled advantages over traditional DNA extraction methods, primarily in its ability to achieve automated, high-volume operations, meeting the high-throughput requirements of biology and enabling rapid and timely responses to outbreaks of infectious diseases.

[0003] Magnetic bead-based nucleic acid extraction is a novel nucleic acid extraction technology that uses nano-biological magnetic beads as carriers. Magnetic bead-based nucleic acid extraction generally includes four main steps: lysis, binding, washing, and elution. Each step can be achieved individually or in combination by various different methods. To improve viral nucleic acid recovery efficiency, the nucleic acid extraction reagent in the lysis step is crucial in determining the quality of nucleic acid extraction. However, the extraction effect of existing nucleic acid extraction reagents is relatively average, resulting in reduced viral nucleic acid recovery efficiency, increased extraction time, and decreased work efficiency. Therefore, there is a need in this field to develop more specific capture probes and extraction systems to effectively improve the extraction efficiency of viral nucleic acids. Summary of the Invention

[0004] The first objective of this invention is to provide a universal dual-trap DNA capture probe, which has better specificity in capturing nucleic acids, and the magnetic beads do not inhibit the PCR reaction. It can be placed in the PCR reaction system to participate in the PCR reaction without eluting the nucleic acids on the magnetic beads, thus effectively saving process steps.

[0005] The second objective of this invention is to provide a magnetic bead extraction reagent system based on a DNA capture probe with a universal dual-trap structure;

[0006] The third objective of this invention is to provide a nucleic acid extraction method for extracting target DNA using magnetic beads based on the aforementioned magnetic bead extraction reagent.

[0007] To address the aforementioned technical problems, this invention provides a capture probe, which is a dual-capture structure probe with the following sequence structure:

[0008] Target C1-poly (CGT) 5-15-Target C2; where,

[0009] The sequence of Target C1 is as follows:

[0010] TTTTTTTTCTTTTCTTTTCTTTCTTTCTTTCTTTTCTTTTCTTTTTTT;

[0011] The sequence of Target C2 is as follows:

[0012] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0013] Preferably, the capture probe has the following sequence structure: Target C1-poly (CGT) 8-12 -Target C2; a more preferred structure is Target C1-poly(CGT). 10 -Target C2.

[0014] The present invention also provides a magnetic bead extraction reagent for extracting nucleic acids based on the magnetic bead method, comprising the aforementioned capture probe and corresponding magnetic beads.

[0015] Specifically, the general grasping sequence of the magnetic bead is poly(GCA). 4-14 The preferred sequence is poly(GCA). 7-11 The preferred sequence is poly(GCA)9.

[0016] Specifically, the extraction reagents include: lysis binding buffer, first washing buffer, second washing buffer, elution buffer, the capture probe and corresponding magnetic beads, protease and protein digestion solution.

[0017] Specifically, the extraction reagent includes:

[0018] The lysis binding solution comprises: a guanidine salt compound, a surfactant, a buffer solution, EDTA, and a thiol reducing agent; wherein the final application concentrations of each component in the lysis binding solution are as follows: 3-8 mol / L guanidine salt compound, 0.2-5% surfactant, 5-50 mmol / L buffer solution, 10-50 mmol / L EDTA, and 10-100 mmol / L thiol reducing agent; and / or,

[0019] The first washing solution comprises: a guanidine salt compound, a surfactant, and ethanol; wherein the final application concentrations of each component in the first washing solution are as follows: 1-4 mol / L guanidine salt compound, 0.4-4% surfactant, and 30-70% ethanol; and / or,

[0020] The second washing solution comprises: ethanol; the final concentration of the ethanol used is 70-80%; and / or,

[0021] The eluent comprises: buffer solution and EDTA; wherein the final application concentrations of each component in the eluent are as follows: 5-20 mmol / L buffer solution, 0.5-2 mmol / L EDTA; and / or,

[0022] The magnetic beads contain the following concentration of components: 5-15 mg / mL poly(GCA) 4-14 Magnetic beads;

[0023] The capture probe comprises the following components at concentrations: 5-15 mg / mL Target C1-poly (CGT). 5-15 -Target C2;

[0024] The protease includes: proteinase K; the final application concentration of proteinase K is 10-30 mg / mL; and / or,

[0025] The protein digestion solution comprises: buffer, EDTA, surfactant and NaCl; wherein the final application concentrations of each component in the protein digestion solution are as follows: 5-15 mmol / L buffer, 5-15 mmol / L EDTA (pH 8), 0.1-1% surfactant, and 100-200 mmol / L NaCl.

[0026] Specifically, the magnetic bead extraction reagent, based on the final application concentration of each component, includes:

[0027] The lysis binding solution comprises the following components at the following concentrations: 6 mol / L guanidine salt compound, 2% surfactant, 10 mmol / L buffer, 20 mmol / L EDTA, 20 mmol / L thiol reducing agent; and / or,

[0028] The first washing solution comprises the following components at the following concentrations: 3 mol / L guanidine salt compound, 1% surfactant, and 50% ethanol; and / or,

[0029] The second washing solution comprises the following components at the following concentrations: 75% ethanol; and / or,

[0030] The eluent comprises the following components at the following concentrations: 10 mmol / L buffer, 1 mmol / L EDTA; and / or,

[0031] The magnetic beads contain the following concentration of component: 10 mg / mL poly(GCA) 4-14 Magnetic beads; and / or,

[0032] The capture probe comprises the following concentration: 10 mg / mL Target C1-poly (CGT) 5-15 -TargetC2; and / or,

[0033] The protease comprises the following concentrations: 20 mg / mL proteinase K; and / or,

[0034] The protein digestion solution comprises the following components at the following concentrations: 10 mmol / L buffer, 10 mmol / L EDTA (pH 8), 0.5% surfactant, and 150 mmol / L NaCl.

[0035] Specifically, in the magnetic bead extraction reagent:

[0036] The guanidine salt compound includes at least one of guanidine isothiocyanate (GITC) or guanidine hydrochloride; and / or,

[0037] The thiol reducing agent includes at least one of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or b-ME; and / or,

[0038] The surfactant includes at least one of SDS, Triton X-100, or Tween 20; and / or,

[0039] The buffer solution includes at least one of Tris buffer or sodium citrate buffer.

[0040] In the magnetic bead extraction reagent of this invention, each component can be stored independently. When using the extraction reagent, it can be mixed with the sample to be tested at a 1:1 volume ratio according to the selected final application concentration.

[0041] In the magnetic bead extraction reagent of the present invention, the probe and the magnetic beads can be directly coupled during application without special coupling treatment. During application, they can be mixed and shaken at room temperature based on the selected application concentration.

[0042] The present invention also provides a nucleic acid extraction method based on magnetic beads, including the step of extracting nucleic acid using the magnetic bead extraction reagent.

[0043] Specifically, the nucleic acid extraction method includes the following steps:

[0044] S1. Take the sample to be tested, add the lysis binding solution, magnetic beads, and protease, mix well, and perform a temperature bath treatment at 50-70℃.

[0045] S2. Separate the magnetic beads by centrifugation or magnetic racking, discard the liquid and continue to add the first washing solution, mix until the magnetic beads are completely dispersed;

[0046] S3. Separate the magnetic beads by centrifugation or magnetic racking, discard the liquid and continue to add the second washing solution, mix until the magnetic beads are completely dispersed;

[0047] S4. Separate the magnetic beads by centrifugation or magnetic racking, discard the liquid and dry at room temperature; continue to add the eluent and mix well.

[0048] S5. Extract the magnetic beads and liquid and perform PCR detection.

[0049] The present invention also provides the application of the capture probe or the magnetic bead extraction reagent in the field of nucleic acid extraction by magnetic bead method.

[0050] The capture probe described in this invention is a universal dual-capture DNA capture probe with a designed sequence structure of Target C1-poly (CGT). 5-15 -Target C2; where,

[0051] The sequence of Target C1 is designed as follows: TTTTTTTTCTTTTCTTTTCTTTTTCTTTTTCTTTTTTCTTTTCTTTTTTT;

[0052] The sequence of Target C2 is designed as follows: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0053] The capture probe described in this invention is a universal dual-capture DNA capture probe. Under this structure, the probe can rapidly hybridize with the easily unwinding portion of DNA and capture the DNA.

[0054] The magnetic bead extraction reagent for nucleic acid extraction based on magnetic beads described in this invention, on the basis of the traditional magnetic bead reagent system, by designing a DNA capture probe with a universal dual-trap structure, the magnetic beads that capture nucleic acid do not inhibit the PCR reaction, can be put into the PCR reaction system to participate in the PCR reaction, without the need to wash the nucleic acid on the magnetic beads, making the automated operation easier. Attached Figure Description

[0055] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0056] Figure 1 The amplification curve of HBV DNA extraction in Example 1;

[0057] Figure 2 This is the amplification curve of DNA extracted from urine sediment in Example 2. Detailed Implementation

[0058] In the following embodiments of the present invention, a capture probe is provided, the capture probe having a dual-capture structure and a sequence structure as shown below:

[0059] Target C1-poly (CGT) 5-15 -Target C2; where,

[0060] The sequence of Target C1 is as follows:

[0061] TTTTTTTTCTTTTCTTTTCTTTCTTTCTTTCTTTTCTTTTCTTTTTTT;

[0062] The sequence of Target C2 is as follows:

[0063] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0064] In the following embodiments of the present invention, a magnetic bead extraction reagent for extracting nucleic acids based on the magnetic bead method is also provided, the extraction reagent comprising:

[0065] The lysis binding solution comprises the following components at concentrations (based on the final application concentration): 3-8 mol / L guanidine salt compound, 0.2-5% surfactant, 5-50 mmol / L buffer, 10-50 mmol / L EDTA (pH 7.5), and 10-100 mmol / L thiol reducing agent;

[0066] The first washing solution comprises the following components at concentrations (based on the final concentration for application): 1-4 mol / L guanidine salt compound, 0.4-4% surfactant, and 30-70% ethanol;

[0067] The second washing solution comprises the following components at the following concentrations (based on the final concentration applied): 70-80% ethanol;

[0068] The eluent comprises the following components at concentrations (based on the final concentration for application): 5-20 mmol / L buffer and 0.5-2 mmol / L EDTA (pH 8.0).

[0069] The magnetic beads comprise the following components at concentrations (based on the final application concentration): 5-15 mg / mL poly(GCA)9 magnetic beads;

[0070] The capture probe comprises the following components at concentrations (based on the final application concentration): 5-15 mg / mL Target C1-poly(CGT)10-Target C2;

[0071] The protease comprises the following concentrations (based on the final application concentration): 10-30 mg / mL proteinase K;

[0072] The protein digestion solution comprises the following components at concentrations (based on the final application concentration): 5-15 mmol / L buffer, 5-15 mmol / L EDTA (pH 8), 0.1-1% surfactant, and 100-200 mmol / L NaCl.

[0073] As an example, the present invention also provides a magnetic bead extraction reagent for nucleic acid extraction based on magnetic bead method in the following embodiments, the extraction reagent comprising:

[0074] The lysis binding solution comprises the following components at the following concentrations (based on the final application concentration): 6 mol / L guanidine isothiocyanate (GITC), 2% SDS, 10 mmol / L Tris (pH 6.7), 20 mmol / L EDTA (pH 7.5), and 20 mmol / L dithiothreitol (DTT).

[0075] The first washing solution comprises the following components at the following concentrations (based on the final concentration applied): 3 mol / L guanidine isothiocyanate (GITC), 0.5% Triton X-100, 0.5% Tween 20, and 50% ethanol;

[0076] The second washing solution comprises the following components at the following concentrations (based on the final concentration applied): 75% ethanol;

[0077] The eluent comprises the following components at concentrations (based on the final concentration applied): 10 mmol / L Tris (pH 8.0) and 1 mmol / L EDTA (pH 8.0).

[0078] The magnetic beads comprise the following components at the following concentrations (based on the final concentration applied): 10 mg / mL poly(GCA) 9 magnetic beads;

[0079] The capture probe comprises the following components at concentrations (based on the final application concentration): 10 mg / mL Target C1-poly(CGT)10-Target C2;

[0080] The protease comprises the following concentration (based on the final application concentration): 20 mg / mL proteinase K;

[0081] The protein digestion solution comprises the following components at the following concentrations (based on the final application concentration): 10 mmol / L Tris (pH 8), 10 mmol / L EDTA (pH 8), 0.5% SDS, and 150 mmol / L NaCl.

[0082] In the following embodiments of the present invention, all reagents selected are conventional reagents in the art.

[0083] Example 1

[0084] In this embodiment, HBV (hepatitis B virus) is used as an example for HBV DNA extraction and qualitative detection.

[0085] Take the HBV standard from the China National Institutes for Food and Drug Control and dilute it with HBV DNA negative plasma to 100,000 copies / mL, 10,000 copies / mL, and 1,000 copies / mL. Take the diluted samples and HBV DNA negative plasma and perform nucleic acid extraction according to the following procedure. Each sample is repeated twice.

[0086] In this embodiment, the reagent system for HBV DNA extraction includes:

[0087] The lysis binding solution comprises the following components at the following concentrations (based on their final application concentration): 6 mol / L guanidine isothiocyanate (GITC), 2% SDS, 10 mmol / L Tris (pH 6.7), 20 mmol / L EDTA (pH 7.5), and 20 mmol / L dithiothreitol (DTT).

[0088] The first washing solution comprises the following components at the following concentrations (based on their final application concentration): 3 mol / L guanidine isothiocyanate (GITC), 0.5% Triton X-100, 0.5% Tween 20, and 50% ethanol;

[0089] The second washing solution comprises the following components at the following concentrations (based on their final application concentration): 75% ethanol;

[0090] The eluent comprises the following components at concentrations (based on their final application concentration): 10 mmol / L Tris (pH 8.0) and 1 mmol / L EDTA (pH 8.0).

[0091] The magnetic beads comprise the following components at the following concentrations (based on their final application concentration): 10 mg / mL poly(GCA) 9 magnetic beads;

[0092] The capture probe comprises components at the following concentrations (using its final concentration meter): ;in,

[0093] The sequence of Target C1 is as follows:

[0094] TTTTTTTTCTTTTCTTTTCTTTCTTTCTTTCTTTTCTTTTCTTTTTTT;

[0095] The sequence of Target C2 is as follows:

[0096] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT;

[0097] The protease comprises the following concentration: 20 mg / mL proteinase K;

[0098] The protein digestion solution comprises the following components at the following concentrations (based on their final application concentration): 10 mmol / L Tris (pH 8), 10 mmol / L EDTA (pH 8), 0.5% SDS, and 150 mmol / L NaCl.

[0099] In this embodiment, the HBV DNA extraction method includes the following steps:

[0100] S1. Take a 1.5mL centrifuge tube, add 400μL of the lysis binding solution, 200μL of plasma, 10μL of magnetic beads, 10μL of capture probe, and 30μL of proteinase K. After mixing, incubate at 65℃ for 10 minutes, mix well, and let stand at room temperature for 10 minutes.

[0101] S2. Separate the magnetic beads by conventional centrifugation or magnetic racking and discard the liquid; add 500 μL of the first washing solution and mix until the magnetic beads are completely dispersed.

[0102] S3. Separate the magnetic beads by conventional centrifugation or magnetic racking and discard the liquid; add 500 μL of the second washing solution and mix until the magnetic beads are completely dispersed.

[0103] S4. Separate the magnetic beads by conventional centrifugation or magnetic racking and discard the liquid; dry at room temperature for 10 minutes; add 50 μL of HBV reaction solution of various concentrations and mix well;

[0104] S5. Aspirate the magnetic beads and liquid into a PCR reaction tube and place it in a fluorescence PCR instrument for detection.

[0105] In this embodiment, the HBV detection procedure is as follows:

[0106] 95℃, 5 min; (95℃, 10 sec; 60℃, 25 sec) × 45, fluorescence signal detected at 60℃.

[0107] In this embodiment, the detection results of each sample are shown in Table 1 below, and the amplification curves of the detected samples are shown in the attached figure. Figure 1 As shown.

[0108] Table 1 Test Results

[0109]

[0110] As can be seen, this extraction method can effectively extract HBV DNA with good homogeneity between wells. In particular, the operation eliminates the need for HBV DNA elution, allowing HBV DNA from 200 μL of plasma to be loaded for detection, which improves the sensitivity of HBV DNA detection and simplifies the experimental procedure.

[0111] Example 2

[0112] This embodiment uses DNA extraction and PCR detection based on urine sediment.

[0113] Take 50 mL of midstream morning urine from three random individuals, centrifuge at 6000 rpm for 10 min, discard the supernatant, and mix the sediment with 1 mL of physiological saline for later use. For testing, perform two replicates for each sediment and two replicates for each physiological saline solution.

[0114] In this embodiment, the reagent system for extracting DNA from urine sediment includes:

[0115] The lysis binding solution comprises the following components at the following concentrations (based on their final application concentration): 6 mol / L guanidine isothiocyanate (GITC), 2% SDS, 10 mmol / L Tris (pH 6.7), 20 mmol / L EDTA (pH 7.5), and 20 mmol / L dithiothreitol (DTT).

[0116] The first washing solution comprises the following components at the following concentrations (based on their final application concentration): 3 mol / L guanidine isothiocyanate (GITC), 0.5% Triton X-100, 0.5% Tween 20, and 50% ethanol;

[0117] The second washing solution comprises the following components at the following concentrations (based on their final application concentration): 75% ethanol;

[0118] The eluent comprises the following components at concentrations (based on their final application concentration): 10 mmol / L Tris (pH 8.0) and 1 mmol / L EDTA (pH 8.0).

[0119] The magnetic beads comprise the following components at the following concentrations (based on their final application concentration): 10 mg / ml poly(GCA) 9 magnetic beads;

[0120] The capture probe comprises components at the following concentrations (using its final concentration meter): ;in,

[0121] The sequence of Target C1 is as follows:

[0122] TTTTTTTTCTTTTCTTTTCTTTCTTTCTTTCTTTTCTTTTCTTTTTTT;

[0123] The sequence of Target C2 is as follows:

[0124] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT;

[0125] The protease comprises the following components at the following concentrations (based on their final application concentration): 20 mg / mL proteinase K;

[0126] The protein digestion solution comprises the following components at the following concentrations (based on their final application concentration): 10 mmol / L Tris (pH 8), 10 mmol / L EDTA (pH 8), 0.5% SDS, and 150 mmol / L NaCl.

[0127] In this embodiment, the method for extracting DNA from urine sediment includes the following steps:

[0128] S1. Take a 1.5 mL centrifuge tube, add 200 μL of urine sediment / physiological saline, 400 μL of lysis binding buffer, and 30 μL of proteinase K. Mix well and incubate at 56 °C for 10 minutes, then centrifuge at 6000 rpm for 1 minute. Take another 1.5 mL centrifuge tube, add 10 μL of magnetic beads, 10 μL of capture probe, and 600 μL of the above lysis supernatant. Mix well and let stand for 10 minutes. Invert the tube three times during the incubation period.

[0129] S2. Separate the magnetic beads by centrifugation or magnetic racking, discard the liquid, and continue to add 500μL of the first washing solution, mix well until the magnetic beads are completely dispersed;

[0130] S3. Separate the magnetic beads by centrifugation or magnetic rack, discard the liquid, and continue to add 500μL of the second washing solution, mix well until the magnetic beads are completely dispersed;

[0131] S4. Separate the magnetic beads by centrifugation or magnetic racking, discard the liquid, and dry at room temperature for 10 minutes; continue to add 10μL-50μL of the eluent, mix well and set aside.

[0132] S5. Aspirate the magnetic beads and liquid into a PCR reaction tube and place it in a fluorescence PCR instrument for detection.

[0133] In this embodiment, the PCR detection procedure is as follows: 95℃, 5min; (95℃, 10sec; 60℃, 25sec) × 45, detecting fluorescence signal at 60℃.

[0134] In this embodiment, the detection results of each sample are shown in Table 2 below, and the amplification curves of the detected samples are attached. Figure 2As shown.

[0135] Table 2 Test Results

[0136]

[0137] As can be seen, the capture probe structure and extraction reagent system described in this invention can effectively extract even human genomes, and the operation is simple, requires no elution, and is user-friendly for subsequent operations.

[0138] In summary, the magnetic bead extraction reagent for nucleic acid extraction based on magnetic beads described in this invention, on the basis of the traditional magnetic bead reagent system, by designing a DNA capture probe with a universal dual-trap structure, the magnetic beads that capture nucleic acid do not inhibit the PCR reaction and can be put into the PCR reaction system to participate in the PCR reaction without the need to wash the nucleic acid on the magnetic beads, making the automated operation easier.

[0139] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A capture probe, characterized in that, The capture probe is a dual-capture structure probe, with the following sequence structure: Target C1-poly (CGT) 5-15 -Target C2; where, The sequence of Target C1 is as follows: TTTTTTTTCTTTTCTTTTCTTTCTTTCTTTCTTTTCTTTTCTTTTTTT; The sequence of Target C2 is as follows: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

2. A magnetic bead extraction reagent for nucleic acid extraction based on magnetic bead method, characterized in that, It includes the capture probe and the corresponding magnetic bead as described in claim 1.

3. The magnetic bead extraction reagent according to claim 2, characterized in that, The general grasping sequence for the magnetic beads is poly(GCA). 4-14 .

4. The magnetic bead extraction reagent according to claim 3, characterized in that, The magnetic bead extraction reagent includes: lysis binding buffer, first washing solution, second washing solution, elution solution, the capture probe and corresponding magnetic beads, protease and protein digestion solution; In the extraction reagent: The lysis binding solution comprises: a guanidine salt compound, a surfactant, a buffer solution, EDTA, and a thiol reducing agent; wherein the final application concentrations of each component in the lysis binding solution are as follows: 3-8 mol / L guanidine salt compound, 0.2-5% surfactant, 5-50 mmol / L buffer solution, 10-50 mmol / L EDTA, and 10-100 mmol / L thiol reducing agent; and / or, The first washing solution comprises: a guanidine salt compound, a surfactant, and ethanol; wherein the final application concentrations of each component in the first washing solution are as follows: 1-4 mol / L guanidine salt compound, 0.4-4% surfactant, and 30-70% ethanol; and / or, The second washing solution comprises: ethanol; the final concentration of the ethanol used is 70-80%; and / or, The eluent comprises: buffer solution and EDTA; wherein the final application concentrations of each component in the eluent are as follows: 5-20 mmol / L buffer solution, 0.5-2 mmol / L EDTA; and / or, The magnetic beads contain the following concentration of poly(GCA): 5-15 mg / mL. 4-14 Magnetic beads; The capture probe comprises the following concentrations of the ingredient: 5-15 mg / mL Target C1-poly (CGT). 5-15 -Target C2; The protease includes: proteinase K; the final application concentration of proteinase K is 10-30 mg / mL; and / or, The protein digestion solution comprises: buffer, EDTA, surfactant and NaCl; wherein the final application concentrations of each component in the protein digestion solution are as follows: 5-15 mmol / L buffer, 5-15 mmol / L EDTA (pH 8), 0.1-1% surfactant, and 100-200 mmol / L NaCl.

5. The magnetic bead extraction reagent according to claim 4, characterized in that, Based on the final concentration of each component, the extraction reagent includes: The lysis binding solution comprises the following components at the following concentrations: 6 mol / L guanidine salt compound, 2% surfactant, 10 mmol / L buffer, 20 mmol / L EDTA, 20 mmol / L thiol reducing agent; and / or, The first washing solution comprises the following components at the following concentrations: 3 mol / L guanidine salt compound, 1% surfactant, and 50% ethanol; and / or, The second washing solution comprises the following components at the following concentrations: 75% ethanol; and / or, The eluent comprises the following components at the following concentrations: 10 mmol / L buffer, 1 mmol / L EDTA; and / or, The magnetic beads contain the following concentration of component: 10 mg / mL poly(GCA) 4-14 Magnetic beads; and / or, The capture probe comprises the following concentration: 10 mg / mL Target C1-poly (CGT) 5-15 -Target C2; and / or, The protease comprises the following concentrations: 20 mg / mL proteinase K; and / or, The protein digestion solution comprises the following components at the following concentrations: 10 mmol / L buffer, 10 mmol / L EDTA (pH 8), 0.5% surfactant, and 150 mmol / L NaCl.

6. The magnetic bead extraction reagent according to claim 4 or 5, characterized in that: The guanidine salt compound includes at least one of guanidine isothiocyanate (GITC) or guanidine hydrochloride; and / or, The thiol reducing agent includes at least one of dithiothreitol (DTT), TCEP, or b-ME; and / or, The surfactant includes at least one of SDS, Triton X-100, or Tween 20; and / or, The buffer solution includes at least one of Tris buffer or sodium citrate buffer.

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