Reagent combination or kit for rapidly extracting free nucleic acid from trace plasma sample and extraction method

Abstract

The invention relates to a reagent combination or kit for rapidly extracting free nucleic acid from a trace plasma sample and an extraction method, and belongs to the technical field of biological diagnosis. The reagent combination or the kit comprises the following components: a sample treatment solution, a rinsing solution, magnetic beads and an eluent, the sample treatment liquid is prepared from the following main components: guanidine isothiocyanate, urea, Tris-HCl, EDTA (Ethylene Diamine Tetraacetic Acid), NaCl, Tween-20, Triton-100, ammonium sulfate and isopropanol; the main components of the rinsing liquid are Tris-HCl and absolute ethyl alcohol. The main component of the eluent is water. The method for extracting the free nucleic acid in the trace plasma sample is simple and convenient to operate, high in efficiency, less in reagent combination, more economical and capable of realizing automation, the content of inhibitors in the obtained nucleic acid is low, and downstream experimental analysis is facilitated.

Description

Technical Field

[0001] The invention relates to a reagent combination or a kit and an extraction method for quickly extracting free nucleic acid from a trace plasma sample, belonging to the technical field of biological diagnosis. Background Art

[0002] In recent years, early diagnosis and personalized treatment of cancer have received increasing attention. Circulating tumor cells and free nucleic acids, as important components of liquid biopsy, can provide valuable information for early screening, efficacy monitoring and prognosis evaluation of tumors. Compared with traditional tissue biopsy, liquid biopsy has the advantages of less trauma, easy repeated sampling and real-time monitoring, and is particularly suitable for monitoring metastatic cancer and evaluating treatment effects.

[0003] At present, the technology of extracting circulating tumor nucleic acids or free nucleic acids from trace plasma samples (<1mL) has gradually become a research hotspot. However, the existing extraction methods still have limitations in terms of sensitivity, specificity, and ease of operation. Most methods rely on larger volumes of samples (≥2mL), and the extraction process is cumbersome and time-consuming, which limits their efficiency in clinical applications. In addition, the concentration of free nucleic acids in plasma is low, and how to quickly extract sufficient nucleic acids while maintaining high purity remains a technical challenge.

[0004] The prior art also discloses some plasma free nucleic acid extraction kits, such as Chinese patent applications CN107663521A, CN114058616A, CN115386576A, CN116904442A and CN117947018A, all of which disclose plasma free nucleic acid extraction kits or reagent combinations. However, the prior art has the following deficiencies: the kits or reagent combinations involve too many solutions, including proteinase K or SDS sodium dodecyl sulfate. In addition, the extraction methods of the prior art require additional heating, ice bathing or incubation processes, which are complicated to operate and affect the detection efficiency.

[0005] In summary, developing an efficient, rapid and sensitive method for extracting circulating tumor nucleic acids or free nucleic acids from trace plasma samples has important clinical significance. This can not only enhance the application value of liquid biopsy methods, but also promote the development of precision medicine for tumors. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a reagent combination or kit and extraction method for quickly extracting free nucleic acids from trace plasma samples. The present invention aims to explore a method for quickly, sensitively and specifically extracting circulating tumor nucleic acids or free nucleic acids. The reagent combination or kit of the present invention uses a magnetic bead method for extraction, and is matched with a sample treatment solution, a rinse solution and an eluent, wherein the sample treatment solution is equivalent to a lysate and a first rinse solution, that is, it contains a lysate function and a rinse function, and the rinse solution is equivalent to a second rinse solution, omitting the use of proteinase K or SDS sodium dodecyl sulfate. The extraction method of the present invention can be carried out at room temperature, without additional heating, ice bath and incubation processes, with strong consistency and simple operating steps.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] In a first aspect of the present invention, a reagent combination or a kit for rapidly extracting free nucleic acid from a trace amount of plasma sample is provided.

[0009] The reagent combination or kit of the present invention comprises the following components: a sample treatment solution, a rinse solution, magnetic beads and an eluent;

[0010] The main components of the sample treatment solution are: guanidine thiocyanate, urea, Tris-HCl, EDTA, NaCl, Tween-20, Trition-100, ammonium sulfate and isopropanol;

[0011] The main components of the rinsing solution are: Tris-HCl and anhydrous ethanol.

[0012] The main component of the eluent is water.

[0013] Preferably, the main components of the sample treatment solution are: 1-5M guanidine thiocyanate, 1-5M urea, 10-100mM Tris-HCl, 10-100mM EDTA, 10-200mM NaCl, 10-200mM ammonium sulfate, 10%-30% isopropanol, 1%-10% Trition-100 and 0.01%-1% Tween-20. More preferably, the main components of the sample treatment solution are: 5M guanidine thiocyanate, 2M urea, 20mM Tris-HCl, 10mM EDTA, 100mM NaCl, 0.05% Tween 20, 150mM ammonium sulfate, 5% Triton-100 and 20% isopropanol.

[0014] Preferably, the main components of the rinse solution are: 0.1-0.5M Tris-HCl and 75%-90% anhydrous ethanol. More preferably, the main components of the rinse solution are: 0.15M Tris-HCl and 80% anhydrous ethanol.

[0015] Preferably, the magnetic beads are silica-modified magnetic beads. More preferably, the magnetic bead particle size is less than 1 um, and the magnetic bead content in the magnetic bead solution is not less than 20 mg / mL. It is recommended to use magnetic microspheres with a particle size of 800 nm, a magnetic bead solid content of 20 mg / mL, and a surface modified with silica groups.

[0016] Preferably, the main component of the eluent is enzyme-free water. More preferably, the eluent comprises 10 mM EDTA and enzyme-free water.

[0017] The functions of the various components in the reagent combination or kit of the present invention are as follows:

[0018] In the sample processing solution, the role of guanidine isothiocyanate is to lyse hemoglobin in trace samples, etc., the role of urea includes dissolving, binding and precipitating proteins, the role of Tris-HCl, EDTA and NaCl is to serve as a buffer system to provide a stable solution environment for extraction, the role of Tween-20 and Trition-100 includes acting as surfactants to precipitate proteins and stabilize the nucleic acid structure, and the role of isopropanol is to assist the binding of small molecule nucleic acids to magnetic beads and dissolve some impurities.

[0019] In the rinse solution, Tris-HCl serves as a buffer system to provide a stable solution environment, and anhydrous ethanol serves to dissolve impurities such as organic proteins and components in the sample processing solution.

[0020] The function of magnetic beads is to bind to nucleic acid molecules.

[0021] The main component of the eluent is water.

[0022] In the second aspect of the present invention, a method for rapidly extracting free nucleic acid from a trace amount of plasma sample using the reagent combination or kit as described in the first aspect is provided, and the method is for non-disease diagnosis purposes.

[0023] The method of the present invention comprises the following steps: taking a separated trace plasma sample, adding a sample treatment solution and magnetic beads thereto, shaking and mixing, allowing the hemoglobin in the trace plasma to be fully lysed (because the sample treatment solution contains a large amount of salt particles, which denatures the protein), and promoting the binding of free nucleic acids in the sample to the magnetic beads (at this time, in addition to the free nucleic acids in the plasma, some protein impurities, etc., are bound to the magnetic beads). Subsequently, the mixed solution containing the magnetic beads is placed on a magnetic rack, and after removing the supernatant, the sample treatment solution is added again to mix with the magnetic beads. At this time, the sample treatment solution not only allows the magnetic beads to bind to the nucleic acids, but also has the function of dissolving the impurities bound to the surface of the magnetic beads (because the sample treatment solution contains alcohol, which can dissolve some organic impurities, that is, rinsing to remove impurities). Then, the magnetic beads are rinsed twice with a rinsing solution, and after the impurities are completely removed, the free nucleic acids bound to the surface of the magnetic beads can be eluted using an eluting solution.

[0024] The present invention has the following technical effects:

[0025] 1) The present invention provides a reagent combination or a kit and an extraction method for quickly extracting free nucleic acids from trace plasma samples. The reagent combination or kit of the present invention requires only 4 components, including: a sample treatment solution, a rinse solution, magnetic beads and an eluent. The sample treatment solution can cause the protein and other substances in the plasma sample to precipitate, and promote the binding of free nucleic acids to the magnetic beads. In addition, because the sample treatment solution contains alcohol, some impurities can be dissolved, that is, after the cleavage and binding process is completed, the sample treatment solution is used to remove the impurities bound to the surface of the magnetic beads. The magnetic beads are then rinsed twice with the rinse solution, and after the impurities are completely removed, the free nucleic acids on the surface of the magnetic beads are eluted with the eluent. The present invention is used to extract free nucleic acids from trace plasma samples, which is simple to operate, efficient, requires fewer reagent combinations, is more economical, can be automated, and has a low inhibitor content in the obtained nucleic acid, which is beneficial to downstream experimental analysis.

[0026] 2) The reagent combination or kit and extraction method of the present invention are used to extract free nucleic acids from trace plasma samples, which is simple to operate, highly efficient, requires fewer reagent combinations, is more economical, and can be automated. Compared with competing products, the library construction is unstable, resulting in low concentrations of individual samples; the operation time is long after adding proteinase K digestion, and the average concentration of the library product is low; the sample treatment solution used in the present invention has both lysis and rinsing functions, and is better than ethanol-containing rinsing solutions; compared with manual column extraction, the method of the present invention can be fully automated, improving experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Gel run images of nanogram (ng) library products of nucleic acids extracted by different methods (the brighter the band in the gel image, the better the product quality, and vice versa, the darker the band, the worse the product quality.) DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the materials used in the present invention are all commercially available products.

[0029] Magnetic beads and magnetic bead buffer were purchased from Promega.

[0030] Example 1

[0031] The reagent for rapidly extracting free nucleic acid from a trace amount of plasma sample of this embodiment comprises the following components: a sample processing solution, a rinsing solution, magnetic beads and an elution solution;

[0032] The specific components of the sample treatment solution are as follows: 5 M guanidine thiocyanate, 2 M urea, 20 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 0.05% Tween 20, 150 mM ammonium sulfate, 5% Triton-100, and 20% isopropanol.

[0033] The specific composition of the rinse solution is as follows: 0.15 M Tris-HCl and 80% anhydrous ethanol.

[0034] The magnetic beads have a particle size of 800nm, a solid content of 20mg / mL, and are magnetic microspheres modified with silica groups on their surface.

[0035] The elution buffer included 10 mM EDTA and enzyme-free water.

[0036] Example 2

[0037] The reagents and consumables of Example 1 are used to extract free nucleic acids from trace plasma, and the extraction method includes the following steps:

[0038] 1. Prepare the sample. Centrifuge the blood at 16000×g for 10 min. Take 100uL of the supernatant. Fill the part less than 100uL with PBS to form the sample required for the experiment.

[0039] 2. Take a 1.5mL centrifuge tube, add 100uL of plasma sample, 150uL of sample treatment solution, and 10uL of magnetic bead suspension (magnetic bead suspension is a mixture of magnetic beads and magnetic bead buffer, and the magnetic beads need to be mixed before adding magnetic beads), and place it on a constant temperature mixer at room temperature and vortex at 1200rpm for 8 minutes.

[0040] 3. Place the centrifuge tube from the previous step on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, discard the solution.

[0041] 4. Add 200uL of sample treatment solution to the centrifuge tube in the previous step and vortex at room temperature for 1 minute.

[0042] 5. Place the centrifuge tube from the previous step on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, discard the solution.

[0043] 6. Add 500uL of rinse solution to the centrifuge tube in the previous step and vortex at room temperature for 1 minute.

[0044] 7. Place the centrifuge tube from the previous step on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, discard the solution.

[0045] 8. Repeat steps 6-7.

[0046] 9. Centrifuge the tube immediately, place it back on the magnetic rack, discard the solution, and let it dry for 2 minutes.

[0047] 10. Add 100uL of elution buffer into the centrifuge tube, vortex to resuspend the magnetic beads in the elution buffer, and place it on a constant temperature mixer and vortex at room temperature and 1200rpm for 8 minutes.

[0048] 11. After the centrifuge tube from the previous step is centrifuged, place it on a magnetic rack and let it stand for 30 seconds. After the magnetic beads are adsorbed, transfer the eluent.

[0049] The whole process of manual operation using the present invention does not exceed half an hour. The present invention is applicable to trace plasma samples with a nucleic acid volume of no more than 1 mL. If the sample volume is greater than 100 uL, the amount of sample treatment solution and magnetic bead suspension solution needs to be adjusted proportionally, and the amount of magnetic bead suspension solution does not exceed 50 uL. The amount of rinsing solution and eluent remains unchanged.

[0050] Example 3

[0051] Automated extraction (used with Kangwei CWE320032 channel nucleic acid extractor)

[0052] 1. Sample preparation: centrifuge the plasma sample at 12000×g for 10 min, take 100uL of the supernatant, and fill the portion less than 100uL with PBS.

[0053] 2. Add the following reagents in Example 1 to the 96DW extraction deep well plate according to the table below:

[0054]

[0055]

[0056] 3. Set the program for CWE3200 according to the table below, put the magnetic sleeve into the magnetic sleeve rack of the extractor and put it on the extraction plate rack of the deep well plate extractor.

[0057]

[0058] 4. The program runs for about 27 minutes and ends. After the program runs, take out the deep-well plate, seal it with sealing film, and store it in the freezer.

[0059] Example 4

[0060] Manual extraction and automated extraction were performed simultaneously for 300uL plasma samples according to the experimental methods of Examples 2 and 3 of the present invention.

[0061] Sample preparation: 6 healthy volunteers were drawn blood using 5mL free nucleic acid storage tubes (CW2814M), and the blood was centrifuged at 12000×g for 10 min to separate plasma (about 2000uL plasma per person). 300uL of sample was extracted according to the manual extraction operation steps in Example 2 and the automated extraction requirements in Example 3.

[0062] Because the concentration of cfDNA in 2mL plasma samples is close to or even lower than the detection limit of the Qubit fluorometer, the nucleic acid obtained in this experiment was not measured by the Qubit fluorometer. The extracted nucleic acid was amplified by short-fragment fluorescence using the Bio-Rad CFX, and the amplification Ct values ​​were compared. Water was set as a negative control. The Ct value of the extracted nucleic acid as a template amplification should be smaller than the control, indicating the relative content of the template. The smaller the Ct value, the more template, that is, the higher the concentration. In addition, the nucleic acid was extracted and the library was constructed with reference to CWY014M-A. The concentration of the nucleic acid product after library construction can be determined by Qubit, and the concentration of the nucleic acid product determined by Qubit can be verified by running the gel through agarose gel electrophoresis.

[0063] The primer sequences used for CFX fluorescence quantitative amplification are as follows:

[0064] F:aggtgctggagaggatgt

[0065] R: ggaatcgccacactgact

[0066] Fluorescence amplification was performed using Convi UltraSYBR Mixture (Cat. No.: CW0957).

[0067] The fluorescence amplification system is as follows:

[0068] Reagent name volume UltraSYBR Mixture 12.5 F / R 0.5 / 0.5 Temp 5 <![CDATA[H2O]]> 6.5 Total 25

[0069] The fluorescence amplification program was set up as follows:

[0070]

[0071] The amplification results are as follows:

[0072] Ct Manual extraction Automated extraction Sample 1 19.53 18.56 Sample 2 19.63 19.45 Sample 3 18.20 17.77 Sample 4 18.22 17.64 Sample 5 15.50 15.03 Sample 6 15.46 15.37 average 17.76 17.30

[0073] The concentrations of the library products are as follows:

[0074] ng / uL Manual extraction Automated extraction Sample 1 3.89 4.63 Sample 2 4.64 4.51 Sample 3 7.06 7.97 Sample 4 8.18 8.96 Sample 5 7.87 9.23 Sample 6 9.44 10.1 average 6.85 7.57

[0075] Conclusion: The average relative content of nucleic acids obtained by the automated extraction of 300uL plasma using the present invention is higher than that obtained by manual operation, and the concentration of the corresponding nucleic acid product for library construction is also slightly higher.

[0076] Example 5 Control Test

[0077] Sample preparation: refer to the sample preparation method in Example 4. Another 8 volunteers provided samples of this example.

[0078] Option A: Thermo

[0079] The extraction method was performed using the free nucleic acid extraction kit of Thermo Company, catalog number A29319, the corresponding Kingfisher extractor and the provided procedure. The sample volume was 300uL.

[0080] Scheme B: Adding protease K in the present invention

[0081] Before extraction, the sample pretreatment was performed according to the Thermo kit operation steps, and after adding proteinase K, the plasma protein was heated and digested (the process was about 20 minutes). The extraction process was performed according to the machine extraction in Example 4 of the present invention, and the sample volume was 300uL.

[0082] Scheme C: The present invention

[0083] The extraction process was similar to that in Example 4 of the present invention, with a sample volume of 300 uL.

[0084] Solution D: Replace the first rinse solution

[0085] The sample processing solution of the present invention was replaced with another rinsing solution (rinsing solution 1 in patent CN116286794A). The extraction process was similar to that of Example 4 of the present invention, and the sample volume was 300uL.

[0086] Solution E: CW2603

[0087] The extraction was performed using the Kangwei Century Column Method Micro-plasma Free Nucleic Acid Extraction Kit (Cat. No.: CW2603) with reference to the operating instructions. The sample volume was 300uL.

[0088] The experimental results of the concentration of nucleic acid products in the AE library are as follows:

[0089]

[0090] It can be seen from the concentration results in Table 1 that the average concentration of the product after extraction in Scheme C of the present invention is the highest.

[0091] Figure 1 Gel run diagram of nanogram (ng) library products of nucleic acids extracted by different methods, such as Figure 1 As shown, there are eight samples in total, and each sample corresponds to scheme AE from left to right, namely Thermo, proteinase K added in the present invention, the present invention, replacement of the first step rinsing solution and CW2603. Figure 1It can be seen that after sample 7 was extracted using the Thermo competitor's product, there was no band in the library construction product run on the gel, and the band of the library construction product of this sample was not obvious after the first step of rinsing solution was replaced for extraction. In addition, the gel bands of the library construction products of other samples corresponded to the measured product concentrations one by one, and the band sizes were in line with expectations (250bp-500bp).

[0092] in conclusion:

[0093] The present invention is used to extract free nucleic acids from trace plasma samples, which is simple to operate, highly efficient, requires fewer reagent combinations, is more economical, and can be automated.

[0094] Compared with competing products, the library construction is unstable, resulting in low concentration of individual samples; the operation time is long after adding proteinase K digestion, and the average concentration of the library product is low; the sample processing solution used in the present invention has both lysis and rinsing functions, and has better effect than ethanol-containing rinsing solutions; compared with manual column extraction, the method of the present invention can be fully automated, thereby improving experimental efficiency.

[0095] In summary, the present invention is used to extract free nucleic acids from trace plasma samples, which is simple to operate, highly efficient, requires fewer reagent combinations, is more economical, can be automated, and has a low inhibitory content in the obtained nucleic acids, which is beneficial for downstream experimental analysis.

[0096] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A reagent combination or kit for rapidly extracting free nucleic acid from a trace amount of plasma sample, characterized in that: The reagent combination or kit includes the following components: sample treatment solution, rinse solution, magnetic beads and elution solution; The main components of the sample treatment solution are: guanidine thiocyanate, urea, Tris-HCl, EDTA, NaCl, Tween-20, Trition-100, ammonium sulfate and isopropanol; The main components of the rinsing solution are: Tris-HCl and anhydrous ethanol; The main component of the eluent is water.

2. The reagent combination or kit according to claim 1, characterized in that: The main components of the sample treatment solution are: 1-5M guanidine thiocyanate, 1-5M urea, 10-100mM Tris-HCl, 10-100mM EDTA, 10-200mM NaCl, 10-200mM ammonium sulfate, 10%-30% isopropanol, 1%-10% Trition-100 and 0.01%-1% Tween-20.

3. The reagent combination or kit according to claim 1, characterized in that: The main components of the rinsing solution are: 0.1-0.5M Tris-HCl and 75%-90% anhydrous ethanol.

4. The reagent combination or kit according to claim 1, characterized in that: The magnetic beads are silica-modified magnetic beads.

5. The reagent combination or kit according to claim 1, characterized in that: The main component of the eluent is enzyme-free water.

6. The reagent combination or kit according to claim 2, characterized in that: The main components of the sample treatment solution are: 5M guanidine thiocyanate, 2M urea, 20mM Tris-HCl, 10mM EDTA, 100mM NaCl, 0.05% Tween 20, 150mM ammonium sulfate, 5% Triton-100 and 20% isopropanol.

7. The reagent combination or kit according to claim 3, characterized in that: The main components of the rinsing solution are: 0.15M Tris-HCl and 80% anhydrous ethanol.

8. The reagent combination or kit according to claim 4, characterized in that: The magnetic bead particle size is less than 1 um, and the magnetic bead content in the magnetic bead liquid is not less than 20 mg / mL.

9. The reagent combination or kit according to claim 5, characterized in that: The elution buffer included 10 mM EDTA and enzyme-free water.

10. A method for rapidly extracting free nucleic acid from a trace amount of plasma sample using the reagent combination or kit as described in any one of claims 1 to 9, wherein the method is for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: taking a separated trace plasma sample, adding a sample processing solution and magnetic beads thereto, shaking and mixing, allowing hemoglobin and the like in the trace plasma to be fully lysed, and promoting the binding of free nucleic acids in the sample to the magnetic beads; then placing the mixed solution containing the magnetic beads on a magnetic rack, removing the supernatant, and adding the sample processing solution again to mix with the magnetic beads; The magnetic beads are then rinsed twice with the rinse solution to completely remove impurities, and then the free nucleic acids bound to the surface of the magnetic beads are eluted with the elution solution.

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