Kit and method for rapidly extracting large-volume sample virus nucleic acid

Abstract

The invention relates to the technical field of virus extraction kits, in particular to a large-volume sample virus nucleic acid rapid extraction kit and method.The large-volume sample virus nucleic acid rapid extraction kit comprises a protease K solution, magnetic beads, a lysis solution, a rinsing solution 1, a rinsing solution 2 and an eluent; the magnetic beads are hydroxyl magnetic beads; the lysate is prepared from guanidine isothiocyanate, Tris alkali, Tris hydrochloric acid, triton X-100, isopropyl alcohol and polyoxyethylene ether; the rinsing liquid 1 is composed of a guanidine hydrochloride solution and isopropanol; the rinsing liquid 2 is composed of absolute ethyl alcohol and purified water; the kit provided by the invention can easily obtain viral nucleic acid from a large-volume sample, is simple to operate, can extract a plurality of samples at one time, realizes rapid diagnosis, and makes up the vacancy of a kit for automatically extracting viral nucleic acid from a large-volume sample at high flux in the current market.

Description

Technical Field

[0001] This invention relates to the field of virus extraction kit technology, and in particular to a rapid viral nucleic acid extraction kit and method for large-volume samples. Background Technology

[0002] Viral nucleic acid extraction is a core step in pathogen detection (such as HIV and influenza viruses), directly affecting detection sensitivity and accuracy. Unpurified samples may contain interfering substances such as RNases, host proteins, and environmental impurities, leading to nucleic acid degradation or false negative / false positive results. Common nucleic acid extraction methods include solution extraction, centrifugation column extraction, and magnetic bead extraction. Magnetic bead extraction has been widely used in in vitro diagnostics in recent years due to its high efficiency, high throughput, and ease of commercialization. Magnetic beads are nanomagnetic materials that specifically adsorb nucleic acids in high-salt buffer solutions and release them in low-salt buffer solutions, achieving nucleic acid enrichment and separation.

[0003] Currently, commercially available magnetic bead-based viral nucleic acid extraction methods employ small-volume sample loading, such as 200μL or 300μL. While this has no impact on samples with high viral nucleic acid loads, it is less effective for environmental monitoring (e.g., virus detection in water bodies) or large volumes of clinical body fluids (e.g., saliva, blood) with low viral loads, leading to a higher risk of missed detections. Common classic lysis buffers include TRIzol, CATB-LiCl, and alkaline lysis buffers. However, these solutions are cumbersome, time-consuming, have limitations in sample processing, and are prone to leaving residues that can affect downstream experiments. Most magnetic bead-based nucleic acid extraction lysis buffers currently use high-salt solutions. For large-volume samples, pretreatment is often required to improve efficiency, making the process cumbersome when dealing with large sample volumes. Therefore, optimizing reagent formulations and improving kit extraction efficiency is crucial.

[0004] Therefore, we propose a rapid viral nucleic acid extraction kit and method for large-volume samples to solve the above problems. Summary of the Invention

[0005] The technical problem this invention aims to solve is the current situation where the automation of large-volume sample extraction is not widespread and requires cumbersome steps such as sample pretreatment. This invention provides a high-throughput automated nucleic acid extraction kit for large-volume samples, which can obtain products directly by adding samples without sample pretreatment. This provides high-quality, high-yield nucleic acids for downstream quantitative fluorescence detection, and provides an effective and reliable guarantee for rapid and accurate diagnosis of disease types and prevention and blocking of disease transmission.

[0006] A rapid viral nucleic acid extraction kit for large-volume samples includes proteinase K solution, magnetic beads, lysis buffer, wash buffer 1, wash buffer 2 and elution buffer;

[0007] The magnetic beads are hydroxyl magnetic beads;

[0008] The lysis solution consists of guanidine isothiocyanate, Tris base, Tris hydrochloric acid, Triton X-100, isopropanol and polyoxyethylene ether.

[0009] The rinsing solution 1 is composed of guanidine hydrochloride solution and isopropanol;

[0010] The rinsing solution 2 is composed of anhydrous ethanol and purified water;

[0011] The eluent is purified water.

[0012] Preferably, the composition and content of the lysis buffer are as follows:

[0013] Guanidine isothiocyanate 40%-45%

[0014] Tris base 0.4%-0.7%

[0015] Tris hydrochloric acid 0.5%-0.8%

[0016] Qulaton X-100 3%-4%

[0017] Isopropanol 18%-22%

[0018] Polyoxyethylene ether 4%-6%;

[0019] The remainder is purified water.

[0020] Preferably, the rinsing solution 1 is a mixture of guanidine hydrochloride solution and isopropanol, wherein the concentration of guanidine hydrochloride solution is 7 mol / L and the volume ratio of guanidine hydrochloride solution to isopropanol is (3-5):1.

[0021] Preferably, the mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

[0022] Preferably, the concentration of proteinase K in the proteinase K solution is 15-25 mg / mL.

[0023] Preferably, a rapid method for extracting viral nucleic acid from large-volume samples is provided, which uses the aforementioned rapid viral nucleic acid extraction kit for large-volume samples to extract viral nucleic acid.

[0024] Preferably, it includes the following steps:

[0025] Step 1: Mix the lysis buffer with the proteinase K solution at a volume ratio of (4-6):1 to obtain a mixed lysis buffer;

[0026] Step 2: Separate the reagents: Columns 1 and 5: lysis buffer; Columns 2 and 6: wash buffer 1; Columns 3 and 7: wash buffer 2 and magnetic beads; Columns 4 and 8: elution buffer;

[0027] Step 3: Add 2ml of sample to columns 1 and 5, and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor;

[0028] Step 4: Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor;

[0029] Step 5: Select and run the program. After the automated program finishes, remove the nucleic acid samples from columns 4 and 8 to obtain a large volume of viral nucleic acid samples.

[0030] The beneficial effects of this invention are: the kit of this invention can easily obtain viral nucleic acid from large-volume samples, and the operation is simple. It can extract multiple samples at once, realize rapid diagnosis, and fill the gap in the current market for automated high-throughput viral nucleic acid extraction kits for large-volume samples. Detailed Implementation

[0031] The present invention will be further explained below with reference to specific embodiments.

[0032] Example 1

[0033] The kit for extracting viral nucleic acid from large-volume samples includes the following components and their concentrations:

[0034] Lysis buffer: guanidine isothiocyanate 20 g

[0035] Tris base 0.2 g

[0036] Tris hydrochloric acid 0.25 g

[0037] Triton X-100 1.5g

[0038] Isopropanol 9 g

[0039] 2 g of polyoxyethylene ether

[0040] 17.05 ml of purified water;

[0041] Rinse solution 1: 7M guanidine hydrochloride 30 ml

[0042] 10 ml of isopropanol;

[0043] Rinse solution 2: 30 ml purified water

[0044] 30 g of anhydrous ethanol;

[0045] Eluent: Purified water

[0046] Magnetic beads: Commercial silica magnetic beads

[0047] Proteinase K: A commercially available product, dissolved in purified water (DEPC water) to obtain a final proteinase K solution concentration of 20 mg / mL.

[0048] Example 2

[0049] The kit for extracting viral nucleic acid from large-volume samples includes the following components and their concentrations:

[0050] Lysis buffer: guanidine isothiocyanate 22.5 g

[0051] Tris base 0.35 g

[0052] Tris hydrochloric acid 0.4 g

[0053] Triton X-100 2 g

[0054] Isopropanol 11 g

[0055] 3 g of polyoxyethylene ether

[0056] 10.75 ml of purified water;

[0057] Rinse solution 1: 7M guanidine hydrochloride 50 ml

[0058] 10 ml of isopropanol;

[0059] Rinse solution 2: 12.5 ml purified water

[0060] 37.5 g of anhydrous ethanol;

[0061] Eluent: Purified water

[0062] Magnetic beads: Commercial silica magnetic beads

[0063] Proteinase K: A commercially available product, dissolved in purified water (DEPC water) to obtain a final proteinase K solution concentration of 20 mg / mL.

[0064] Example 3

[0065] The kit for extracting viral nucleic acid from large-volume samples includes the following components and their concentrations:

[0066] Lysis buffer: guanidine isothiocyanate 22 g

[0067] Tris base 0.3 g

[0068] Tris hydrochloric acid 0.4 g

[0069] Triton X-100 2 g

[0070] 10 g of isopropanol

[0071] 3 g of polyoxyethylene ether

[0072] 12.3 ml of purified water;

[0073] Rinse solution 1: 7M guanidine hydrochloride 40 ml

[0074] 11 ml of isopropanol;

[0075] Rinse solution 2: 11 ml purified water

[0076] 35 g of anhydrous ethanol;

[0077] Eluent: Purified water

[0078] Magnetic beads: Commercial silica magnetic beads

[0079] Proteinase K: A commercially available product, dissolved in purified water (DEPC water) to obtain a final proteinase K solution concentration of 20 mg / mL.

[0080] In Examples 1-3, a rapid method for extracting viral nucleic acid from large-volume samples is described, using the aforementioned rapid viral nucleic acid extraction kit for large-volume samples to extract viral nucleic acid.

[0081] Includes the following steps:

[0082] Step 1: Mix the lysis buffer with the proteinase K solution at a volume ratio of (4-6):1 to obtain a mixed lysis buffer;

[0083] Step 2: Separate the reagents: Columns 1 and 5: lysis buffer; Columns 2 and 6: wash buffer 1; Columns 3 and 7: wash buffer 2 and magnetic beads; Columns 4 and 8: elution buffer;

[0084] Step 3: Add 2ml of sample to columns 1 and 5, and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor;

[0085] Step 4: Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor;

[0086] Step 5: Select and run the program. After the automated program finishes, remove the nucleic acid samples from columns 4 and 8 to obtain a large volume of viral nucleic acid samples.

[0087] The extraction instrument was a fully automated nucleic acid extractor developed by Xingchun (Changzhou) Biotechnology Co., Ltd., model Purfirer32; the total extraction time was 30 minutes.

[0088] The following examines the efficiency of DNA and RNA virus extraction at different lysis buffer volumes:

[0089] Reagent preparation: Prepare the solutions required for the experiment according to Example 3, including lysis buffer, wash buffer 1, wash buffer 2, elution buffer, proteinase K solution, and magnetic beads;

[0090] In this embodiment, the lysis buffer volume is 2.5 ml.

[0091] Comparative lysis buffer 1 volume: 3 ml

[0092] Comparison of lysis buffer 2 volumes: 2 ml

[0093] Comparison of lysis buffer volume 3: 1.5 ml

[0094] Nucleic acid extraction stage:

[0095] The lysis buffer was mixed with proteinase K solution at a volume ratio of 5:1 to obtain a mixed lysis buffer.

[0096] Reagents are listed as follows: Columns 1 and 5: Lysis buffer; Columns 2 and 6: Wash buffer 1; Columns 3 and 7: Wash buffer 2 and magnetic beads; Columns 4 and 8: Elution buffer;

[0097] Add 2ml of sample to columns 1 and 5, and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor.

[0098] Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor.

[0099] Select the program and run it. After the automated program finishes, remove the nucleic acid samples from columns 4 and 8 for the next step of qPCR amplification.

[0100] Table 1. Results of DNA virus extraction efficiency with different lysis buffer volumes in Example 3 of this invention.

[0101]

[0102] Table 2. Results of RNA virus extraction efficiency with different lysis buffer volumes in Example 3 of this invention.

[0103]

[0104] As can be seen from Tables 1 and 2, the lysis buffer volume in the examples and the comparative lysis buffers 1-3 are all relatively large (commercially available small sample lysis buffers typically use a volume in the μL range), and the detection results are accurate.

[0105] The following is a comparison of virus sample extraction using the kit of this invention with commercially available conventional small-sample kits;

[0106] Nucleic acid extraction was performed according to the above procedure, with a lysis buffer volume of 2.5 ml.

[0107] Table 3 Results of DNA virus extraction from the invented kit and commercially available routine small-sample kits

[0108]

[0109] Table 4. Results of DNA virus extraction from the invented kit and commercially available routine small-sample kits.

[0110]

[0111] As can be seen from Tables 3 and 4, the viral DNA extracted by this invention has a higher concentration (lower Ct value) than that extracted by commercially available viral extraction kits.

[0112] A comparison of the reagent kit of this invention with commercially available reagent kits for extracting real samples of African swine fever virus (DNA virus).

[0113] Reagent preparation: Prepare the experimental solutions according to Example 3, including lysis buffer, wash buffer 1, wash buffer 2, elution buffer, and magnetic beads;

[0114] Quantitative real-time PCR detection of samples:

[0115] (1) Take the commercially available ASFV African Swine Fever Detection Kit (Fluorescent Quantitative PCR Probe Method), prepare the reaction solution system according to the product instructions, and dispense it into 8-tube quantitative PCR kits;

[0116] Reaction solution: 20 μL

[0117] (2) Add 5 μL of nucleic acid sample to each sample, mix well, and centrifuge.

[0118] (3) Turn on the PCR instrument and set the program parameters as shown in Table 5 below;

[0119] Table 5 PCR Procedure

[0120]

[0121] Set the channels and sample information, and set the reaction system to 25 μl; select the fluorescence channel: Detection channel (Reporter Dye) FAM, Quencher channel (Quencher Dye) NONE;

[0122] Once the settings are complete, save the file and run the reaction program.

[0123] The process is complete, and the analysis results are shown.

[0124] Table 6 Results of ASFV African swine fever sample extraction detected by real-time PCR

[0125]

[0126] Experimental results: As can be seen from Table 6, the viral DNA extracted by this invention has a higher concentration (lower Ct value) than that extracted by commercially available viral extraction kits.

[0127] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A large volume sample viral nucleic acid rapid extraction kit, characterized by, The proteinase K solution, the magnetic beads, the lysis solution, the rinsing solution 1, the rinsing solution 2 and the elution solution are included. The magnetic beads are hydroxyl magnetic beads. The lysis solution is composed of guanidine isothiocyanate, Tris base, Tris hydrochloride, Triton X-100, isopropyl alcohol and polyoxyethylene ether. The rinsing solution 1 is composed of guanidine hydrochloride solution and isopropyl alcohol. The rinsing solution 2 is composed of anhydrous ethanol and purified water. The elution solution is purified water.

2. The rapid extraction kit for virus nucleic acid of a large volume sample according to claim 1, characterized in that, The components and contents of the lysis solution are as follows: Guanidine isothiocyanate 40%-45% Tris base 0.4%-0.7% Tris hydrochloride 0.5%-0.8% Triton X-100 3%-4% Isopropyl alcohol 18%-22% Polyoxyethylene ether 4%-6% The rest is purified water.

3. The rapid extraction kit for virus nucleic acid of large volume sample according to claim 1, characterized in that, The rinsing solution 1 is a mixture of guanidine hydrochloride solution and isopropyl alcohol, wherein the concentration of the guanidine hydrochloride solution is 7 mol / L, and the volume ratio of the guanidine hydrochloride solution to the isopropyl alcohol is (3-5):

1.

4. The rapid extraction kit for virus nucleic acid of large volume sample according to claim 1, characterized in that, The mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

5. The rapid extraction kit for virus nucleic acid of large volume sample according to claim 1, characterized in that, The concentration of proteinase K in the proteinase K solution is 15-25 mg / mL.

6. The method for rapid extraction of viral nucleic acid from a large volume sample according to any one of claims 1 to 5, wherein The virus nucleic acid is extracted by using the large-volume sample virus nucleic acid rapid extraction kit.

7. The rapid extraction method for viral nucleic acid from large-volume samples according to claim 6, characterized in that, The method comprises the following steps: Step 1, mixing the lysis solution and the proteinase K solution, wherein the volume ratio of the lysis solution to the proteinase K solution is (4-6):1, to obtain a mixed lysis solution; Step 2, arranging the reagents: column 1, 5: lysis solution; column 2, 6: rinsing solution 1; column 3, 7: rinsing solution 2 and magnetic beads; column 4, 8: elution solution; Step 3, adding 2 ml of sample to column 1, 5, and placing the 96-deep-well plate on the base of the 32-channel automatic nucleic acid extractor; Step 4, inserting the magnetic rod sleeve into the card slot of the 32-channel automatic nucleic acid extractor magnetic rod sleeve frame; Step 5, selecting a program and running, and after the automatic program is completed, taking out the nucleic acid sample in column 4, 8, to obtain the large-volume sample virus nucleic acid.

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