Kit and method for extracting high-purity viral nucleic acid

Abstract

The invention relates to the technical field of nucleic acid extraction kits, in particular to a high-purity viral nucleic acid extraction kit, which comprises 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 prepared from guanidine isothiocyanate, Tris alkali, Tris hydrochloric acid, triton X-100 and isopropyl alcohol; the rinsing liquid 2 is composed of absolute ethyl alcohol and purified water; the method for extracting the nucleic acid by utilizing the magnetic bead method based extraction kit is simple and convenient to operate, free of toxic reagents such as phenol chloroform and the like, simple and convenient in steps and capable of extracting the nucleic acid on a machine only by adding a sample, so that the experimental operation steps are simplified to the greatest extent, and the safety of experimenters is ensured.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction kit technology, and in particular to a high-purity viral nucleic acid extraction kit and method. Background Technology

[0002] Viral nucleic acid extraction is the process of isolating viral nucleic acids (DNA or RNA) from viral samples. It is widely used in clinical pathogen detection, infectious diseases, and gene diagnosis. Nucleic acid extraction is the most critical step in diagnostic testing, directly affecting the results of subsequent experiments. 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 both enrichment and separation of nucleic acids.

[0003] my country is a major livestock farming country, and early prevention of various livestock viral diseases mainly relies on qPCR methods, such as for detecting African swine fever and porcine reproductive and respiratory syndrome virus (PRRSV). Currently, downstream experimental reagents on the market, in order to improve detection efficiency, not only optimize the performance of the reagents themselves but also increase the amount of nucleic acid samples. This places higher demands on the quality of the nucleic acid samples. The more impurities in the nucleic acid, the more inhibitory the amplification reagent will be, leading to inaccurate test results and unnecessary losses. Therefore, developing a high-purity nucleic acid extraction reagent is particularly important.

[0004] The method of nucleic acid purification is a crucial factor affecting the quality of extracted nucleic acids, as only high-quality nucleic acids can meet the requirements of various downstream applications. This process includes two main steps: lysis and separation purification. The reagent formulation has a significant impact on obtaining high-quality nucleic acids. Common classic lysis buffers include TRIzol, CATB-LiCl, and alkaline lysis buffers. However, these lysis buffers are cumbersome, time-consuming, have limitations in sample processing, and are prone to leaving residues that can affect downstream experiments. Currently, most magnetic bead-based nucleic acid extraction lysis buffers use high-salt solutions, and some kits require the addition of proteinase K for sample digestion, which is cumbersome when dealing with large sample volumes. Furthermore, there are numerous types of viral samples, such as serum, plasma, swabs (oropharyngeal swabs, environmental swabs), gauze, and tissue homogenates, and some kits are not compatible with the extraction of multiple samples. Therefore, optimizing reagent formulations, improving kit extraction efficiency, and enhancing sample compatibility are crucial for supporting disease prevention and control in livestock farming. To address these issues, we propose a high-purity viral nucleic acid extraction kit and method. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a high-purity viral nucleic acid extraction kit and method.

[0006] A high-purity viral nucleic acid extraction kit includes magnetic beads, lysis buffer, washing buffer 1, washing 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 isothiocyanate, Tris base, Tris hydrochloric acid, Triton X-100 and isopropanol;

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

[0011] The eluent is purified water.

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

[0013]

[0014] The solvent is purified water.

[0015] The composition and content of the rinsing solution 1 are as follows:

[0016]

[0017] The solvent is purified water.

[0018] The mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

[0019] A method for extracting high-purity viral nucleic acid, using the aforementioned high-purity viral nucleic acid extraction kit to extract nucleic acid.

[0020] Includes the following steps:

[0021] Step 1: Using a 96-well plate, separate the lysis buffer, rinse buffer 1, rinse buffer 2, magnetic beads, and eluent. Columns 2 and 8 contain the lysis buffer; columns 3 and 9 contain rinse buffer 1; columns 4 and 10 contain rinse buffer 2 and magnetic beads; and columns 6 and 12 contain the eluent.

[0022] Step 2: Add 200 μL of sample to columns 2 and 8, and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor.

[0023] Step 3: Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor;

[0024] Step 4: Select and run the program. After the automated program finishes, remove the nucleic acid samples from columns 6 and 12 to obtain high-purity viral nucleic acid.

[0025] The beneficial effects of this invention are:

[0026] 1. The present invention utilizes a magnetic bead-based extraction kit for nucleic acid extraction, which is simple to operate, does not contain toxic reagents such as phenol and chloroform, and has a simple procedure. It only requires adding the sample to extract nucleic acid, which simplifies the experimental operation steps to the greatest extent and ensures the safety of the experimental personnel.

[0027] 2. In response to the needs of livestock breeding enterprises for nucleic acid extraction speed, throughput and quality when there are a large number of clinical samples, this kit can meet the requirements of simple operation, high sample throughput, good nucleic acid extraction quality, and the entire extraction process only takes 16 minutes. Detailed Implementation

[0028] The present invention will be further explained below with reference to specific embodiments.

[0029] A high-purity viral nucleic acid extraction kit includes magnetic beads, lysis buffer, washing buffer 1, washing buffer 2, and elution buffer;

[0030] The magnetic beads are hydroxyl magnetic beads;

[0031] The lysis solution consists of guanidine isothiocyanate, Tris base, Tris hydrochloric acid, Triton X-100, isopropanol and polyoxyethylene ether.

[0032] The rinsing solution 1 is composed of guanidine isothiocyanate, Tris base, Tris hydrochloric acid, Triton X-100 and isopropanol;

[0033] The rinsing solution 2 is composed of anhydrous ethanol and purified water;

[0034] The eluent is purified water.

[0035] The composition and content of the lysis buffer are as follows:

[0036]

[0037] The solvent is purified water.

[0038] The composition and content of the rinsing solution 1 are as follows:

[0039]

[0040] The solvent is purified water.

[0041] The mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

[0042] A method for extracting high-purity viral nucleic acid, using the aforementioned high-purity viral nucleic acid extraction kit to extract nucleic acid.

[0043] Includes the following steps:

[0044] Step 1: Using a 96-well plate, separate the lysis buffer, rinse buffer 1, rinse buffer 2, magnetic beads, and eluent. Columns 2 and 8 contain the lysis buffer; columns 3 and 9 contain rinse buffer 1; columns 4 and 10 contain rinse buffer 2 and magnetic beads; and columns 6 and 12 contain the eluent.

[0045] Step 2: Add 200 μL of sample to columns 2 and 8, and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor.

[0046] Step 3: Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor;

[0047] Step 4: Select and run the program. After the automated program finishes, remove the nucleic acid samples from columns 6 and 12 to obtain high-purity viral nucleic acid.

[0048] The composition of the magnetic beads, lysis buffer, rinsing solution 1, rinsing solution 2, and eluent in each embodiment is described in detail below.

[0049] In Example 1,

[0050] The composition and content of the lysis buffer are as follows:

[0051]

[0052] The solvent is purified water, and the amount of purified water is 22g.

[0053] The composition and content of the rinsing solution 1 are as follows:

[0054]

[0055] The solvent is purified water, and the amount of purified water is 22g.

[0056] The mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%.

[0057] Eluent: Purified water;

[0058] Magnetic beads: Commercially available silica magnetic beads.

[0059] In Example 2,

[0060] The composition and content of the lysis buffer are as follows:

[0061]

[0062] The solvent is purified water, and the amount of purified water is 10g.

[0063] The composition and content of the rinsing solution 1 are as follows:

[0064]

[0065] The solvent is purified water, and the amount of purified water is 10g.

[0066] The rinsing solution 2 contains 70% anhydrous ethanol by mass. Eluent: purified water.

[0067] Magnetic beads: Commercially available silica magnetic beads.

[0068] In Example 3,

[0069] The composition and content of the lysis buffer are as follows:

[0070]

[0071] The composition and content of rinsing solution 1 are as follows:

[0072]

[0073] The mass fraction of anhydrous ethanol in rinsing solution 2 is 60%.

[0074] Eluent: Purified water;

[0075] Magnetic beads: Commercially available silica magnetic beads.

[0076] Comparative Example 1: In Comparative Example 1, the lysis solution differs from that in Example 3, but the rest is the same;

[0077] lysis buffer:

[0078]

[0079] Comparative Example 2: In Comparative Example 2, the lysis solution differs from that in Example 3, but the rest is the same;

[0080] lysis buffer:

[0081]

[0082] The high-purity viral nucleic acid extraction kits from Example 3, Comparative Example 1, and Comparative Example 2 were used for viral nucleic acid extraction. The procedure was as follows:

[0083] Reagents were separated into columns: using a 96-well plate, the lysis buffer, wash buffer 1, wash buffer 2, magnetic beads, and eluent were separated into columns 2 and 8, respectively; the lysis buffer was in columns 3 and 9; the wash buffer 1 was in columns 4 and 10; the wash buffer 2 and magnetic beads were in columns 4 and 10; and the eluent was in columns 6 and 12.

[0084] Add 200 μL of sample to column 2 / 8 and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor.

[0085] Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor.

[0086] Select the program and run it. After the automated program finishes, remove the nucleic acid samples from columns 6 and 12 for the next step of qPCR amplification.

[0087] Table 1. Results of RNA virus extraction efficiency using different lysis buffers

[0088]

[0089] Table 2. Results of DNA virus extraction efficiency with different lysis buffers

[0090]

[0091]

[0092] A comparison of the extraction of real samples of African swine fever virus (DNA virus) using the kit of this invention and competing kits:

[0093] Reagent preparation: Prepare the solutions required for the experiment according to Example 3, including lysis buffer, wash buffer 1, wash buffer 2, elution buffer, and magnetic beads;

[0094] Nucleic acid extraction:

[0095] Reagents are listed as follows: Columns 2 / 8: lysis buffer; Columns 3 / 9: wash buffer 1; Columns 4 / 10: wash buffer 2 and magnetic beads; Columns 6 / 12: elution buffer.

[0096] Add 200 μL of sample to column 2 / 8 and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor.

[0097] Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor.

[0098] Select the program and run it. After the automated program finishes, remove the nucleic acid samples from columns 6 and 12.

[0099] Quantitative real-time PCR detection of samples:

[0100] (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 the quantitative PCR eight-tube strip;

[0101] Reaction solution: 20 μL

[0102] (2) Add 5 μL of the nucleic acid sample extracted in step 3, mix well, and centrifuge briefly;

[0103] (3) Turn on the PCR instrument and set the program parameters as shown in Table 3 below;

[0104] Table 3 PCR Procedure

[0105]

[0106] Set the channels and sample information, and set the reaction system to 25 μL; select the fluorescence channel: Detection channel (ReporterDye) FAM, Quencher channel (QuencherDye) NONE;

[0107] Once the settings are complete, save the file and run the reaction program.

[0108] The process is complete, and the analysis results are shown.

[0109] Table 4. Results of ASFV (African Swine Fever) Sample Extraction Detection by Quantitative Real-Time PCR

[0110]

[0111] Experimental results: As shown in Table 4, it can be seen that the viral DNA extracted by this invention has a higher concentration (lower Ct value) than that extracted by a viral extraction kit from a domestic company.

[0112] An embodiment of the present invention and competing products for extracting African swine fever virus from different samples and detecting the extracted products using real-time quantitative PCR with different sample loading volumes includes the following steps:

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

[0114] Nucleic acid extraction:

[0115] Reagents are listed as follows: Columns 2 / 8: lysis buffer; Columns 3 / 9: wash buffer 1; Columns 4 / 10: wash buffer 2 and magnetic beads; Columns 6 / 12: elution buffer.

[0116] Add 200 μL of sample to column 2 / 8 and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor.

[0117] Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor.

[0118] Select the program and run it. After the automated program finishes, remove the nucleic acid samples from columns 6 and 12.

[0119] Quantitative real-time PCR detection of samples:

[0120] Group 1: Reaction system 20 μL + 5 μL

[0121] (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 the quantitative PCR eight-tube strip;

[0122] Reaction solution: 20 μL

[0123] (2) Add 5 μL of the nucleic acid sample extracted in step 3, mix well, and centrifuge briefly;

[0124] (3) Turn on the PCR instrument and set the program parameters as shown in Table 3;

[0125] After setting the channels and sample information, the reaction system is set to 25 μL; the fluorescence channel selection is: Detector Dye FAM, Quencher Dye NONE.

[0126] Once the settings are complete, save the file and run the reaction program.

[0127] The process is complete, and the analysis results are shown.

[0128] Group 2: Reaction system 20 μL + 10 μL

[0129] (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 the quantitative PCR eight-tube strip;

[0130] Reaction solution: 20 μL

[0131] (2) Add 10 μL of the nucleic acid sample extracted in step 3, mix well, and centrifuge briefly;

[0132] (3) Turn on the PCR instrument and set the program parameters as shown in Table 3;

[0133] After setting the channels and sample information, the reaction system is set to 30ul; the fluorescence channel selection is: Detection channel (Reporter Dye) FAM, Quencher channel (Quencher Dye) NONE;

[0134] Once the settings are complete, save the file and run the reaction program.

[0135] The process is complete, and the analysis results are shown.

[0136] Table 5. Ct values ​​of 5 μL samples amplified by real-time quantitative PCR for ASFV (African Swine Fever) detection.

[0137]

[0138] Table 6. Ct values ​​of 10 μL samples amplified by real-time quantitative PCR for ASFV (African Swine Fever) detection.

[0139]

[0140] Experimental results: As shown in Tables 5 and 6, the Ct values ​​of the kit of the present invention are all lower than those of the competing kits. Furthermore, as the sample volume increases, the extract of the kit of the present invention does not show a significant inhibitory effect in the real-time PCR system, while the competing kits show a certain inhibitory effect as the sample volume increases.

[0141] 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 high-purity viral nucleic acid extraction kit, characterized in that, Includes magnetic beads, lysis buffer, rinse buffer 1, rinse buffer 2, and elution buffer; The magnetic beads are hydroxyl magnetic beads; The lysis solution consists of guanidine isothiocyanate, Tris base, Tris hydrochloric acid, Triton X-100, isopropanol and polyoxyethylene ether. The rinsing solution 1 is composed of guanidine isothiocyanate, Tris base, Tris hydrochloric acid, Triton X-100 and isopropanol; The rinsing solution 2 is composed of anhydrous ethanol and purified water; The eluent is purified water.

2. The high-purity viral nucleic acid extraction kit according to claim 1, characterized in that, The composition and content of the lysis buffer are as follows: The solvent is purified water.

3. The high-purity viral nucleic acid extraction kit according to claim 1, characterized in that, The composition and content of the rinsing solution 1 are as follows: The solvent is purified water.

4. The high-purity viral nucleic acid extraction kit according to claim 1, characterized in that, The mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

5. A method for extracting high-purity viral nucleic acid according to any one of claims 1-4, characterized in that, Viral nucleic acid was extracted using the high-purity viral nucleic acid extraction kit described above.

6. The method for extracting high-purity viral nucleic acid according to claim 5, characterized in that, Includes the following steps: Step 1: Using a 96-well plate, separate the lysis buffer, rinse buffer 1, rinse buffer 2, magnetic beads, and eluent. Columns 2 and 8 contain the lysis buffer; columns 3 and 9 contain rinse buffer 1; columns 4 and 10 contain rinse buffer 2 and magnetic beads; and columns 6 and 12 contain the eluent. Step 2: Add 200 μL of sample to columns 2 and 8, and place the 96-well plate on the base of the 32-channel automated nucleic acid extractor. Step 3: Insert the magnetic rod holder into the slot of the magnetic rod holder of the 32-channel automated nucleic acid extractor; Step 4: Select and run the program. After the automated program finishes, remove the nucleic acid samples from columns 6 and 12 to obtain high-purity viral nucleic acid.

Images