Kit for extracting viral nucleic acid and application thereof

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a kit for extracting viral nucleic acid and application of the kit. The kit comprises a lysis solution, a cleaning solution 1, a cleaning solution 2, an eluent, protease K dry powder, a protease K diluent and a magnetic bead solution. The kit adopts a magnetic bead nucleic acid extraction method to solve the problems of tedious extraction steps, complex operation, large harm to human and the like of a traditional method, and compared with an existing extraction kit, the kit has higher nucleic acid extraction efficiency.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a kit for viral nucleic acid extraction and its application. Background Technology

[0002] In molecular biology, nucleic acids, as indispensable biomolecules in living organisms, include two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They are carriers of genetic information and play a crucial role in cell growth, division, and function. High-quality nucleic acids are essential for molecular biology applications because nucleic acid extraction is a fundamental component of molecular biology and a basic step in many research and applications. For example, cloning, qRT-PCR, and next-generation sequencing technologies in the fields of whole genome and transcriptomics all require high-quality nucleic acids as templates. Furthermore, the significance of nucleic acid extraction lies in its wide range of applications. In disease diagnosis and research, extracting and detecting specific nucleic acid sequences from patient samples can assist doctors in making accurate diagnoses and developing personalized treatment plans. In forensic identification, nucleic acid extraction technology is the basis of DNA fingerprinting, aiding in crime investigation. In agricultural and food science, nucleic acid extraction is used in research on gene-edited crops and food safety testing. In ecological environment monitoring, extracting DNA from environmental samples allows for the identification and quantification of species composition in ecosystems. In genomics research, nucleic acid extraction is a fundamental and crucial step, helping to unravel the mysteries of evolutionary relationships.

[0003] With the rapid development of gene diagnostics, genetically modified food testing, and personalized medicine, traditional nucleic acid extraction methods such as phenol / chloroform extraction, alcohol precipitation, affinity chromatography, density gradient centrifugation, and silica gel column chromatography are no longer sufficient to meet the needs of modern biotechnology. These methods involve complex extraction steps. Phenol / chloroform extraction requires multiple intricate steps, including cell lysis, organic phase extraction, centrifugation, aqueous phase transfer, and alcohol precipitation. The entire process involves toxic reagents, is cumbersome, and takes 2-3 hours. While silica gel column chromatography is relatively simple, it still requires repeated centrifugation, washing, and elution, posing a risk of nucleic acid shearing. Alcohol precipitation is simple to operate but has low recovery rates and is prone to leaving residual salts and other inhibitors.

[0004] In contrast, the magnetic bead extraction method significantly simplifies the process: first, nucleic acids are released using a lysis buffer, then surface-modified magnetic beads are added to specifically bind the nucleic acids. Under an external magnetic field, the magnetic bead-nucleic acid complex is rapidly separated. After simple washing, pure nucleic acids are obtained by elution with a low-salt buffer. The entire process requires no centrifugation, can be completed within 20-40 minutes, and can be fully automated. This simplified procedure not only improves experimental efficiency but also reduces human error, making it particularly suitable for clinical testing and large-scale screening applications. From an operational perspective, the magnetic bead method revolutionarily simplifies the nucleic acid extraction process by replacing complex steps such as phase separation and centrifugation in traditional methods with specific binding of magnetic beads and magnetic field separation. This is a key advantage enabling its widespread application in modern molecular diagnostics. Therefore, there is an urgent need for high-throughput, automated nucleic acid extraction methods. Against this backdrop, the magnetic bead nucleic acid extraction method has emerged. Magnetic bead-based nucleic acid extraction is a modern nucleic acid separation and purification method based on superparamagnetic nanospheres and surface modification technology. Its core principle involves magnetic microspheres coated with functional groups such as silanol and carboxyl groups specifically binding to nucleic acid molecules in a high-salt, low-pH buffer system. Solid-liquid separation is achieved under an external magnetic field, followed by the release of pure nucleic acids through a low-salt, high-pH elution buffer. This method overcomes the limitations of traditional organic solvent methods and silica membrane centrifugation column technology in terms of cumbersome operation, throughput limitations, and toxicity risks, offering significant advantages such as ease of operation, high extraction efficiency, easy automation, and high-throughput processing.

[0005] Currently, this technology has been widely applied in various fields such as clinical pathogen detection, tumor gene screening, next-generation sequencing library construction, and forensic identification, and has become a core supporting technology for large-scale nucleic acid testing of emerging infectious diseases such as COVID-19. With the development of nanomaterials science and molecular biology, the magnetic bead method is evolving towards integration and intelligence, continuously driving technological innovation in the fields of molecular diagnostics and precision medicine through its integration with new technologies such as microfluidic chips and CRISPR detection.

[0006] Although the magnetic bead method simplifies the process through magnetic separation, it still has some problems in the application of viral nucleic acid extraction: such as insufficient lysis efficiency: the viral envelope structure is dense, and conventional lysis buffers cannot effectively release nucleic acids, resulting in a decrease in the detection rate of low-concentration samples; and lack of pH control: existing technologies often ignore the pH coordination in the lysis / washing process, which affects the nucleic acid extraction efficiency. Summary of the Invention

[0007] To address the problems existing in the magnetic bead method, this invention provides a kit for viral nucleic acid extraction and its application.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution solution, and magnetic bead solution.

[0010] The lysis buffer contains guanidine hydrochloride, polyethylene glycol octylphenyl ether (Triton X-100), tris(hydroxymethyl)aminomethane (Tris), hexadecyltrimethylammonium bromide (CTAB), disodium ethylenediaminetetraacetate (EDTA-2Na), and fatty alcohol polyoxyethylene ether, with a pH of 6.0-6.5.

[0011] The cleaning solution 1 contains guanidine hydrochloride, sodium chloride, isopropanol, and polysorbate-20 (Tween 20), with a pH value of 6.0-6.5;

[0012] The cleaning solution 2 contains 75% anhydrous ethanol;

[0013] The eluent contains DEPC water;

[0014] The proteinase K dilution contains tris(hydroxymethyl)aminomethane.

[0015] The magnetic beads in the magnetic bead liquid are bio-magnetic beads, and their components include iron(III) oxide.

[0016] Furthermore, the surface of the magnetic beads in the magnetic bead liquid is modified with silanol or carboxyl groups.

[0017] Furthermore, the magnetic beads in the magnetic bead liquid have a particle size of 800 nm, optimized specific surface area, and 40% more binding sites compared to magnetic beads with a particle size of >1 μm; the magnetic responsiveness is balanced, avoiding the problem of slow separation of magnetic beads with a particle size of <500 nm.

[0018] Furthermore, the magnetic saturation intensity of the magnetic beads in the magnetic bead solution is 55-65 emu / g. The high magnetic saturation intensity ensures rapid magnetic separation, avoids nucleic acid retention and loss, and improves the detection rate of low-concentration samples.

[0019] Furthermore, the lysis buffer contains 350-365g (366-382mM) of guanidine hydrochloride, 196ml of polyethylene glycol octylphenyl ether, 80g (66mM) of tris(hydroxymethyl)aminomethane, 4.5-5.5g (12.3-15.1mM) of hexadecyltrimethylammonium bromide, 10mM of disodium ethylenediaminetetraacetate, and 1.56% by volume of fatty alcohol polyoxyethylene ether.

[0020] Furthermore, the cleaning solution 1 contains 45g (471mM) guanidine hydrochloride, 35g (599mM) sodium chloride, 410ml (5.36mol / L) isopropanol, and 2.9ml (2.6mM) polysorbate-20.

[0021] Furthermore, the lysis buffer contains 360g of guanidine hydrochloride and 5g of hexadecyltrimethylammonium bromide, and the pH value of the lysis buffer is 6.0, which enhances the stability of the CTAB-surfactant complex.

[0022] Furthermore, the pH value of the cleaning solution 1 is 6.0 to maintain a consistent low-salt environment and avoid pH fluctuations that could cause nucleic acid shedding.

[0023] The present invention also provides the application of the kit in viral nucleic acid extraction.

[0024] The present invention also provides an extraction method for the aforementioned reagent kit, comprising the following steps:

[0025] (1) Add 450 μL of lysis buffer to deep well plate 1, add 20 μL of magnetic bead solution and 500 μL of isopropanol mixture to deep well plate 2, add 500 μL of cleaning solution 1 to deep well plate 3, add 500 μL of cleaning solution 2 to deep well plate 4, leave deep well plate 5 empty, and add 100 μL of elution solution to deep well plate 6.

[0026] (2) Add the sample to be tested into the deep well plate 1;

[0027] (3) Use a nucleic acid extraction instrument to extract nucleic acid from the sample according to the following extraction procedure until the extraction is complete.

[0028]

[0029] The nucleic acid extraction instrument is the Tianlong NP968 nucleic acid extractor or a similar automated device.

[0030] Compared with existing technologies, the kit for viral nucleic acid extraction provided by this invention uses magnetic bead nucleic acid extraction method to solve the problems of cumbersome extraction steps, complicated operation and great harm to humans in traditional methods. Moreover, it has higher nucleic acid extraction efficiency than existing extraction kits, solving the problem of low extraction efficiency.

[0031] The high-concentration salt environment of guanidine hydrochloride in the lysis buffer of this invention disrupts the viral envelope protein structure while stabilizing nucleic acids and preventing degradation. CTAB and fatty alcohol polyoxyethylene ether synergistically break down the viral membrane; the combination of the cationic surfactant CTAB and the nonionic surfactant fatty alcohol polyoxyethylene ether works together to dissolve the viral lipid envelope in a weakly acidic environment, improving lysis efficiency. The lysis buffer and washing buffer are both controlled at pH 6.0-6.5; the acidic environment enhances the binding force between nucleic acids and magnetic beads, solving industry pain points such as low lysis efficiency and low extraction efficiency in viral nucleic acid extraction. Experimental data show that this invention improves nucleic acid yield and reduces CT values, making it particularly suitable for clinical testing scenarios with low viral loads. Attached Figure Description

[0032] Figure 1The fluorescence curve is shown in Experiment 1 of this invention, which is a real-time quantitative PCR test.

[0033] Figure 2 The fluorescence curve is shown in Experiment 1 of this invention, which is a real-time PCR fluorescence curve. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] To better illustrate the kit for viral nucleic acid extraction and its application provided in the embodiments of the present invention, further examples are given below.

[0036] Example 1

[0037] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0038] The lysis buffer consisted of 350g guanidine hydrochloride, 196ml Triton X-100, 80g Tris, 4.5g CTAB, 10mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0039] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0040] Cleaning solution 2: 75% anhydrous ethanol;

[0041] Elution buffer: DEPC water;

[0042] Proteinase K solution: Tris solution of proteinase K dry powder.

[0043] Example 2

[0044] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0045] The lysis buffer consisted of 350g guanidine hydrochloride, 196ml Triton X-100, 80g Tris, 5.0g CTAB, 10mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0046] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0047] Cleaning solution 2: 75% anhydrous ethanol;

[0048] Elution buffer: DEPC water;

[0049] Proteinase K solution: Tris solution of proteinase K dry powder.

[0050] Example 3

[0051] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0052] The lysis buffer consisted of 350g guanidine hydrochloride, 196ml Triton X-100, 80g Tris, 5.5g CTAB, 10mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0053] Cleaning solution 1: Guanidine hydrochloride 45g, NaCl 35g, isopropanol 410ml, Tween 20 volume 2.9ml, pH value 6.0;

[0054] Cleaning solution 2: 75% anhydrous ethanol;

[0055] Elution buffer: DEPC water;

[0056] Proteinase K solution: Tris solution of proteinase K dry powder.

[0057] Example 4

[0058] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0059] The lysis buffer consisted of 355g guanidine hydrochloride, 196ml Triton X-100, 80g Tris, 4.5g CTAB, 10mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0060] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0061] Cleaning solution 2: 75% anhydrous ethanol;

[0062] Elution buffer: DEPC water;

[0063] Proteinase K solution: Tris solution of proteinase K dry powder.

[0064] Example 5

[0065] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0066] The lysis buffer consisted of 355g guanidine hydrochloride, 196ml Triton X-100, 80g Tris, 5.0g CTAB, 10mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0067] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0068] Cleaning solution 2: 75% anhydrous ethanol;

[0069] Elution buffer: DEPC water;

[0070] Proteinase K solution: Tris solution of proteinase K dry powder.

[0071] Example 6

[0072] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0073] The lysis buffer consisted of 355g guanidine hydrochloride, 196ml Triton X-100, 80g Tris, 5.5g CTAB, 10mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0074] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0075] Cleaning solution 2: 75% anhydrous ethanol;

[0076] Elution buffer: DEPC water;

[0077] Proteinase K solution: Tris solution of proteinase K dry powder.

[0078] Example 7

[0079] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0080] The lysis buffer consisted of 360 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 4.5 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0081] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0082] Cleaning solution 2: 75% anhydrous ethanol;

[0083] Elution buffer: DEPC water;

[0084] Proteinase K solution: Tris solution of proteinase K dry powder.

[0085] Example 8

[0086] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0087] The lysis buffer consisted of 360 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.0 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0088] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0089] Cleaning solution 2: 75% anhydrous ethanol;

[0090] Elution buffer: DEPC water;

[0091] Proteinase K solution: Tris solution of proteinase K dry powder.

[0092] Example 9

[0093] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0094] The lysis buffer consisted of 360 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.5 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0095] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0096] Cleaning solution 2: 75% anhydrous ethanol;

[0097] Elution buffer: DEPC water;

[0098] Proteinase K solution: Tris solution of proteinase K dry powder.

[0099] Example 10

[0100] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0101] The lysis buffer consisted of 365 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 4.5 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0102] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0103] Cleaning solution 2: 75% anhydrous ethanol;

[0104] Elution buffer: DEPC water;

[0105] Proteinase K solution: Tris solution of proteinase K dry powder.

[0106] Example 11

[0107] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0108] The lysis buffer consisted of 365 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.0 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0109] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0110] Cleaning solution 2: 75% anhydrous ethanol;

[0111] Elution buffer: DEPC water;

[0112] Proteinase K solution: Tris solution of proteinase K dry powder.

[0113] Example 12

[0114] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0115] The lysis buffer consisted of 365 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.5 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0116] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0117] Cleaning solution 2: 75% anhydrous ethanol;

[0118] Elution buffer: DEPC water;

[0119] Proteinase K solution: Tris solution of proteinase K dry powder.

[0120] Example 13

[0121] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0122] The lysis buffer consisted of 360 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.0 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.0.

[0123] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.5;

[0124] Cleaning solution 2: 75% anhydrous ethanol;

[0125] Elution buffer: DEPC water;

[0126] Proteinase K solution: Tris solution of proteinase K dry powder.

[0127] Example 14

[0128] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0129] The lysis buffer consisted of 360 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.0 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.5.

[0130] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.0;

[0131] Cleaning solution 2: 75% anhydrous ethanol;

[0132] Elution buffer: DEPC water;

[0133] Proteinase K solution: Tris solution of proteinase K dry powder.

[0134] Example 15

[0135] A kit for viral nucleic acid extraction includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution buffer, and magnetic bead solution; details are as follows:

[0136] The lysis buffer consisted of 360 g guanidine hydrochloride, 196 ml Triton X-100, 80 g Tris, 5.0 g CTAB, 10 mM EDTA-2Na, 1.56% fatty alcohol polyoxyethylene ether, and a pH of 6.5.

[0137] Cleaning solution 1: 45g guanidine hydrochloride, 35g NaCl, 410ml isopropanol, 2.9ml Tween 20, pH 6.5;

[0138] Cleaning solution 2: 75% anhydrous ethanol;

[0139] Elution buffer: DEPC water;

[0140] Proteinase K solution: Tris solution of proteinase K dry powder.

[0141] Experimental Example 1

[0142] To better illustrate the characteristics of the kits for viral nucleic acid extraction provided in the embodiments of the present invention, the kits for viral nucleic acid extraction provided in Examples 1-12 will be subjected to corresponding nucleic acid extraction tests below.

[0143] Experimental methods:

[0144] (1) Sample preparation

[0145] Syncytosis A pseudovirus was collected at an initial concentration of 10. 4 copies / ml, diluted to 10 3 Copies / ml concentration for future reference.

[0146] (2) Sample processing and detection

[0147] Nucleic acid extraction was performed on the prepared syncytial A pseudovirus samples using the kits provided in Examples 1-12.

[0148] The results are shown in Table 1 and Figure 1 As shown, the smallest detected CT value indicates the largest amount of nucleic acid extracted. The kit of the present invention has high efficiency in extracting viral nucleic acid, especially the kit provided in Example 8, which has the highest efficiency in extracting viral nucleic acid (as shown in Table 1).

[0149] Table 1

[0150]

[0151]

[0152] Figure 1 This is the fluorescence curve in quantitative real-time PCR. The number of cycles where the threshold line (horizontal straight line) intersects the amplification curve is the CT value of the curve. The CT value represents the concentration of the target nucleic acid being amplified. The smaller the CT value, the more target nucleic acid is extracted and the better the effect. The more delayed the CT value, the less target nucleic acid is extracted and the worse the effect.

[0153] Experimental Example 2

[0154] To better illustrate the characteristics of the viral nucleic acid extraction kits provided in the embodiments of the present invention, the corresponding nucleic acid extraction tests were performed on the kits provided in Examples 8 and 13, 14, and 15. The experimental methods are the same as in Experiment 1.

[0155] The results are shown in Table 2 and Figure 2 As shown, the smallest CT value indicates the largest amount of nucleic acid extracted, suggesting higher efficiency in viral nucleic acid extraction.

[0156] Table 2

[0157] Group CT value Example 8 26.01 Example 13 29.21 Example 14 28.65 Example 15 29.19

[0158] Experimental Example 3

[0159] The reagent kit provided in Example 8 of this invention was compared with the commercially available Baochuang nucleic acid extraction kit for nucleic acid extraction testing.

[0160] Experimental methods:

[0161] (1) Sample preparation

[0162] The concentration of pseudovirus syncytial A was 10. 4 10 5 10 6 The copies / mL and the pseudovirus syncytial B concentration were 10. 4 10 5 10 6 copies / mL;

[0163] (2) Sample processing and detection

[0164] Nucleic acid extraction was performed using the kit provided in Example 8 of this invention and the commercially available Baochuang nucleic acid extraction kit, respectively.

[0165] The results are shown in the table. Under the conditions of two targets and three different concentrations, the Ct value of nucleic acid extraction by the kit provided by this invention is lower than that of the kit provided by Baochuang. This indicates that the kit provided by this invention has a stronger ability to extract nucleic acids, can extract more nucleic acids, has higher extraction efficiency, and has obvious advantages.

[0166] Table 3

[0167] Target concentration Ct value of the present invention Baochuang Ct value <![CDATA[Pseudovirus Syncytin A-10 4 copies / mL]]> 19.23 20.99 <![CDATA[Pseudovirus Syncytin A-10 5 copies / mL]]> 15.95 17.38 <![CDATA[Pseudovirus Syncytin A-10 6 copies / mL]]> 13.37 14.68 <![CDATA[Pseudovirus syncytin B-10 4 copies / mL]]> 20.93 22.37 <![CDATA[Pseudovirus Syncytin B-10 5 copies / mL]]> 17.18 18.47 <![CDATA[Pseudovirus RSV B-10 6 copies / mL]]> 14.93 16.25

[0168] This invention, through the limitation of specific component combinations and synergistic ratios of lysis buffer and washing solution 1, as well as the regulation of a dual pH system, enables the nucleic acid extraction efficiency of the kit to be significantly superior to existing technologies, and has great application value in viral nucleic acid extraction.

[0169] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kit for viral nucleic acid extraction, characterized in that, Includes lysis buffer, washing buffer 1, washing buffer 2, elution buffer, proteinase K dry powder, proteinase K dilution solution, and magnetic bead solution; The lysis buffer contains guanidine hydrochloride, polyethylene glycol octylphenyl ether, tris(hydroxymethyl)aminomethane, hexadecyltrimethylammonium bromide, disodium ethylenediaminetetraacetate, and fatty alcohol polyoxyethylene ether, with a pH of 6.0-6.

5. The cleaning solution 1 contains guanidine hydrochloride, sodium chloride, isopropanol, and polysorbate-20, with a pH value of 6.0-6.5; The cleaning solution 2 contains 75% anhydrous ethanol; The eluent contains DEPC water; The proteinase K dilution contains tris(hydroxymethyl)aminomethane.

2. The reagent kit according to claim 1, characterized in that, The magnetic beads in the liquid are modified with silanol or carboxyl groups on their surface.

3. The reagent kit according to claim 1, characterized in that, The magnetic beads in the liquid have a particle size of 800 nm.

4. The reagent kit according to claim 1, characterized in that, The magnetic saturation intensity of the magnetic beads in the magnetic bead liquid is 55-65 emu / g.

5. The reagent kit according to claim 1, characterized in that, The lysis solution contains 350-365g of guanidine hydrochloride, 196ml of polyethylene glycol octylphenyl ether, 80g of tris(hydroxymethyl)aminomethane, 4.5-5.5g of hexadecyltrimethylammonium bromide, 10mM of disodium ethylenediaminetetraacetate, and 1.56% fatty alcohol polyoxyethylene ether by volume.

6. The reagent kit according to claim 5, characterized in that, The cleaning solution 1 contains 45g of guanidine hydrochloride, 35g of sodium chloride, 410ml of isopropanol, and 2.9ml of polysorbate-20.

7. The reagent kit according to claim 6, characterized in that, The lysis buffer contained 360g of guanidine hydrochloride and 5g of cetyltrimethylammonium bromide, and the pH value of the lysis buffer was 6.

0.

8. The reagent kit according to claim 7, characterized in that, The pH value of the cleaning solution 1 is 6.

0.

9. The use of the kit according to any one of claims 1 to 8 in viral nucleic acid extraction.

10. The extraction method of the reagent kit according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Add 450 μL of lysis buffer to deep well plate 1, add 20 μL of magnetic bead solution and 500 μL of isopropanol mixture to deep well plate 2, add 500 μL of cleaning solution 1 to deep well plate 3, add 500 μL of cleaning solution 2 to deep well plate 4, and add 100 μL of elution solution to deep well plate 6. (2) Add the sample to be tested into the deep well plate 1; (3) Use a nucleic acid extraction instrument to extract nucleic acid from the sample according to the set extraction program.

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