Nucleic acid extraction composition, reagent, kit and application thereof

Abstract

The invention provides a nucleic acid extraction composition, a reagent, a kit and application thereof, and belongs to the technical field of molecular biology. The nucleic acid extraction composition comprises at least five of sodium hydroxide, lithium chloride, lauryl sodium sulfate, polylysine, tris (2-formyl ethyl) phosphine hydrochloride, Chelex-100 and bovine serum albumin. Compared with an existing nucleic acid extraction reagent, the nucleic acid extraction composition is used for nucleic acid extraction without complex extraction and purification steps, a split product can be directly used for downstream reactions such as subsequent isothermal amplification, reaction inhibition is not caused, the process can be simplified, the needed nucleic acid can be extracted in one step at normal temperature within a short time, and the method is simple and convenient to operate. The extracted nucleic acid sample does not need to be further treated and can be directly used for downstream nucleic acid amplification detection.

Description

Technical Field

[0001] The present application relates to the technical field of molecular biology, and specifically, to nucleic acid extraction compositions, reagents, kits and their applications. More specifically, the present application relates to a nucleic acid extraction composition, a nucleic acid extraction reagent, a nucleic acid extraction kit, a nucleic acid extraction method and a nucleic acid detection method for non-diagnostic purposes. Background Art

[0002] As an accurate disease diagnosis method, nucleic acid detection has now been widely used in the detection of various pathogen samples. Among them, nucleic acid extraction is an important link in the nucleic acid analysis process, which has an important impact on the accuracy, sensitivity, etc. of the final results. Since the nucleic acid located inside the cell is not only protected by the cell wall and cell membrane on the outside, but for eukaryotes, it is also protected by the nuclear membrane of the cell nucleus. Even for prokaryotic viruses, the outside is wrapped with a nucleocapsid. When performing nucleic acid detection, it is necessary to destroy the cell structure and separate the nucleic acid from the protein to completely expose the nucleic acid.

[0003] Common nucleic acid extraction methods include boiling method, thermal lysis method, proteinase K method, etc. These methods all require heating during the operation process, that is, nucleic acid extraction needs to be realized with the help of a constant temperature incubation device. In addition, existing nucleic acid lysis reagents usually need to perform extraction and purification operations after lysis to reduce the adverse effects of interfering components in the lysed samples on subsequent nucleic acid detection (such as PCR, qPCR). For example, the mainstream magnetic bead method extraction usually requires steps such as lysis, washing, and elution, with high cost, many operation steps, and long time consumption, and is not suitable for use under experimental conditions lacking professional technical personnel such as in primary hospitals, detection sites, and home self-examinations. Summary of the Invention

[0004] The present application aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present application is to propose a nucleic acid extraction composition, reagent, kit and their applications, which are used to extract nucleic acid in a test sample at room temperature in one step, with high extraction efficiency, simple operation and low cost, and are suitable for efficient lysis of various pathogenic microorganisms.

[0005] Specifically, the technical solution of the present application is as follows:

[0006] In the first aspect of the present application, the present application proposes a nucleic acid extraction composition. According to an embodiment of the present application, the nucleic acid extraction composition includes at least five of sodium hydroxide, lithium chloride, sodium dodecyl sulfate, polylysine, tris(2-carboxyethyl)phosphine hydrochloride, Chelex-100 and bovine serum albumin.

[0007] Compared with existing nucleic acid extraction reagents, nucleic acid extraction using the aforementioned nucleic acid extraction composition does not require complex extraction and purification steps. The lysis products can be directly used for subsequent downstream reactions such as isothermal amplification without causing reaction inhibition, enabling process simplification. Moreover, the desired nucleic acid can be extracted in one step at room temperature within a short time, and the extracted nucleic acid sample does not require further treatment and can be directly used for downstream nucleic acid amplification detection.

[0008] Sodium hydroxide (NaOH) in the aforementioned components, as a strong base, can provide a high pH environment, rapidly disrupt the primary structure of proteins, and promote the rupture of cell membranes and nuclear membranes. This process helps to accelerate the release of nucleic acids and, at the same time, ensures the relative structural integrity of nucleic acids during extraction by inactivating nucleases.

[0009] Lithium chloride (LiCl) in the aforementioned components can effectively strip proteins from chromatin, separating proteins from nucleic acids. In addition, lithium ions can change the charged properties on the surfaces of cell walls and cell membranes, thereby enhancing the ability of cells to be rapidly lysed. Lithium chloride also has the excellent property of preserving DNA for a long time and can effectively stabilize the structure of nucleic acids.

[0010] Sodium dodecyl sulfate (SDS) in the aforementioned components binds to proteins, disrupting the secondary structure of proteins (such as hydrogen bonds, salt bonds, and hydrophobic interactions), resulting in the disintegration of the native conformation of proteins, thereby dissolving membrane proteins, depolymerizing nuclear proteins, and destroying the structures of cell membranes and nuclear membranes, ultimately releasing nucleic acids. The mechanism of action of SDS is based on the denaturation of proteins by non-covalent forces, but this denaturation is reversible. After removing SDS, some proteins may recover their original structure and function, thus affecting the nucleic acid extraction effect and subsequent PCR reactions.

[0011] Polylysine in the aforementioned components, as an amphoteric polymer, has an isoelectric point of 9.04. When the pH value of the nucleic acid extraction solution is greater than 9.04, polylysine carries a negative charge and plays a role in disrupting cell membranes similar to SDS, promoting the release of nucleic acids; when the pH value is less than 9.04, polylysine carries a positive charge and can bind to SDS through hydrophobic and electrostatic interactions, thereby preventing the destruction of polymerase activity by SDS and ensuring the efficient progress of subsequent amplification reactions.

[0012] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in the aforementioned components, as a strong reducing agent, has good water solubility and can break the disulfide bonds within and between proteins, restoring them to the thiol state. This process destroys the spatial structure and activity of proteins, helps to rupture cell membranes, and inhibits the activity of nucleases by breaking their disulfide bonds, reducing interference with subsequent nucleic acid amplification reactions. At the same time, TCEP can effectively act within a wide pH range, further enhancing the cell lysis efficiency during nucleic acid extraction.

[0013] Chelex-100 in the aforementioned components is a chelating resin, mainly composed of styrene and divinylbenzene, which can efficiently chelate divalent metal ions, inhibit the activity of nucleases in the sample, and protect nucleic acids from degradation. Chelex-100 also promotes the rupture of cell membranes and helps protein precipitation, contributing to the efficient release of nucleic acids. By centrifugation or static precipitation, Chelex-100 particles can be separated from divalent metal ions and other impurities in the sample, avoiding interference in the PCR reaction.

[0014] Bovine serum albumin (BSA) in the aforementioned components, as a PCR enhancer, can improve the polymerization ability of the PCR reaction, help open the secondary structure of primers, and enhance the specificity of primers to templates. At the same time, BSA can neutralize impurities such as hemoglobin that may exist in crude samples, reduce the negative impact of these impurities on the PCR reaction, and further improve the efficiency and accuracy of nucleic acid amplification.

[0015] In some examples of this application, the aforementioned nucleic acid extraction composition includes at least six of sodium hydroxide, lithium chloride, sodium dodecyl sulfate, polylysine, tris(2-carboxyethyl)phosphine hydrochloride, Chelex-100, and bovine serum albumin. By adjusting and optimizing the components of the composition, it shows more stability in various practical application scenarios, can effectively extract nucleic acids at room temperature, ensuring high efficiency and simplicity of operation.

[0016] In some preferred examples of this application, the aforementioned nucleic acid extraction composition includes sodium hydroxide, lithium chloride, sodium dodecyl sulfate, polylysine, tris(2-carboxyethyl)phosphine hydrochloride, Chelex-100, and bovine serum albumin.

[0017] In some preferred examples of this application, the nucleic acid extraction composition includes at least six of 50 mM - 800 mM sodium hydroxide, 5 mM - 100 mM lithium chloride, 0.05 wt% - 0.85 wt% sodium dodecyl sulfate, 0.1 wt% - 2 wt% polylysine, 1 mM - 10 mM tris(2-carboxyethyl)phosphine hydrochloride, 1 wt% - 30 wt% Chelex-100, and 0.01 wt% - 0.5 wt% bovine serum albumin. The ratio of the aforementioned components effectively improves the nucleic acid extraction efficiency, can quickly break cell membranes, release nucleic acids, while inhibiting the activity of nucleases and avoiding nucleic acid degradation. Through the synergistic effect of the ratio of polylysine and BSA, the inhibition of SDS on the PCR reaction is reduced, the polymerase activity is ensured, and the efficiency and specificity of PCR amplification are improved. In addition, Chelex-100 can remove divalent metal ions in the sample, further purify nucleic acids, and ensure its high purity, suitable for downstream analysis.

[0018] It is understandable that, in the aforementioned nucleic acid extraction composition, the concentration of sodium hydroxide can optionally be 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM or 800 mM; the concentration of lithium chloride can optionally be 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM; the weight percentage (wt%) of sodium dodecyl sulfate can optionally be 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt% or 0.5 wt%; the weight percentage of polylysine can optionally be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt% or 2 wt%; the concentration of tris(2-carboxyethyl)phosphine hydrochloride can optionally be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM; the weight percentage of Chelex-100 can optionally be 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt% or 30 wt%; the weight percentage of bovine serum albumin can optionally be 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt% or 0.5 wt%.

[0019] In some preferred examples of the present application, the nucleic acid extraction composition includes at least six of the following: 100 mM - 700 mM sodium hydroxide, 5 mM - 35 mM lithium chloride, 0.1 wt% - 0.85 wt% sodium dodecyl sulfate, 0.2 wt% - 1 wt% polylysine, 3 mM - 15 mM tris(2-carboxyethyl)phosphine hydrochloride, 5 wt% - 20 wt% Chelex-100, and 0.1 wt% - 0.5 wt% bovine serum albumin. The ratio of the foregoing components effectively improves the nucleic acid extraction efficiency, can rapidly disrupt cell membranes, release nucleic acids, while inhibiting nuclease activity and avoiding nucleic acid degradation. Through the synergistic effect of the ratio of polylysine and BSA, the inhibition of the PCR reaction by SDS is reduced, the polymerase activity is ensured, and the efficiency and specificity of PCR amplification are improved. In addition, Chelex-100 can remove divalent metal ions in the sample, further purify nucleic acids, and ensure its high purity, suitable for downstream analysis.

[0020] In some examples of the present application, the foregoing nucleic acid extraction composition is selected from any one of 1) - 21):

[0021] 1) 100 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0022] 2) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0023] 3) 700 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0024] 4) 300 mM sodium hydroxide, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0025] 5) 300 mM sodium hydroxide, 5 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0026] 6) 300 mM sodium hydroxide, 35 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0027] 7) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0028] 8) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.1 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0029] 9) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.85 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0030] 10) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0031] 11) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.2 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0032] 12) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 1 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0033] 13) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0034] 14) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 3 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0035] 15) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 15 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0036] 16) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, and 0.3 wt% bovine serum albumin;

[0037] 17) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 5 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0038] 18) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 20 wt% Chelex-100, and 0.3 wt% bovine serum albumin;

[0039] 19) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, and 10 wt% Chelex-100;

[0040] 20) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.1 wt% bovine serum albumin;

[0041] 21) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.5 wt% bovine serum albumin.

[0042] The nucleic acid extraction compositions with the above different ratios can effectively improve the efficiency of nucleic acid extraction, reduce PCR inhibition, and enhance the accuracy and sensitivity of subsequent amplification reactions. In addition, providing multiple formulations for selection can meet the requirements of various application scenarios.

[0043] In some preferred examples of the present application, the aforementioned nucleic acid extraction composition is selected from any one of 2), 15), 18), or 21). After nucleic acid extraction using any one of the formulations of 2), 15), 18), or 21), the Ct value of quantitative PCR amplification is relatively low, indicating that any one of the aforementioned formulations has a high nucleic acid extraction efficiency, a large amount of extracted nucleic acid, good purity, and is not interfered by inhibitors. At the same time, it also indicates that the aforementioned formulations can effectively remove inhibitors such as metal ions and enzymes during the nucleic acid extraction process.

[0044] In some preferred examples of the present application, the aforementioned nucleic acid extraction composition is selected from 2). In an appropriate alkaline environment, polylysine is negatively charged and can exert a cell lysis effect similar to SDS. At the same time, TCEP can further promote the cell lysis and nucleic acid release process by disrupting the disulfide bonds within and between proteins. However, too high a pH will cause a significant decrease in the reducing ability of TCEP, which has a negative impact on nucleic acid lysis. Additionally, appropriate amounts of LiCl, SDS, and polylysine can increase the cell lysis efficiency while minimizing the negative impact on the subsequent PCR amplification process. Adding appropriate amounts of Chelex-100 and BSA can avoid affecting the subsequent PCR amplification process by removing and shielding impurities and PCR interferents.

[0045] In some examples of the present application, the pH of the aforementioned nucleic acid extraction composition is selected from 9.0 - 13, optionally 9, 10, 11, 12, or 13.

[0046] In some preferred examples of the present application, the pH of the nucleic acid extraction composition is selected from 9.35 - 11.34; optionally 9.35, 10.88, 11.23, 10.72, 10.68, 11.21, 10.86, 11.02, 10.91, 10.52, 10.98, 11.34, 11.13, 10.85, 10.21, 11.02, 11.31, 10.96, 10.97, 10.83, or 10.76.

[0047] In some more preferred examples of the present application, the pH of the aforementioned nucleic acid extraction composition is 10.88. In this alkaline environment, polylysine is negatively charged and can exert a cell lysis effect similar to that of SDS. At the same time, TCEP can further promote the cell lysis and nucleic acid release process by disrupting the disulfide bonds within and between proteins. However, too high a pH will cause a significant decrease in the reducing ability of TCEP, which will have a negative impact on nucleic acid lysis. In addition, appropriate amounts of LiCl, SDS, and polylysine can improve the cell lysis efficiency while minimizing the negative impact on the subsequent PCR amplification process. Adding appropriate amounts of Chelex-100 and BSA can avoid affecting the subsequent PCR amplification process by removing and shielding impurities and PCR interferents.

[0048] In a second aspect of the present application, the present application provides a nucleic acid extraction reagent. According to the embodiments of the present application, the aforementioned nucleic acid extraction reagent includes: the nucleic acid extraction composition of any example of the first aspect. Compared with existing nucleic acid extraction reagents, using the aforementioned nucleic acid extraction reagent for nucleic acid extraction does not require a complex pretreatment process, and the required nucleic acid can be extracted in one step at room temperature. The extracted nucleic acid sample does not require further treatment and can be directly used for downstream nucleic acid amplification detection.

[0049] In a third aspect of the present application, the present application provides a nucleic acid extraction kit. According to the embodiments of the present application, the aforementioned nucleic acid extraction kit includes: the nucleic acid extraction reagent described in the second aspect. Compared with existing nucleic acid extraction reagents, using the aforementioned kit for nucleic acid extraction does not require a complex pretreatment process, and the required nucleic acid can be extracted in one step at room temperature. The extracted nucleic acid sample does not require further treatment and can be directly used for downstream nucleic acid amplification detection.

[0050] In a fourth aspect of the present application, the present application provides a nucleic acid extraction method. According to the embodiments of the present application, the aforementioned method includes: performing nucleic acid extraction treatment on a sample to be tested with the nucleic acid extraction composition described in the first aspect or the nucleic acid extraction reagent described in the second aspect. The nucleic acid extraction method of the present application does not require professional special instruments and can be carried out in a sealed environment throughout the process. While improving the detection efficiency, it can also greatly avoid cross-contamination and environmental pollution generated during the sample treatment process, and can be applied to different environments, especially the environment of outdoor real-time nucleic acid detection. The nucleic acid extraction method using the aforementioned nucleic acid extraction composition has low cost, a simple operation process, a short operation time, and avoids the problems of heating or equipment dependence in traditional methods, and is suitable for environments such as primary hospitals, detection sites, and home self-examination.

[0051] In some examples of the present application, the aforementioned nucleic acid is derived from microorganisms, such as fungi, bacteria, viruses, etc.

[0052] In some preferred examples of the present application, the foregoing method further includes: the step of collecting a sample by an oropharyngeal swab and / or a nasopharyngeal swab.

[0053] In some preferred examples of the present application, the sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice or cerebrospinal fluid.

[0054] In some preferred examples of the present application, the nucleic acid extraction treatment includes: mixing the sample to be tested with a nucleic acid extraction composition or a nucleic acid extraction reagent for 5 min - 10 min; obtaining a supernatant by static settling or centrifugation treatment. The supernatant contains the nucleic acid to be tested and can be directly used for subsequent amplification reactions.

[0055] In some preferred examples of the present application, the sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the preservation solution to the nucleic acid extraction composition or the nucleic acid extraction reagent is 1:(1 - 3), optionally 1:1, 1:2 or 1:3.

[0056] In the fifth aspect of the present application, the present application provides a nucleic acid detection method for non-diagnostic purposes. According to the embodiments of the present application, the foregoing method includes: (1) performing nucleic acid extraction treatment on the sample to be tested with the nucleic acid extraction composition of any example in the first aspect or the nucleic acid extraction reagent described in the second aspect; (2) amplifying and detecting the nucleic acid extracted in step (1). Using the nucleic acid extraction composition or the nucleic acid extraction reagent of the embodiments of the present application can effectively improve the nucleic acid extraction efficiency, reduce nucleic acid loss, ensure the accuracy of the amplification result, and thus improve the accuracy of nucleic acid detection.

[0057] In some examples of the present application, the foregoing nucleic acid is derived from microorganisms, such as fungi, bacteria, viruses, etc.

[0058] In some preferred examples of the present application, the foregoing method further includes: the step of collecting a sample by an oropharyngeal swab and / or a nasopharyngeal swab.

[0059] In some preferred examples of the present application, the sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice or cerebrospinal fluid.

[0060] In some preferred examples of the present application, the nucleic acid extraction treatment includes: mixing the sample to be tested with a nucleic acid extraction composition or a nucleic acid extraction reagent for 5 min - 10 min; obtaining a supernatant by static settling or centrifugation treatment. The supernatant contains the nucleic acid to be tested and can be directly used for subsequent amplification reactions.

[0061] In some preferred examples of the present application, the sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the preservation solution to the nucleic acid extraction composition or the nucleic acid extraction reagent is 1:(1 - 3), optionally 1:1, 1:2 or 1:3.

[0062] Additional aspects and advantages of the present application will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present application. Brief Description of the Drawings

[0063] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other accompanying drawings can be obtained based on these drawings without creative efforts.

[0064] Figure 1 It is a schematic diagram of the results of a nucleic acid release effect comparison curve of nucleic acid extraction reagents with different formulations provided in an embodiment of the present application for nucleic acid extraction of Staphylococcus aureus;

[0065] Figure 2 It is a schematic diagram of the results of a nucleic acid release effect comparison curve of nucleic acid extraction reagents provided in an embodiment of the present application for nucleic acid extraction of different pathogens;

[0066] Figure 3 It is a schematic diagram of the results of a nucleic acid release effect comparison curve of three different nucleic acid extraction reagents used in a comparative experiment provided in an embodiment of the present application. Detailed Embodiments

[0067] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0068] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0069] The term "optionally" is only used for descriptive purposes and cannot be construed as indicating or implying relative importance. Thus, features defined with "optionally" may explicitly or implicitly include or not include this feature.

[0070] The solution of this application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate this application and should not be regarded as limiting the scope of this application. For those without specific techniques or conditions noted in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications shall be followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0071] Example 1: Application of Nucleic Acid Extraction Reagent Optimized Based on Different Component Concentrations in PCR Detection

[0072] The nucleic acid extraction reagent in this example was prepared according to the component concentrations shown in Table 1, and the concentrations of each component were optimized. The specific components and concentrations of the reagents used are as follows:

[0073] Sodium hydroxide (NaOH): 100 mM, 300 mM, 700 mM

[0074] Lithium chloride (LiCl): 0 mM, 5 mM, 15 mM, 35 mM

[0075] Sodium dodecyl sulfate (SDS): 0 wt%, 0.10 wt%, 0.25 wt%, 0.85 wt%

[0076] Poly-L-lysine: 0 wt%, 0.2 wt%, 0.5 wt%, 1.0 wt%

[0077] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP): 0 mM, 3 mM, 7 mM, 15 mM

[0078] Chelex-100: 0 wt%, 5 wt%, 10 wt%, 20 wt%

[0079] Bovine serum albumin (BSA): 0 wt%, 0.1 wt%, 0.3 wt%, 0.5 wt%

[0080] All components were used to prepare the nucleic acid release agent with water as the solvent, and the component concentrations were optimized to ensure the best results during nucleic acid extraction.

[0081] Table 1 Nucleic Acid Extraction Reagent Formulation and Detection Results

[0082]

[0083]

[0084]

[0085] 1. Nucleic Acid Extraction and Detection

[0086] Taking Staphylococcus aureus as an example, nucleic acid extraction and detection were carried out:

[0087] (1) Sample preparation: Staphylococcus aureus (BNCC381462) was purchased from Beijing NaChuangLian Biotechnology Co., Ltd. The above bacterial liquid was inoculated into 5 mL of sterilized tryptone broth medium and cultured at 37 °C on a shaker at 170 rpm for 18 hours. Take 1 mL of the overnight-cultured bacterial liquid, transfer it to a 1.5 mL centrifuge tube, centrifuge at 7000 rpm for 2 minutes at room temperature, discard the supernatant, and collect the bacterial cells. Dilute the bacterial cells with sterile normal saline to 1.0×10^ 6 CFU / mL for standby.

[0088] (2) Nucleic acid extraction: Take 300 μL of the diluted Staphylococcus aureus bacterial liquid and transfer it to a 1.5 mL centrifuge tube. Add 600 μL of nucleic acid extraction reagent (prepared according to the components listed in Table 1). Cover the centrifuge tube cap, gently invert and mix well, and then let it stand at room temperature for 10 minutes. The supernatant after standing is the final nucleic acid extraction product.

[0089] (3) Evaluation of nucleic acid extraction effect: Use the Staphylococcus aureus nucleic acid detection kit (PCR-fluorescent probe method) from Guangdong Huankai Microbial Sci-Tech Co., Ltd. to perform PCR detection and evaluate the lysis effect of different nucleic acid extraction reagent formulations on Staphylococcus aureus.

[0090] The specific operation process is as follows:

[0091] 1. Take out the premix from the kit, fully melt it, then vortex and mix well and centrifuge briefly. Add 20 μL of the premix to the PCR reaction tube;

[0092] 2. Add 5 μL of the nucleic acid extraction supernatant obtained from the above step to the PCR reaction tube, tighten the tube cap, and centrifuge briefly;

[0093] 3. Immediately carry out the PCR amplification reaction. The total volume of the PCR reaction system is 25 μL, and the detection is carried out under the FAM detection channel. In the second stage of the reaction, fluorescence signals are collected at 60 °C. The specific PCR program is shown in Table 2.

[0094] Table 2 PCR program

[0095]

[0096] The nucleic acid detection results are shown in Table 1 and Figure 1As shown, the amplification Ct values were analyzed, and the optimal nucleic acid extraction reagent formulation was the one described in Group 2 of Table 1, i.e., using water as the solvent, and the concentrations of other components were: sodium hydroxide 300 mM, lithium chloride 15 mM, SDS 0.25 wt%, polylysine 0.5 wt%, TCEP 7 mM, Chelex-100 10 wt%, BSA 0.3 wt%. Specific analysis: In an appropriate alkaline environment, polylysine carries a negative charge and can exert a cell lysis effect similar to that of SDS. At the same time, TCEP can further promote the cell lysis and nucleic acid release process by disrupting the disulfide bonds within and between proteins. However, too high a pH will cause a significant decrease in the reducing ability of TCEP and have a negative impact on nucleic acid lysis. In addition, appropriate amounts of LiCl, SDS, and polylysine can improve the cell lysis efficiency while minimizing the negative impact on the subsequent PCR amplification process. Adding appropriate amounts of Chelex-100 and BSA can avoid affecting the subsequent PCR amplification process by removing and shielding impurities and PCR interferents.

[0097] Example 2: Application of Nucleic Acid Extraction Reagent Based on Optimized Formulation in Nucleic Acid Detection of Multiple Pathogens

[0098] 1. Preparation of Nucleic Acid Extraction Reagent

[0099] In this example, the formulation of Group 2 in Table 1 was used to prepare the nucleic acid extraction reagent. The concentrations of the formulation components were as follows: sodium hydroxide 300 mM, lithium chloride 15 mM, SDS 0.25 wt%, polylysine 0.5 wt%, TCEP 7 mM, Chelex-100 10 wt%, BSA 0.3 wt%, using water as the solvent.

[0100] 2. Nucleic Acid Extraction and Detection

[0101] Taking Staphylococcus aureus, Mycobacterium tuberculosis, Escherichia coli, Candida albicans, and the novel coronavirus as examples, nucleic acid extraction and detection were carried out. The sample preparation and nucleic acid extraction methods for each pathogen were the same as those in Example 1, and the specific process was as follows:

[0102] (1) Staphylococcus aureus (BNCC381462), Mycobacterium tuberculosis (H37Ra attenuated strain), Escherichia coli (BNCC133264), and Candida albicans (BNCC299343) were all purchased from Beijing Na Biotechnology Co., Ltd., and the same nucleic acid extraction process as in Example 1 was used.

[0103] (2) The novel coronavirus pseudovirus was purchased from Guangdong Hexin Health Technology Co., Ltd. During the sample preparation process, it was diluted to 5000 copies / mL with sterile normal saline for standby.

[0104] The nucleic acid extraction products of all pathogens were subjected to PCR amplification using corresponding commercially available nucleic acid detection kits, and the detection results are shown in Table 3 and Figure 2 as follows. The kits are: Mycobacterium tuberculosis nucleic acid detection kit (fluorescent PCR method, purchased from Jiangsu Hongweitesi Medical Technology Co., Ltd.); Candida albicans nucleic acid detection kit (PCR-fluorescent probe method, purchased from Kunpeng Gene (Beijing) Scientific Instruments Co., Ltd.); Escherichia coli nucleic acid detection kit (PCR-fluorescent probe method, purchased from Shengxiang Biotech Co., Ltd.); 2019-nCoV nucleic acid detection kit (fluorescent PCR method, purchased from Shengxiang Biotech Co., Ltd.).

[0105] Table 3: Nucleic acid extraction and detection results of different pathogens

[0106] Pathogen type Average Ct value Staphylococcus aureus 22.3 Mycobacterium tuberculosis 23.4 Escherichia coli 21.5 Candida albicans 23.6 SARS-CoV-2 20.3

[0107] Comparative experiment

[0108] To further verify the effectiveness of the nucleic acid extraction reagent of the present invention, a commercial nucleic acid extraction kit was used as a comparison. The same nucleic acid extraction was performed on Staphylococcus aureus, and the commercial kits used included:

[0109] (1) "Sample Release Agent" (FA04001) of Zhenshi Biotech, which is a nucleic acid rapid extraction product, and its main components include NaOH, yeast total RNA, grinding beads and water. The extraction method is the same as the process described in Example 1;

[0110] (2) "Magnetic Bead Method Pathogenic Microorganism DNA / RNA Extraction Kit" (CW3061S) of Jiangsu Kangwei Century Biotechnology Co., Ltd., and the extraction method is carried out according to the kit instructions.

[0111] The detection results are shown in Table 4 and Figure 3 as follows. The nucleic acid extraction reagent of the present application has achieved results close to those of commercial nucleic acid extraction kits that require multiple steps and are time-consuming, with greatly simplified operation steps, and is superior to other commercially available nucleic acid rapid extraction products.

[0112] Table 4: Comparison results of commercial nucleic acid extraction reagents and the nucleic acid extraction reagent of the present invention

[0113] Nucleic acid extraction reagent type Average Ct value The nucleic acid extraction reagent of the present invention 22.4 Zhenshi Biotechnology - Sample Release Agent 25.4 CW Biotech - Magnetic Bead Extraction Kit 22.0

[0114] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0115] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application without departing from the principles and purposes of the present application.

Claims

1. A nucleic acid extraction composition, characterized in that, Comprising: at least five of sodium hydroxide, lithium chloride, sodium dodecyl sulfate, polylysine, tris(2-carboxyethyl)phosphine hydrochloride, Chelex-100, and bovine serum albumin.

2. The nucleic acid extraction composition according to claim 1, wherein Comprising: at least six of sodium hydroxide, lithium chloride, sodium dodecyl sulfate, polylysine, tris(2-carboxyethyl)phosphine hydrochloride, Chelex-100, and bovine serum albumin; Optionally, the nucleic acid extraction composition comprises: at least six of 50 mM - 800 mM sodium hydroxide, 5 mM - 100 mM lithium chloride, 0.05 wt% - 0.85 wt% sodium dodecyl sulfate, 0.1 wt% - 2 wt% polylysine, 1 mM - 10 mM tris(2-carboxyethyl)phosphine hydrochloride, 1 wt% - 30 wt% Chelex-100, and 0.01 wt% - 0.5 wt% bovine serum albumin; Preferably, the nucleic acid extraction composition comprises: at least six of 100 mM - 700 mM sodium hydroxide, 5 mM - 35 mM lithium chloride, 0.1 wt% - 0.85 wt% sodium dodecyl sulfate, 0.2 wt% - 1 wt% polylysine, 3 mM - 15 mM tris(2-carboxyethyl)phosphine hydrochloride, 5 wt% - 20 wt% Chelex-100, and 0.1 wt% - 0.5 wt% bovine serum albumin.

3. The nucleic acid extraction composition according to claim 2, wherein The nucleic acid extraction composition is selected from any one of 1) - 21): 1) 100 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 2) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 3) 700 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 4) 300 mM sodium hydroxide, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 5) 300 mM sodium hydroxide, 5 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 6) 300 mM sodium hydroxide, 35 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 7) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 8) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.1 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 9) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.85 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 10) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 11) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.2 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 12) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 1 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 13) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 14) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 3 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 15) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 15 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 16) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, and 0.3 wt% bovine serum albumin; 17) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 5 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 18) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 20 wt% Chelex-100, and 0.3 wt% bovine serum albumin; 19) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, and 10 wt% Chelex-100; 20) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.1 wt% bovine serum albumin; 21) 300 mM sodium hydroxide, 15 mM lithium chloride, 0.25 wt% sodium dodecyl sulfate, 0.5 wt% polylysine, 7 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 wt% Chelex-100, and 0.5 wt% bovine serum albumin.

4. The nucleic acid extraction composition according to claim 3, wherein The nucleic acid extraction composition is selected from any one of 2), 15), 18), or 21).

5. The nucleic acid extraction composition according to claim 4, wherein The nucleic acid extraction composition is selected from 2).

6. The nucleic acid extraction composition according to any one of claims 1-5, characterized in that The pH of the nucleic acid extraction composition is selected from 9.0 - 13; Preferably, the pH of the nucleic acid extraction composition is selected from 9.35 - 11.34; More preferably, the pH of the nucleic acid extraction composition is 10.

88.

7. A nucleic acid extraction reagent, characterized in that, Comprising: The nucleic acid extraction composition according to any one of claims 1 - 6.

8. A nucleic acid extraction kit, characterized in that, Comprising: The nucleic acid extraction composition according to any one of claims 1 - 6 or the nucleic acid extraction reagent according to claim 7.

9. A nucleic acid extraction method, characterized in that, Comprising: Performing nucleic acid extraction treatment on the sample to be tested with the nucleic acid extraction composition according to any one of claims 1 - 6 or the nucleic acid extraction reagent according to claim 7; Optionally, the nucleic acid is derived from a microorganism; Preferably, the method further comprises the step of collecting the sample by oropharyngeal swab and / or nasopharyngeal swab; Preferably, the sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice, or cerebrospinal fluid; Preferably, the nucleic acid extraction treatment comprises: mixing the sample to be tested with the nucleic acid extraction composition or the nucleic acid extraction reagent for 5 min - 10 min; obtaining the supernatant by static settling or centrifugation; Preferably, the sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the preservation solution to the nucleic acid extraction composition or the nucleic acid extraction reagent is 1:(1 - 3).

10. A nucleic acid detection method for non-diagnostic purposes, characterized in that, Comprising: (1) Performing nucleic acid extraction treatment on the sample to be tested with the nucleic acid extraction composition according to any one of claims 1 - 6 or the nucleic acid extraction reagent according to claim 7; (2) Amplifying and detecting the nucleic acid extracted in step (1); Optionally, the nucleic acid is derived from a microorganism; Preferably, the method further comprises the step of collecting the sample by oropharyngeal swab and / or nasopharyngeal swab; Preferably, the sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice, or cerebrospinal fluid; Preferably, the nucleic acid extraction process includes: mixing the sample to be tested with a nucleic acid extraction composition or a nucleic acid extraction reagent for 5 min - 10 min; obtaining a supernatant by static settling or centrifugation; Preferably, the sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the preservation solution to the nucleic acid extraction composition or the nucleic acid extraction reagent is 1:1 - 3.

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