Gram-positive bacterium nucleic acid extraction lysis buffer, kit and application thereof

Abstract

The invention provides a gram-positive bacterium nucleic acid extraction lysis buffer, a kit and application thereof, and belongs to the technical field of biology. The lysate comprises cell wall hydrolase and a cell lysis buffer solution, wherein the cell wall hydrolase comprises endolysin, lysozyme and autolytic enzyme; in the lysis solution, the concentration of the endolysin is 100 [mu] g / mL to 1000 [mu] g / mL; the concentration of the lysozyme is 50 [mu] g / mL to 200 [mu] g / mL; and the concentration of the autolytic enzyme is 200 [mu] g / mL to 1000 [mu] g / mL. Compared with the traditional gram positive bacterium nucleic acid extraction lysate, the use process of the lysate does not need high-temperature heating, mechanical vibration, ultrasound and other additional steps, equipment assistance is not needed, the operation is convenient, the energy consumption is low, the lysate does not contain high-risk chemical reagents, the safety is high, and the dependency on professional environments and professionals is low.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and specifically, to a lysis solution for extracting nucleic acids from Gram-positive bacteria, a kit and its application. More specifically, the present application relates to a lysis solution for extracting nucleic acids from Gram-positive bacteria, a kit for extracting nucleic acids from Gram-positive bacteria, a method for extracting nucleic acids from Gram-positive bacteria, and a method for detecting nucleic acids of Gram-positive bacteria for non-diagnostic purposes. Background Art

[0002] Gram-positive bacilli are widely present in the natural environment, and infections can occur at any age. Common Gram-positive bacilli include Staphylococcus aureus, Clostridium tetani, Bacillus anthracis, Bacillus subtilis, etc. After being infected with bacteria, a series of inflammatory diseases can be triggered, which can lead to mucosal swelling, congestion, accompanied by discomfort such as pain, fever, and chills.

[0003] For diseases caused by Gram-positive bacteria, molecular diagnosis is a rapid, accurate, and efficient method, but pretreatment of bacteria lysis is required. The cell wall of Gram-positive bacteria is composed of a thick peptidoglycan layer (20 - 80 nm), accounting for 50% - 90% of the dry weight of the cell wall. This highly cross-linked network structure provides strong mechanical strength. In addition, teichoic acid in the cell wall further enhances the stability of the cell wall by binding to divalent cations (such as Mg2+, Ca 2 +). Gram-positive bacteria lack an outer membrane, but their thick peptidoglycan layer is directly exposed outside, forming a strong barrier, making it difficult for ordinary chemicals to penetrate. Therefore, pretreatment for lysis of Gram-positive bacteria usually requires a combination of mechanical methods (such as ultrasonic disruption), chemical methods (such as strong acids and strong bases), or thermal methods (high-temperature boiling) and other methods to be effectively achieved, but these methods are relatively cumbersome and have low safety. Summary of the Invention

[0004] The present application aims to at least solve one of the technical problems in the related art to some extent. For this reason, one object of the present application is to provide a lysis solution for extracting nucleic acids from Gram-positive bacteria, a kit and its application. The lysis solution has high safety; is simple to operate and has wide applicability; the process of nucleic acid lysis and release is mild and efficient, which is beneficial to the amplification and detection of downstream nucleic acids.

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

[0006] In the first aspect of the present application, the present application provides a lysis solution for extracting Gram-positive bacterial nucleic acids. According to an embodiment of the present application, the lysis solution includes: a cell wall hydrolase and a cell lysis buffer; wherein, the aforementioned cell wall hydrolase includes: endolysin, lysozyme and autolysin; in the aforementioned lysis solution, the concentration of the aforementioned endolysin is 100 μg / mL - 1000 μg / mL; the concentration of the aforementioned lysozyme is 50 μg / mL - 200 μg / mL; the concentration of the aforementioned autolysin is 200 μg / mL - 1000 μg / mL.

[0007] Compared with the traditional lysis solution for extracting Gram-positive bacterial nucleic acids, the aforementioned lysis solution does not require additional steps such as high-temperature heating, mechanical vibration, and ultrasound during use, does not require equipment assistance, is convenient to operate and has low energy consumption, and does not contain high-risk chemical reagents, has high safety, and has low dependence on a professional environment and professional personnel.

[0008] In some examples of the present application, the functions of the components in the aforementioned cell wall hydrolase are as follows:

[0009] Endolysin recognizes and binds to peptidoglycan through its domain, and hydrolyzes the glycosidic bond or peptide bond in peptidoglycan to destroy the integrity of the cell wall; lysozyme can specifically cleave the β-1,4-glycosidic bond in peptidoglycan to destroy the network structure of peptidoglycan; autolysin can destroy the cell membrane, recognize peptidoglycan in the cell wall and act specifically.

[0010] It can be understood that the concentration of the aforementioned endolysin can optionally be 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL or 1000 μg / mL; the concentration of the aforementioned lysozyme can optionally be 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL or 200 μg / mL; the concentration of the aforementioned autolysin can optionally be 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL or 1000 μg / mL.

[0011] In some preferred examples of the present application, in the aforementioned lysis solution, the concentration of the aforementioned endolysin is 200 μg / mL; the concentration of the aforementioned lysozyme is 100 μg / mL; the concentration of the aforementioned autolysin is 500 μg / mL.

[0012] In some examples of the present application, the components of the foregoing cell lysis buffer include at least one of Triton-100, Brij-100, NaCl, Tris-HCl, EDTA, and PEG-2000.

[0013] In some preferred examples of the present application, the components of the foregoing cell lysis buffer include Triton-100, Brij-100, NaCl, Tris-HCl, EDTA, and PEG-2000. The functions of the components in the foregoing cell lysis buffer are as follows: Triton X-100 (Triton) is a non-ionic surfactant that can promote the release of nucleic acids by disrupting the cell membrane or viral envelope structure; Brij-100 is a polyoxyethylene ether surfactant that can stabilize nucleic acids in an alkaline environment and prevent their degradation; NaCl can adjust the ionic strength to promote cell lysis and nucleic acid release; Tris-HCl can maintain the pH stability of the reaction system, protect the integrity of nucleic acids, promote cell lysis, and improve the efficiency of nucleic acid extraction; EDTA is a metal ion chelator whose main function is to inhibit the activity of nucleases and protect nucleic acids from degradation; PEG-2000 interacts with nucleic acid molecules at high salt concentrations, causing the nucleic acids to change from a hydrated linear conformation to a compact spherical conformation. This conformational change exposes the negatively charged phosphate groups on the surface of nucleic acid molecules, increasing the electrostatic interaction between nucleic acid molecules and thus promoting nucleic acid release.

[0014] In some examples of the present application, in the foregoing cell lysis buffer, the volume percentage of the foregoing Triton-100 is 0.02%-0.5%; the volume percentage of the foregoing Brij-100 is 0.05%-0.5%; the concentration of the foregoing NaCl is 100 mM-1000 mM; the concentration of the foregoing Tris-HCl is 50 mM-500 mM; the concentration of the foregoing EDTA is 50 mM-200 mM; the volume percentage of the foregoing PEG-2000 is 0.05%-0.5%.

[0015] It is understandable that the volume percentage of the aforementioned Triton-100 can optionally be 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%; the volume percentage of the aforementioned Brij-100 can optionally be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%; the concentration of the aforementioned NaCl can optionally be 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM or 1000 mM; the concentration of the aforementioned Tris-HCl can optionally be 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM or 500 mM; the concentration of the aforementioned EDTA can optionally be 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM; the volume percentage of the aforementioned PEG-2000 can optionally be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%.

[0016] In some preferred examples of the present application, in the aforementioned lysis solution, the volume percentage of the aforementioned Triton-100 is 0.1%; the volume percentage of the aforementioned Brij-100 is 0.1%; the concentration of the aforementioned NaCl is 500 mM; the concentration of the aforementioned Tris-HCl is 100 mM; the concentration of the aforementioned EDTA is 50 mM; the volume percentage of the aforementioned PEG-2000 is 0.2%.

[0017] In the second aspect of the present application, the present application provides a kit for extracting Gram-positive bacterial nucleic acid. According to the embodiments of the present application, the kit includes: the Gram-positive bacterial nucleic acid extraction lysis solution of any example in the first aspect. The aforementioned kit can be used for simple and portable extraction of Gram-positive bacterial nucleic acid, with high safety, without the need to rely on professional personnel and professional equipment, and has a wide range of applications.

[0018] In the third aspect of the present application, the present application provides a method for extracting Gram-positive bacterial nucleic acid. According to the embodiments of the present application, the method includes: mixing a sample to be tested with the Gram-positive bacterial nucleic acid extraction lysis solution of any example in the first aspect for nucleic acid extraction treatment. The aforementioned nucleic acid extraction method does not require professional personnel and professional equipment, has a wide range of applications; is simple to operate and has high safety; the reaction is mild and efficient, without violent reactions, and will not damage nucleic acid substances, facilitating downstream nucleic acid amplification and detection.

[0019] In some examples of the present application, the foregoing nucleic acid treatment includes: mixing the foregoing sample to be tested with the foregoing Gram-positive bacterial nucleic acid extraction and lysis solution for 10 min - 60 min, taking the supernatant, and the foregoing supernatant includes: Gram-positive bacterial nucleic acid.

[0020] It can be understood that the foregoing mixing time can optionally be 10 min, 20 min, 30 min, 40 min, 50 min or 60 min. The foregoing mixing is static mixing without vibration or heating. After statically mixing for the foregoing time, taking the supernatant can obtain a Gram-positive bacterial lysis solution (containing Gram-positive bacterial nucleic acid) with the cell wall removed.

[0021] In some examples of the present application, the foregoing sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab dilution, gastric juice or cerebrospinal fluid.

[0022] In some examples of the present application, the foregoing method further includes: the step of collecting a sample by an oropharyngeal swab or a nasopharyngeal swab.

[0023] Exemplarily, for the extraction of Gram-positive bacterial nucleic acid from a sample collected by using an oropharyngeal swab or a nasopharyngeal swab, it includes: immersing the oropharyngeal swab or nasopharyngeal swab containing the sample to be tested into the lysis solution of the first aspect; rotating the swab 5 - 20 times in the lysis solution and ensuring sample elution; discarding the swab, and mixing and standing the lysis solution at room temperature for 10 - 60 min to complete nucleic acid release.

[0024] In some preferred examples of the present application, the foregoing sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the foregoing preservation solution to the foregoing Gram-positive bacterial nucleic acid extraction and lysis solution is 1:(5 - 20). Optionally, it can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0025] In the fourth aspect of the present application, the present application proposes a method for detecting Gram-positive bacterial nucleic acid for non-diagnostic purposes. According to the embodiments of the present application, the method includes: (1) performing nucleic acid extraction treatment on a sample to be tested with the Gram-positive bacterial nucleic acid extraction and lysis solution of any example of the first aspect; (2) amplifying and detecting the nucleic acid extracted in step (1). Compared with mechanical crushing lysis, the nucleic acid release effect of the method of the present application is better; compared with commercial thermal lysis reagents, the performance is comparable, but the lysis solution of the present application does not require heating, the reaction is mild and efficient, there is no violent reaction, it will not damage nucleic acid substances, the operation is simple and fast, and the applicable range is wider.

[0026] In some examples of the present application, the foregoing nucleic acid treatment includes: mixing the foregoing sample to be tested with the foregoing Gram-positive bacterial nucleic acid extraction and lysis solution for 10 min - 60 min, taking the supernatant, and the foregoing supernatant includes: Gram-positive bacterial nucleic acid.

[0027] It can be understood that the foregoing mixing time can be optionally 10 min, 20 min, 30 min, 40 min, 50 min or 60 min. The foregoing mixing is static mixing without vibration or heating. After statically mixing for the foregoing time, take the supernatant to obtain a Gram-positive bacterial lysis solution (containing Gram-positive bacterial nucleic acid) with the cell wall removed.

[0028] In some examples of the present application, the foregoing sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice or cerebrospinal fluid.

[0029] In some examples of the present application, the foregoing method further includes: the step of collecting a sample by an oropharyngeal swab or a nasopharyngeal swab.

[0030] In some preferred examples of the present application, the foregoing sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the foregoing preservation solution to the foregoing Gram-positive bacterial nucleic acid extraction and lysis solution is 1:(5 - 20). It can be optionally 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0031] Additional aspects and advantages of the present application will be partly given in the following description, partly will become obvious from the following description, or be learned through the practice of the present application. Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to these drawings.

[0033] Figure 1 It is a schematic diagram of the comparison results of the nucleic acid release effects of different lysis methods provided by an embodiment of the present application for Staphylococcus aureus;

[0034] Figure 2 It is a schematic diagram of the comparison results of the nucleic acid release effects of different lysis methods provided by an embodiment of the present application for Bacillus subtilis. Detailed Embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part 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.

[0036] 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 such features. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0037] 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 such features.

[0038] Next, the solutions of the present application will be explained in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating the present application and should not be construed as limiting the scope of the present application. For those not specified in the embodiments regarding specific techniques or conditions, the techniques or conditions described in the literature in the art or according to the product specifications shall be followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0039] Example 1: Nucleic Acid Release and Detection of Staphylococcus aureus

[0040] 1. Bacterial Culture of Staphylococcus aureus

[0041] (1) Preparation of Liquid Medium

[0042] Weigh the following components and dissolve them in water, and make up the volume to 1 L:

[0043] Tryptone: 10.0 g

[0044] NaCl: 5 g

[0045] Beef Extract Powder: 5 g

[0046] (2) Bacterial Inoculation and Culture

[0047] Inoculate Staphylococcus aureus (the strain is from Beina Biology BNCC) into the liquid medium and seal it.

[0048] Incubate it at 37 °C with shaking for 24 - 36 h until obvious colony precipitation appears.

[0049] 2. Preparation of Lysis Solution

[0050] Prepare the lysis solution with the final concentrations of each component as follows:

[0051] Endolysin (Sigma-Aldrich, 196000): 200 μg / mL

[0052] Lysozyme (Solarbio, L8120): 100 μg / mL

[0053] Autolysin (Sigma-Aldrich): 500 μg / mL

[0054] Triton X-100: 0.1%

[0055] Brij-100: 0.1%

[0056] NaCl: 500 mM

[0057] Tris-HCl: 100 mM

[0058] EDTA: 50 mM

[0059] PEG-2000: 0.2%

[0060] Dissolve all components in 1 mL of sterile water, mix well and set aside for later use.

[0061] 3. Release of Staphylococcus aureus nucleic acid

[0062] Take 20 μL of the bacterial culture solution (step 1);

[0063] Add 200 μL of the lysis solution (step 2);

[0064] After thorough mixing, let it stand at room temperature for 30 min for lysis.

[0065] 4. Evaluation of nucleic acid release effect

[0066] (1) Comparative experiment design

[0067] Use the following three lysis methods to release nucleic acid from Staphylococcus aureus suspensions of the same concentration, and use the Staphylococcus aureus qPCR detection kit to detect the nucleic acid release effect:

[0068] The lysis solution of this application (without heating, without mechanical vibration);

[0069] Commercial thermal lysis solution (manufacturer: Shengxiang Biotech, product number: S1002);

[0070] Mechanical shock lysis (ultrasonic frequency > 60 kHz).

[0071] (2) qPCR detection

[0072] Primer and probe sequences (for detecting the nuc gene of Staphylococcus aureus):

[0073] Forward primer: 5’-GCGATTGATGGTGATACGGTT-3’ (SEQ ID NO:1);

[0074] Reverse primer: 5’-AGCCAAGCCTTGACGAACTAAAGC-3’ (SEQ ID NO:2);

[0075] Probe: 5'-FAM-TGCTGAACGAACTGAACGACACCA-BHQ1-3' (SEQ ID NO:3).

[0076] The reaction system is shown in Table 1.

[0077] Table 1 Reaction system

[0078] TaqMan qPCR mix(2×) 10 μL Forward primer(10 μM) 2 μL Reverse primer(10 μM) 2 μL Probe(10 μM) 1 μL Template(Lysate) 5 μL

[0079] qPCR reaction procedure:

[0080] UNG enzyme reaction: 50°C, 2 min;

[0081] Taq enzyme activation: 95°C, 5 min;

[0082] PCR amplification cycle (a total of 45 cycles):

[0083] Denaturation: 95°C, 15 s;

[0084] Annealing: 57°C, 30 s (collect fluorescence signal).

[0085] (3) Experimental results

[0086] Nucleic acids were released from the same Staphylococcus aureus bacterial solution using three lysis buffers, and the released bacterial solution was taken as the qPCR sample and detected using the above steps and system. The results are shown in the appendix Figure 1 and Table 2. The lysis buffer provided in this application has basically the same nucleic acid release effect as the commercial thermal lysis agent, but this lysis buffer does not require heating, and the lysis buffer provided in this application has a better lysis effect than mechanical shaking.

[0087] Table 2 Comparison results of Ct values of amplified Staphylococcus aureus lysis products

[0088]

[0089] Example 2: Nucleic acid release and detection of Bacillus subtilis

[0090] 1. Bacillus subtilis bacterial culture

[0091] (1) Preparation of liquid medium

[0092] Weigh the following components and dissolve them in water, then make up the volume to 1 L:

[0093] Tryptone: 10.0 g

[0094] NaCl: 5 g

[0095] Beef extract powder: 5 g

[0096] (2) Inoculation and culture of bacteria

[0097] Inoculate Bacillus subtilis (the strain is from Beijing Na Biotechnology BNCC) into the liquid medium and seal it.

[0098] Incubate it at 37 °C with shaking for 36 - 48 h until obvious colony precipitation appears.

[0099] 2. Preparation of lysis buffer

[0100] Prepare the lysis buffer with the following final concentrations of each component:

[0101] Endolysin (Sigma - Aldrich, 196000): 200 μg / mL

[0102] Lysozyme (Solarbio, L8120): 100 μg / mL

[0103] Autolysin (Sigma - Aldrich): 500 μg / mL

[0104] Triton X - 100: 0.1%

[0105] Brij - 100: 0.1%

[0106] NaCl: 500 mM

[0107] Tris - HCl: 100 mM

[0108] EDTA: 50 mM

[0109] PEG - 2000: 0.2%

[0110] Dissolve all components in 1 mL of bacterial water and mix well for later use.

[0111] 3. Release of Bacillus subtilis nucleic acid

[0112] Take 20 μL of the bacterial culture solution (step 1);

[0113] Add 200 μL of the lysis buffer (step 2);

[0114] After thorough mixing, let it stand at room temperature for 30 min for lysis.

[0115] 4. Evaluation of nucleic acid release effect

[0116] (1) Comparative experiment design

[0117] Use the following three lysis methods to release nucleic acids from a Bacillus subtilis suspension of the same concentration, and use a Bacillus subtilis qPCR detection kit to detect the nucleic acid release effect:

[0118] The lysis solution of this application (without heating and mechanical vibration);

[0119] Commercial thermal lysis solution (manufacturer: Shengxiang Biotech, product number: S1002);

[0120] Lysis by ultrasonic fragmentation.

[0121] (2) qPCR detection

[0122] Primer and probe sequences (for detecting the Bacillus subtilis 16S rRNA gene):

[0123] Forward primer: 5'-CGGCGGTGAATACGTTC-3' (SEQ ID NO:4)

[0124] Reverse primer: 5'-TCCCGCTTCTCCTTCCT-3' (SEQ ID NO:5)

[0125] Probe: 5'-FAM-ACGTCAAAGCGGCGGT-BHQ1-3' (SEQ ID NO:6).

[0126] The qPCR reaction system is shown in Table 3 (total volume of 20 μL).

[0127] Table 3 Reaction system

[0128] TaqMan qPCR mix(2×) 10 μL Forward primer(10 μM) 2 μL Reverse primer(10 μM) 2 μL Probe(10 μM) 1 μL Template(Lysate) 5 μL

[0129] qPCR reaction procedure:

[0130] UNG enzyme reaction: 50°C, 2 min;

[0131] Taq enzyme activation: 95°C, 5 min;

[0132] PCR amplification cycle (a total of 45 cycles):

[0133] Denaturation: 95°C, 15 s;

[0134] Annealing: 57°C, 30 s (collect fluorescence signal).

[0135] (3) Experimental results

[0136] The nucleic acids of the same Bacillus subtilis bacterial solution were released by three lysis methods. The lysed bacterial solution after the release was taken as a qPCR sample and detected using the above steps and system. The results are shown in the appendix Figure 2 and Table 4. The nucleic acid release effect of the lysis solution provided in this application is basically the same as that of the commercial lysis agent. However, this lysis solution does not require heating, and the lysis solution provided by the present invention has a better lysis effect than mechanical shaking.

[0137] Table 4 Comparison results of Ct values of amplified products of Bacillus subtilis lysates

[0138]

[0139] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" 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 this 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 can be combined in a suitable manner in any one or more embodiments or examples.

[0140] Although the embodiments of this application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting this application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application without departing from the principle and purpose of this application.

Claims

1. A nucleic acid extraction and lysis solution for Gram-positive bacteria, characterized in that, Comprising: Cell wall hydrolase and cell lysis buffer; Wherein, the cell wall hydrolase includes: endolysin, lysozyme and autolysin; in the lysis solution, the concentration of the endolysin is 100 μg / mL - 1000 μg / mL; the concentration of the lysozyme is 50 μg / mL - 200 μg / mL; the concentration of the autolysin is 200 μg / mL - 1000 μg / mL.

2. The lysate according to claim 1, wherein The concentration of the endolysin is 200 μg / mL; the concentration of the lysozyme is 100 μg / mL; the concentration of the autolysin is 500 μg / mL.

3. The lysis solution according to claim 1 or 2, characterized in that, The components of the cell lysis buffer include at least one of Triton-100, Brij-100, NaCl, Tris-HCl, EDTA and PEG-2000; Preferably, the components of the cell lysis buffer include Triton-100, Brij-100, NaCl, Tris-HCl, EDTA and PEG-2000.

4. The lysate according to claim 3, characterized in that, In the cell lysis buffer, the volume percentage of Triton-100 is 0.02% - 0.5%; the volume percentage of Brij-100 is 0.05% - 0.5%; the concentration of NaCl is 100 mM - 1000 mM; the concentration of Tris-HCl is 50 mM - 500 mM; the concentration of EDTA is 50 mM - 200 mM; the volume percentage of PEG-2000 is 0.05% - 0.5%.

5. The lysis solution according to claim 4, wherein In the lysis solution, the volume percentage of Triton-100 is 0.1%; the volume percentage of Brij-100 is 0.1%; the concentration of NaCl is 500 mM; the concentration of Tris-HCl is 100 mM; the concentration of EDTA is 50 mM; the volume percentage of PEG-2000 is 0.2%.

6. A nucleic acid extraction kit for Gram-positive bacteria, characterized in that, Comprising: The Gram-positive bacterial nucleic acid extraction lysis solution according to any one of claims 1 - 5.

7. A method for extracting nucleic acid from Gram-positive bacteria, characterized in that, Comprising: Mixing the sample to be tested with the Gram-positive bacterial nucleic acid extraction lysis solution according to any one of claims 1 - 5 for nucleic acid extraction treatment.

8. The method according to claim 7, wherein The nucleic acid treatment includes: Mixing the sample to be tested with the Gram-positive bacterial nucleic acid extraction lysis solution for 10 min - 60 min, taking the supernatant, and the supernatant includes Gram-positive bacterial nucleic acid; Preferably, the sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice or cerebrospinal fluid; Preferably, the method further includes the step of collecting the sample by oropharyngeal swab or nasopharyngeal swab; Preferably, the sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the preservation solution to the Gram-positive bacterial nucleic acid extraction lysis solution is 1:(5 - 20).

9. A method for detecting Gram-positive bacterial nucleic acids for non-diagnostic purposes, characterized in that, Comprising: (1) Performing nucleic acid extraction treatment on the sample to be tested with the Gram-positive bacterial nucleic acid extraction lysis solution according to any one of claims 1 - 5; (2) Amplifying and detecting the nucleic acid extracted in step (1).

10. The method according to claim 9, characterized in that, The nucleic acid treatment includes: Mix the sample to be tested with the Gram-positive bacterial nucleic acid extraction lysis solution for 10 min - 60 min, and take the supernatant. The supernatant contains Gram-positive bacterial nucleic acid; Preferably, the sample to be tested is selected from blood, urine, saliva, sputum, nasal / pharyngeal swab diluent, gastric juice or cerebrospinal fluid; Preferably, the method further includes the step of collecting the sample by an oropharyngeal swab and / or a nasopharyngeal swab; Preferably, the sample to be tested is placed in a preservation solution; more preferably, the volume ratio of the preservation solution to the Gram-positive bacterial nucleic acid extraction lysis solution is 1:(5 - 20).

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