A lysis solution, kit and method for extracting microbial nucleic acids

Abstract

This invention provides a lysis buffer, kit, and method for extracting microbial nucleic acids. The lysis buffer contains guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, tris(hydroxymethyl)aminomethane, and phosphite. By using phosphite in combination with other components in the lysis buffer, the lysis effect on structurally complex microorganisms such as fungi is improved without affecting the nucleic acids of structurally simple microorganisms such as viruses released earlier. This invention also provides a nucleic acid extraction kit containing the lysis buffer, enabling the co-extraction of nucleic acids from multiple microorganisms. The kit and extraction procedure of this invention, when used together, can obtain high-quality nucleic acids from different microorganisms in various infected samples; it shortens the extraction time for different microbial nucleic acids, simplifies the operation process, and reduces extraction costs.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction technology, and in particular to a lysis buffer, kit, and method for extracting microbial nucleic acids. Background Technology

[0002] Nucleic acid detection technology has wide applications in the field of biology, serving not only as an important tool for scientific research but also as a crucial source of information for clinical management. Before conducting nucleic acid analysis, it is necessary to obtain the nucleic acid from the microorganisms, which is then required for subsequent downstream experiments such as PCR, isothermal amplification, and sequencing. Nucleic acid acquisition is the initial step in the entire detection system, and the quality and yield of the isolated nucleic acid have a decisive impact on the subsequent detection results.

[0003] The natural environment is rich in microorganisms, and their cell or virus structures vary. Viruses have a relatively simple structure, consisting only of a protein coat enclosing DNA or RNA. In addition to the matrix, organelles and nucleus structures enclosed by the cell membrane, bacteria also have cell walls and capsules outside the plasma membrane, and are rich in peptidoglycan, teichoic acid and other substances. The cell walls of fungi are divided into tangible microfibrils and amorphous matrix, mainly composed of chitin, glucan and mannan.

[0004] The difficulty in co-extracting nucleic acids from various microorganisms during nucleic acid isolation lies in the fact that microorganisms with different structures have different requirements for lysis conditions, making it difficult to simultaneously achieve the same nucleic acid extraction effect for different microorganisms. Microorganisms with complex cell structures, such as fungi, require high lysis intensity, otherwise the nucleic acid yield will be affected; while pathogenic microorganisms such as viruses have simple structures, and normal lysis conditions can release nucleic acids. Conversely, excessively strong lysis environments or lysis times will damage the released nucleic acids.

[0005] To address this issue, current methods mostly employ different extraction reagents or different procedures using the same extraction reagent to obtain nucleic acids from microorganisms such as viruses, bacteria, and fungi. However, these methods are complex, require large sample volumes, and have unsatisfactory reproducibility in practice. Therefore, ensuring the nucleic acid yield of different microorganisms within the same procedure is a key research focus in this field. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a lysis buffer and kit that can guarantee the nucleic acid yield of different microorganisms in the same operation process, so as to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0008] In a first aspect, the present invention provides a lysis buffer comprising: a guanidine salt, an anionic surfactant, an inorganic salt, a nonionic surfactant, tris(hydroxymethyl)aminomethane, and a phosphite.

[0009] In some embodiments, the concentration of the guanidine salt in the lysis buffer is 1-5 M.

[0010] In some embodiments, the guanidine salt includes one or more of guanidine hydrochloride and guanidine isothiocyanate.

[0011] In some embodiments, the concentration of the anionic surfactant in the lysis buffer is 1–10 w / w.

[0012] In some embodiments, the anionic surfactant includes at least one of sodium dodecyl sulfate, sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium dodecyl sulfonate.

[0013] In some embodiments, the inorganic salt includes one or both of sodium chloride and potassium chloride.

[0014] In some embodiments, the concentration of the inorganic salt in the lysis solution is 0.1–5 w / v%.

[0015] In some embodiments, the nonionic surfactant includes one or more of polyethylene glycol, Triton, Tween, nonylphenol polyoxyethylene ether, and hexadecyl polyoxyethylene ether.

[0016] In some embodiments, the nonionic surfactant includes one or more of polyethylene glycol, Triton, and Tween.

[0017] In some preferred embodiments, the nonionic surfactant includes polyethylene glycol, Triton, and Tween.

[0018] In some embodiments, the polyethylene glycol has a molecular weight of 2,000-10,000; further, the polyethylene glycol has a molecular weight of 6,000-8,000.

[0019] In some embodiments, the concentration of polyethylene glycol in the pyrolysis solution is 5–15 w / w.

[0020] In some embodiments, the concentration of the triatomine in the lysis solution is 0.1–10 v / v%.

[0021] In some embodiments, the Triton includes one or more of Triton X-100, Triton X-114, and Triton X-305.

[0022] In some embodiments, the Tween includes one or more of Tween-20 and Tween-80.

[0023] In some embodiments, the concentration of Tween in the lysis solution is 0.1–4 v / v%.

[0024] In some embodiments, the concentration of the trihydroxymethylaminomethane in the pyrolysis solution is 0.1 to 1 M.

[0025] In some embodiments, the concentration of the phosphite in the lysis solution is 0.01–2 M.

[0026] In some embodiments, the phosphite includes one or more of sodium phosphite, potassium phosphite, and magnesium phosphite.

[0027] In a second aspect, the present invention provides a nucleic acid extraction kit, the kit comprising the lysis buffer as described above.

[0028] In some embodiments, the kit further includes one or more of proteinase K, an adsorbent, a first washing solution, a second washing solution, and an elution solution.

[0029] In some embodiments, the working concentration of proteinase K is 1–100 mg / mL, based on the volume of the mixture of the test sample and the lysis buffer.

[0030] In some embodiments, the first washing solution includes guanidine salts, alcohols, and tris(hydroxymethyl)aminomethane; the first washing solution tends to remove impurities such as proteins and lipids.

[0031] In some embodiments, the concentration of guanidine salt in the first washing solution is 1–5 M.

[0032] In some embodiments, the guanidine salt includes one or more of guanidine hydrochloride and guanidine isothiocyanate.

[0033] In some embodiments, the concentration of the alcohol is 60–80 v / v%.

[0034] In some embodiments, the alcohol is ethanol; preferably anhydrous ethanol.

[0035] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the first washing solution is 1–50 mM.

[0036] In some embodiments, the first washing solution comprises the following components: guanidine salt 1-5M, alcohol 60-80 v / v%, tris(hydroxymethyl)aminomethane 1-50mM.

[0037] In some embodiments, the second washing solution includes tris(hydroxymethyl)aminomethane and alcohols. The second washing solution tends to remove impurities such as sugars and salts.

[0038] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the second washing solution is 1-100 mM.

[0039] In some embodiments, the concentration of the alcohol is 60–80 v / v%.

[0040] In some embodiments, the alcohol is ethanol, preferably anhydrous ethanol.

[0041] In some embodiments, the eluent is tris(hydroxymethyl)aminomethane. The eluent elutes nucleic acids from the magnetic beads and serves as a matrix for nucleic acid preservation, ready for use in downstream experiments.

[0042] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the eluent is 1–100 mM.

[0043] In some embodiments, the adsorbent is a magnetic bead.

[0044] In some embodiments, the working concentration of the magnetic beads is 0.5-5 mg / mL, based on the volume of the mixture of the sample to be tested and the lysis buffer.

[0045] In some embodiments, the magnetic beads include silanol-modified magnetic beads.

[0046] In some embodiments, the magnetic beads have a particle size of 100–500 nm.

[0047] The present invention also provides the use of the lysis buffer or the kit described above in the extraction of microbial nucleic acids or the preparation of products containing extracted microbial nucleic acids.

[0048] In some embodiments, the microorganisms include one or more of the following: bacteria, viruses, rickettsiae, mycoplasma, chlamydia, spirochetes, fungi, and actinomycetes.

[0049] In some embodiments, the product is preferably a reagent, reagent kit, or system;

[0050] In some embodiments, the system is preferably a nucleic acid extraction and / or detection system.

[0051] The present invention also provides a method for extracting microbial nucleic acids, comprising using a kit as described above to extract microbial nucleic acids from a sample to be tested.

[0052] In some embodiments, the method specifically includes: mixing lysis buffer, proteinase K, adsorbent and test sample to obtain adsorbent with adsorbed nucleic acid; washing the adsorbent with adsorbed nucleic acid with a first washing solution and a second washing solution, and then eluting with an elution buffer to obtain a microbial nucleic acid solution.

[0053] In some implementations, the mixing time is 15-25 minutes.

[0054] In some embodiments, the mixing temperature is 90–98°C.

[0055] In some embodiments, the sample to be tested includes a microbial culture, an environmental sample, or a human sample; wherein the human sample is a microbial infection sample.

[0056] Preferably, the microbial infection sample includes one or more of the following: whole blood, serum, plasma, saliva, urine, pleural effusion, peritoneal fluid, sputum, tissue fluid, follicular fluid, and tissue samples.

[0057] Beneficial effects:

[0058] This invention provides a lysis buffer comprising guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, tris(hydroxymethyl)aminomethane, and phosphite. By using phosphite in combination with other components in the lysis buffer, the lysis effect on structurally complex microorganisms such as fungi is improved without affecting the nucleic acids of structurally simple microorganisms such as viruses that are released first. This invention also provides a nucleic acid extraction kit containing the lysis buffer, enabling the co-extraction of nucleic acids from multiple microorganisms. The kit and extraction procedure of this invention, when used together, can obtain high-quality nucleic acids from different microorganisms in various infected samples; it shortens the extraction time for different microbial nucleic acids, simplifies the operation process, and reduces extraction costs. Attached image description:

[0059] Figure 1 The PCR results for Candida auris in sample 1 of Example 5 are shown.

[0060] Figure 2 The PCR results for Candida auris in sample 2 of Example 5 are shown.

[0061] Figure 3 The PCR results for Candida auris in sample 3 of Example 5 are shown.

[0062] Figure 4 This is the PCR result of influenza A virus from sample 1 in Example 5.

[0063] Figure 5 This is the PCR result of influenza A virus in sample 2 of Example 5.

[0064] Figure 6This is the PCR result of influenza A virus in sample 3 of Example 5.

[0065] Figure 7 The result is the PCR result of Klebsiella pneumoniae in sample 1 of Example 5.

[0066] Figure 8 The result is the PCR result of Klebsiella pneumoniae in sample 2 of Example 5.

[0067] Figure 9 This is the PCR result of Klebsiella pneumoniae in sample 3 of Example 5. Detailed Implementation

[0068] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0069] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0070] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0071] In a first aspect, the present invention provides a lysis buffer comprising: a guanidine salt, an anionic surfactant, an inorganic salt, a nonionic surfactant, tris(hydroxymethyl)aminomethane, and a phosphite. The lysis buffer is used to disrupt the structure of microbial cells and viruses, release nucleic acids, maintain the stability of nucleic acid structures, and provide a favorable environment for the binding of nucleic acids to magnetic beads. The present invention optimizes the formulation of the lysis buffer to ensure the release of nucleic acids from structurally complex microorganisms such as fungi, without affecting the nucleic acids of structurally simple microorganisms such as viruses that are released prematurely. Sodium phosphite in the lysis buffer has good solubility and stability in aqueous solution; sodium phosphite has strong reducing properties, providing a reducing environment, and when used in combination with guanidine salt, it can improve the lysis effect on complex cell structures; the anionic surfactant can disrupt cell membranes and nuclear membranes, and separate histones from DNA; the nonionic surfactant works synergistically with the anionic surfactant to promote the release of nucleic acids; tris(hydroxymethyl)aminomethane provides a stable liquid environment for the nucleic acid extraction process, preventing large fluctuations in the pH of the extraction liquid environment from affecting the nucleic acid extraction efficiency.

[0072] In some embodiments, the concentration of the guanidine salt in the lysis solution is 1-5M; it can also be 2-4M, 1-3M, 3-5M; or 1M, 2M, 3M, 4M or 5M.

[0073] In some embodiments, the guanidine salt includes one or more of guanidine hydrochloride and guanidine isothiocyanate.

[0074] In some embodiments, the concentration of the anionic surfactant in the lysis solution is 1-10 w / w%; it can also be 1-3 w / w%, 3-6 w / w%, 6-10 w / w%, 2-7 w / w%, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 w / w.

[0075] In some embodiments, the anionic surfactant includes at least one of sodium dodecyl sulfate, sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium dodecyl sulfonate; preferably sodium dodecyl sulfate.

[0076] In some embodiments, the inorganic salt includes one or both of sodium chloride and potassium chloride.

[0077] In some embodiments, the concentration of the inorganic salt in the lysis solution is 0.1–5 w / v%; it can also be 0.1–1 w / v%, 0.1–2 w / v%, 2–3 w / v%, 3–5 w / v%, 1–3 w / v%; or it can be 0.1, 0.5, 1, 2, 3, 4, or 5 w / v.

[0078] In some embodiments, the nonionic surfactant includes one or more of polyethylene glycol, Triton, Tween, nonylphenol polyoxyethylene ether, and hexadecyl polyoxyethylene ether. Further, the nonionic surfactant includes one or more of polyethylene glycol, Triton, and Tween; more preferably, the nonionic surfactant includes polyethylene glycol, Triton, and Tween.

[0079] In some embodiments, the polyethylene glycol has a molecular weight of 2000-10000; further, the polyethylene glycol has a molecular weight of 6000-8000; and, for example, one or more of polyethylene glycol 4000, polyethylene glycol 6000, or polyethylene glycol 8000.

[0080] In some embodiments, the concentration of polyethylene glycol in the pyrolysis solution is 5-15 w / w%; it can also be 5-10 w / w%, 10-15 w / w%, 8-13 w / w%, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 w / w.

[0081] In some embodiments, the concentration of the triton in the lysis solution is 0.1–10 v / v%; it can also be 0.1–1 v / v%, 1–5 v / v%, 5–10 v / v%; or it can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v.

[0082] In some embodiments, the Triton includes one or more of Triton X-100, Triton X-114, and Triton X-305.

[0083] In some embodiments, the Tween includes one or more of Tween-20, Tween-60, and Tween-80.

[0084] In some embodiments, the concentration of Tween in the lysis solution is 0.1–4 v / v%; it can also be 0.1–0.5 v / v%, 0.5–1.5 v / v%, 1.5–3 v / v%, 3–4 v / v%, or 0.1, 0.15, 0.5, 1, 2, 3, or 4 v / v%.

[0085] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the lysis solution is 0.1–1 M; it can also be 0.3–0.8 M, or 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1 M.

[0086] In some embodiments, the concentration of the phosphite in the lysis solution is 0.01–2 M; it can also be 0.01–0.1 M, 0.1–0.5 M, 0.5–1 M, 1–2 M, 0.05–0.5 M, or 0.01 M, 0.1 M, 0.5 M, 1 M, 1.5 M, or 2 M.

[0087] In some embodiments, the phosphite includes one or more of sodium phosphite, potassium phosphite, and magnesium phosphite.

[0088] The present invention also provides a kit for microbial nucleic acid extraction, the kit comprising the lysis buffer as described above.

[0089] In some embodiments, the lysis buffer comprises: 1-5M guanidine salt, 1-10 w / w% anionic surfactant, 0.1-5 w / v% inorganic salt, 5-15 w / w% polyethylene glycol, 0.1-10 v / v% Triton, 0.1-4 v / v% Tween, 0.1-1M trihydroxymethane, and 0.01-2M phosphite.

[0090] In some embodiments, the kit further includes one or more of proteinase K, an adsorbent, a first washing solution, a second washing solution, and an elution solution.

[0091] In some embodiments, the working concentration of proteinase K is 1–100 mg / mL, based on the volume of the mixture of the sample to be tested and the lysis buffer; it can also be 5–80 mg / mL, 10–50 mg / mL, 15–30 mg / mL, or 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL. Proteinase K is a protein hydrolysant that can hydrolyze proteins in cell or viral structures and proteins bound to DNA, thereby fully exposing the DNA and facilitating DNA extraction.

[0092] In some embodiments, the first washing solution includes guanidine salts, alcohols, and tris(hydroxymethyl)aminomethane; the first washing solution tends to remove impurities such as proteins and lipids.

[0093] In some embodiments, the concentration of guanidine salt in the first washing solution is 1-5M; it can also be 2-4M, or 1M, 2M, 3M, 4M or 5M.

[0094] In some embodiments, the guanidine salt includes one or more of guanidine acid and guanidine isothiocyanate.

[0095] In some embodiments, the concentration of alcohol in the first washing solution is 60-80 v / v%; it can also be 65-75 v / v%, or 60 v / v%, 65 v / v%, 70 v / v%, 75 v / v%, or 80 v / v.

[0096] In some embodiments, the alcohol is ethanol; preferably anhydrous ethanol.

[0097] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the first washing solution is 1–50 mM; it can also be 1–25 mM, 25–50 mM, 5–20 mM, or 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

[0098] In some embodiments, the first washing solution comprises the following components at concentrations: guanidine salt 1-5M, alcohol 60-80 v / v%, tris(hydroxymethyl)aminomethane 1-50 mM.

[0099] In some embodiments, the second washing solution includes tris(hydroxymethyl)aminomethane and alcohols. The second washing solution tends to remove impurities such as sugars and salts.

[0100] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the second washing solution is 1-100 mM; it can also be 1-60 mM, 60-100 mM, or 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0101] In some embodiments, the concentration of the alcohol is 60-80 v / v%; it can also be 65-75 v / v%, or 60 v / v%, 65 v / v%, 70 v / v%, 75 v / v%, or 80 v / v.

[0102] In some embodiments, the alcohol is ethanol, preferably anhydrous ethanol.

[0103] In some embodiments, the eluent is tris(hydroxymethyl)aminomethane. The eluent elutes nucleic acids from the magnetic beads and serves as a matrix for nucleic acid preservation, ready for use in downstream experiments.

[0104] In some embodiments, the concentration of the eluent tris(hydroxymethyl)aminomethane is 1–100 mM; it can also be 20–80 mM, 40–60 mM, or 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0105] In some embodiments, the adsorbent includes magnetic beads.

[0106] In some embodiments, the magnetic beads include silanol-modified magnetic beads.

[0107] In some embodiments, the working concentration of the magnetic beads is 0.5–5 mg / mL, based on the volume of the mixture of the sample to be tested and the lysis buffer; it can also be 1–3 mg / mL, 2–5 mg / mL, 0.8–4.5 mg / mL, or 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 2 mg / mL, 4.5 mg / mL, or 5 mg / mL.

[0108] In some embodiments, the particle size of the magnetic beads is 100-500nm, or it can be 100-300nm, or it can be 300-500nm, or it can be 150-400nm, or it can be 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, or 500nm.

[0109] The present invention also provides a product for extracting microbial nucleic acids, the product comprising the lysis buffer or kit as described above.

[0110] In some embodiments, the product is preferably a reagent, kit, or system.

[0111] In some implementations, the product is a nucleic acid extraction and / or detection system.

[0112] In some embodiments, the nucleic acid extractor is a fully automated nucleic acid extractor or a manual nucleic acid extraction device.

[0113] In some embodiments, the product also includes other tools that can provide an external magnetic field.

[0114] The present invention also provides the use of the lysis buffer, kit or product as described above in the extraction of microbial nucleic acid or in the preparation of products containing extracted microbial nucleic acid.

[0115] In some embodiments, the microorganisms include one or more of the following: bacteria, viruses, rickettsiae, mycoplasma, chlamydia, spirochetes, fungi, and actinomycetes.

[0116] In some embodiments, the bacteria include, but are not limited to: Acinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis, Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Haemophilus influenzae type b, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae (MDR / CRE), Legionella pneumophila, and Neisseria meningitidis. The following bacteria are listed: meningitides, Neisseriagon orrhoeae, Pseudomonas aeruginosa, Salmonella typhi, paratyphi, typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridium neoformans, Clostridium difficile, Clostridium perfringens, Corynebacterium spp., Enterococcus faecalis, and Enterococcus faecalis. (Faecium), vancomycin-resistant Enterococci (VRE), Listeria monocytogenes, Mycobacterium avium, Mycobacterium tuberculosis (M).The following bacteria are listed: Mycobacterium leprae, Nocardia farcinica, Propionibacterium acnes, Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus pyogenes, Group A Streptococci, Group B Streptococci (agalactiae), and Group C Streptococci.

[0117] Rickettsia include, but are not limited to: Rickettsia proteus, Rickettsia mollusks, Rickettsia konjac, Rickettsia heilongjiangensis, Rickettsia leuciscus, Rickettsia siberiana, Ehrlich. chafie, Ehrlich. canineis, Orientia scrub typhus, and Coxiella beher.

[0118] Viruses include, but are not limited to: dengue virus, Ebola virus, EBV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, HSV-1, HSV-2, HIV, cytomegalovirus (CMV), influenza A virus, influenza B virus, Marburg virus, human respiratory syncytial virus (RSV), SARS coronavirus (SARS-CoV), West Nile virus, human papillomavirus (HPV), human rhinovirus (HRV), and Zika virus.

[0119] Fungi include, but are not limited to: Aspergillus spp., Blastomyces, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus, Fusarium spp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

[0120] Mycoplasma includes, but is not limited to: Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma oralis.

[0121] Chlamydia include, but are not limited to: Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis murineis, and Chlamydia suis.

[0122] Spirochetes include, but are not limited to: Treponema pallidum, Treponema yaws, Treponema pallidum, Treponema pallidum (endemic syphilis), Treponema burgdorferi, Treponema hemsbergensis, or Leptospira question mark.

[0123] Actinomycetes are actinomycetes including, but not limited to, those of the genus Streptomyces. Examples include Streptomyces griseus, Streptomyces fibrinolyticus, and Streptomyces gravidus.

[0124] In some embodiments, the product is preferably a reagent, reagent kit, or system;

[0125] In some embodiments, the system is preferably a nucleic acid extraction and / or detection system.

[0126] The present invention also provides a method for extracting microbial nucleic acids, comprising using the lysis buffer, kit or product described above to extract microbial nucleic acids from a sample to be tested.

[0127] In some embodiments, the method includes the following steps: mixing lysis buffer, proteinase K, adsorbent and sample to be tested to obtain an adsorbent with adsorbed nucleic acid; washing the adsorbent with adsorbed nucleic acid with a first washing solution and a second washing solution, and then eluting with an elution buffer to obtain a microbial nucleic acid solution.

[0128] It should be noted that the above method is not limited to mixing the lysis buffer with the sample to be extracted before adding the magnetic beads for mixing; it is also possible to mix the lysis buffer with the magnetic beads before adding the sample to be extracted for mixing and reaction; or the lysis buffer, magnetic beads and sample to be extracted can be mixed at the same time.

[0129] In some embodiments, the mixing time is 15-25 min, or even 18-23 min, or even 20 min, which yields the best extraction effect for microbial nucleic acids.

[0130] In some embodiments, the mixing temperature is 90-98°C, and may also be 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, or 98°C.

[0131] In some embodiments, the volume ratio of the test sample, lysis buffer, first washing buffer, second washing buffer and elution buffer is 3:3-7:3-7:5-8:1-6; preferably 3:5:5:6:4; the amount of each component added can be adjusted appropriately according to the experimental conditions.

[0132] In some embodiments, the working concentration of the adsorbent is 0.5 to 5 mg / mL, based on the volume of the mixture of the sample to be tested and the lysis buffer.

[0133] In some embodiments, the working concentration of proteinase K is 1–100 mg / mL, based on the volume of the mixture of the test sample and the lysis buffer.

[0134] In some embodiments, the adsorbent is a magnetic bead.

[0135] In some embodiments, the magnetic beads are stored as a magnetic bead suspension. Before use, the magnetic bead suspension and ethanol or DEPC water need to be mixed evenly at a volume ratio of 1:5 to 15 to prepare the working solution for the magnetic beads. The volume ratio can also be 1:8 to 12, or 1:7, 1:8, 1:9, 1:10, or 1:11.

[0136] In some embodiments, the ethanol is 65-85 v / v ethanol, and may also be 70-80 v / v ethanol; it may also be 70 v / v ethanol, 75 v / v ethanol, or 80 v / v ethanol.

[0137] In some embodiments, the volume ratio of the test sample to proteinase K, lysis buffer, magnetic bead working solution, first washing buffer, second washing buffer and elution buffer is 3:0.1-1:3-7:3-7:3-7:5-8:1-6; preferably 3:0.5:5:5:5:6:4; the amount of each component added can be adjusted appropriately according to the experimental conditions.

[0138] In some embodiments, the sample to be tested includes a microbial culture, an environmental sample, or a human sample; wherein the human sample is a microbial infection sample.

[0139] Preferably, the microbial infection sample includes one or more of the following: whole blood, serum, plasma, saliva, urine, pleural effusion, peritoneal fluid, sputum, tissue fluid, follicular fluid, and tissue samples.

[0140] The unit w / v used in this invention refers to the ratio of mass to volume, where the unit of mass is g and the unit of volume is mL, expressed in grams per milliliter (g / mL). For example, 1 w / v% means that 100 mL of solution contains 1 g of the substance.

[0141] Example 1: A kit for extracting microbial nucleic acids

[0142] 1. The reagent kit composition is as follows:

[0143] (1) Decomposition solution: guanidine isothiocyanate 2M, sodium dodecyl sulfate 2w / w, sodium chloride 4w / v, PEG 8000 10w / w, tris(hydroxymethyl)aminomethane (Tris) 0.15M, Triton X-100 7v / v, sodium phosphite 0.1M, Tween 60 0.15v / v.

[0144] (2) First washing solution: guanidine hydrochloride 3M, anhydrous ethanol 70v / v%, tris(hydroxymethyl)aminomethane 10mM;

[0145] (3) Second washing solution: 10mM tris(hydroxymethyl)aminomethane, 70v / v% anhydrous ethanol;

[0146] (4) Eluent: Tris(hydroxymethyl)aminomethane 50mM;

[0147] (5) Proteinase K: The working concentration is 50 mg / mL, where the working concentration of proteinase K is based on the volume of the mixture of sample and lysis buffer.

[0148] (6) Magnetic beads: The magnetic beads are stored in the form of magnetic bead suspension; when using them, the magnetic bead suspension and 75v / v% ethanol are mixed evenly at a volume ratio of 1:9 to form the working solution of the magnetic beads (75v / v% ethanol can also be replaced with DEPC water). The magnetic beads are silanol-modified iron oxide magnetic beads with a particle size of 200nm and a working concentration of 2mg / mL. The working concentration of the magnetic beads is based on the volume of the mixture of sample and lysis buffer.

[0149] This kit can be prepared in a pre-filled state. The reagents are pre-filled into deep-well plates or reagent strips, sealed, and stored at room temperature. The seal is removed before use. The kit preparation can be completed in advance by the production line.

[0150] Specifically, taking the application scenario of a 12*8 deep-well plate (volume of 2.2 mL / well) with a 96-throughput nucleic acid extractor (Mole 96M) as an example, the magnetic bead working solution is aliquoted at 500 μL / well to each well of the first deep-well plate, the lysis buffer at 500 μL / well to each well of the second deep-well plate, the first washing buffer at 500 μL / well to each well of the third deep-well plate, the second washing buffer at 600 μL / well to each well of the fourth deep-well plate, and the elution buffer at 400 μL / well to each well of the fifth deep-well plate.

[0151] 2. Sample processing

[0152] (1) Open the second deep-well plate and add 50 μL of proteinase K and 300 μL of sample to each well. Each well in the same plate corresponds to one sample.

[0153] (2) Open the magnetic beads, first washing solution, second washing solution and elution solution deep well plate, and place each reagent into the corresponding plate position of the nucleic acid extractor according to Table 1.

[0154] Table 1. Reagent Placement Location

[0155] Instrument plate position reagents 1 Magnetic bead working fluid and magnetic rod sleeve 2 The lysis buffer plate with the sample added 3 3rd deep hole plate 4 4th deep hole plate 5 5th deep hole plate

[0156] (3) Set the program according to Table 2 or open the program 1 that has been saved in the instrument, and then start the program.

[0157] Table 2 Program 1

[0158]

[0159] (4) After the procedure is completed, transfer the nucleic acid in the elution plate (plate position 5) to a clean, nuclease-free centrifuge tube for later use.

[0160] Example 2: Optimization of the lysis buffer formulation

[0161] To improve the lysis efficiency against fungal microorganisms without affecting the nucleic acids of previously released viral microorganisms, the formulation of the lysis buffer was adjusted to form different formulation schemes L1-L9, as shown in the table below:

[0162] Table 3 Formulas for Schemes L1-L9

[0163]

[0164] The remaining components and usage procedures remain consistent with Example 1. Cultures of Candida auris, influenza A virus, and Klebsiella pneumoniae are mixed together to simulate a mixed infection sample.

[0165] Three mixed-infection samples (samples 1-3) were extracted using protocols L1-L9 respectively. Influenza A virus nucleic acid was detected using the "Influenza A and B Virus Nucleic Acid Detection Kit (Fluorescent PCR Method)," with the Ct value of the FAM channel reflecting the concentration of Influenza A virus nucleic acid. Candida auris nucleic acid was detected using the "Candida auris Nucleic Acid Detection Kit (Fluorescent PCR Method)," with the Ct value of the FAM channel reflecting the concentration of Candida auris nucleic acid. Klebsiella pneumoniae nucleic acid was detected using the "Klebsiella pneumoniae Detection Kit (Real-time Fluorescent PCR Method)," with the Ct value of the FAM channel reflecting the concentration of Klebsiella pneumoniae nucleic acid. A 2-fold difference in concentration corresponds to one difference in Ct value, and a 2^n-fold difference in concentration corresponds to n differences in Ct values; the lower the concentration, the larger the Ct value. All PCR reagents used were manufactured by Jiangsu Mole Biotechnology Co., Ltd.

[0166] The results are shown in the table below;

[0167] Table 4

[0168]

[0169] The results showed that, compared with the results of schemes L1 to L9 for Candida auris, scheme L7 had the best extraction results, followed by L5 and L6. This indicates that the addition of sodium phosphite played a key role in improving the nucleic acid extraction efficiency of Candida auris and significantly increased the nucleic acid yield of Candida auris. Meanwhile, the use of sodium phosphite had no significant effect on the nucleic acid extraction efficiency of influenza A virus and Klebsiella pneumoniae.

[0170] Comparing the extraction results of schemes L7, L8, and L9, it is shown that after replacing sodium phosphite with other reducing agents commonly used in nucleic acid extraction experiments, such as dithiothreitol or β-mercaptoethanol, the CT value results of Candida auris nucleic acid detection were significantly delayed, indicating that not all substances of the same type can achieve the same effect as sodium phosphite.

[0171] Example 3: Screening of Magnetic Beads

[0172] Except for the magnetic beads, the other components remained the same as in Example 1. Extraction was performed using magnetic beads with different modifications, resulting in different extraction schemes, as shown in the table below:

[0173] Table 5

[0174] Solution Name Magnetic beads Magnetic bead working concentration C1 Silicon hydroxyl magnetic beads 2mg / mL C2 Amino magnetic beads 2mg / mL C3 Thiol-based magnetic beads 2mg / mL C4 Epoxy magnetic beads 2mg / mL

[0175] The remaining components and usage procedures remained consistent with Example 1. Mixed infection samples 1-3 from Example 2 were extracted using protocols C1-C4. Influenza A and B virus nucleic acid detection kits (fluorescent PCR method) were used to detect influenza A virus nucleic acid, with the Ct value of the FAM channel reflecting the concentration of influenza A virus nucleic acid. Candida auris nucleic acid detection kits (fluorescent PCR method) were used to detect Candida auris nucleic acid, with the Ct value of the FAM channel reflecting the concentration of Candida auris nucleic acid. Klebsiella pneumoniae detection kits (real-time fluorescent PCR method) were used to detect Klebsiella pneumoniae nucleic acid, with the Ct value of the FAM channel reflecting the concentration of Klebsiella pneumoniae nucleic acid. A 2-fold difference in concentration corresponds to one difference in Ct value, and a 2^n-fold difference in concentration corresponds to n differences in Ct values; the lower the concentration, the larger the Ct value. All PCR detection reagents mentioned above were manufactured by Jiangsu Mole Biotechnology Co., Ltd.

[0176] The results are shown in the table below:

[0177] Table 6

[0178]

[0179] The results showed that, compared with the previous results, the C1 extraction method yielded the lowest Ct value for nucleic acid products, indicating the highest nucleic acid yield. Therefore, silanol magnetic beads are the preferred choice.

[0180] Example 4: Optimization of the extraction program

[0181] The lysis time in step 3 of the extraction procedure in procedure 1 was adjusted to create different extraction procedures, as shown in the table below:

[0182] Table 7

[0183]

[0184] Keeping the remaining components and usage procedures consistent with Example 1, samples 1-3 of the mixed infection as described in Example 2 were extracted. Influenza A and B virus nucleic acid detection kits (fluorescent PCR method) were used to detect influenza A virus nucleic acid, with the Ct value of the FAM channel reflecting the concentration of influenza A virus nucleic acid. Candida auris nucleic acid detection kits (fluorescent PCR method) were used to detect Candida auris nucleic acid, with the Ct value of the FAM channel reflecting the concentration of Candida auris nucleic acid. Klebsiella pneumoniae detection kits (real-time fluorescent PCR method) were used to detect Klebsiella pneumoniae nucleic acid, with the Ct value of the FAM channel reflecting the concentration of Klebsiella pneumoniae nucleic acid. A 2-fold difference in concentration corresponds to one difference in Ct value, and a 2^n-fold difference in concentration corresponds to n differences in Ct values; the lower the concentration, the larger the Ct value. All the above detection reagents were manufactured by Jiangsu Mole Biotechnology Co., Ltd.

[0185] The results are shown in the table below:

[0186] Table 8

[0187]

[0188] The results showed that: compared with the Klebsiella pneumoniae results, the extraction results of schemes W1-W5 were similar with little difference; compared with the influenza A virus results, the results of schemes W1-W3 were similar and better than schemes W4 and W5; compared with the Candida auris results, the results of schemes W3-W5 were similar and better than schemes W1 and W2; overall, scheme W3 had the best extraction effect, taking into account multiple types of microbial samples, while schemes W1, W2, W4, and W5 had slightly poorer compatibility with sample types.

[0189] Example 5: Comparison with commercially available reagents

[0190] The commercially available fungal microbial extraction reagent, "Yeast Genomic DNA Extraction Kit," from Tiangen was used.

[0191] The mixed infection samples 1-3 as described in Example 2 were extracted using (DP307) (referred to as commercial reagent 1), Novizan's commercial viral microbial extraction reagent "VAMNEVirus DNA / RNA Extraction Kit 3.0" (RM501) (referred to as commercial reagent 2), Thermo Fisher Scientific's commercial bacterial microbial extraction reagent "Genomic DNA Purification Kit" (K0512) (referred to as commercial reagent 3), and the kit method of Example 1.

[0192] Similarly, *Candida auris* nucleic acid was detected using the "Candida auris Nucleic Acid Detection Reagent (Fluorescent PCR Method)," with the Ct value of the FAM channel reflecting the concentration of *Candida auris* nucleic acid (referred to as Detection 1); Influenza A virus nucleic acid was detected using the "Influenza A and B Virus Nucleic Acid Detection Kit (Fluorescent PCR Method)," with the Ct value of the FAM channel reflecting the concentration of Influenza A virus nucleic acid (referred to as Detection 2); and *Klebsiella pneumoniae Detection Kit (Real-Time Fluorescent PCR Method)* was used to detect *Klebsiella pneumoniae* nucleic acid, with the Ct value of the FAM channel reflecting the concentration of *Klebsiella pneumoniae* nucleic acid (referred to as Detection 3). A 2-fold difference in concentration corresponds to one difference in Ct value, and a 2^n-fold difference in concentration corresponds to n differences in Ct values. The lower the concentration, the larger the Ct value. All the PCR reagents mentioned above were manufactured by Jiangsu Mole Biotechnology Co., Ltd.

[0193] The results are shown in Table 9 and Figures 1-9 As shown:

[0194] in Figures 1-3 This is the PCR result for Candida auris. Figures 4-6 This is the PCR result for influenza A virus. Figures 7-9 This is the PCR result for Klebsiella pneumoniae;

[0195] Table 9

[0196]

[0197] The results showed that, compared with the results for Candida auris, the extraction results of commercial reagent 1 and Example 1 were relatively the best; compared with the results for influenza A virus, the extraction results of commercial reagent 2 and Example 1 were relatively the best; compared with the results for Klebsiella pneumoniae, the extraction results of commercial reagents 1, 2, 3 and Example 1 were similar. In summary, this invention combines the extraction advantages of commercial reagents 1, 2 and 3, and can handle nucleic acid extraction from multiple types of microorganisms with good nucleic acid yield.

[0198] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A lysis buffer, characterized in that, The components of the lysis buffer include: guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, tris(hydroxymethyl)aminomethane, and phosphite; The concentration of the phosphite in the lysis solution is 0.1 M; The phosphite is sodium phosphite; The guanidine salt is guanidine isothiocyanate; The inorganic salt is sodium chloride; The anionic surfactant is sodium dodecyl sulfate; The nonionic surfactant includes one or more of PEG8000, Triton X-100, and Tween 60; The concentration of the guanidine salt in the lysis buffer is 2M; The concentration of the anionic surfactant in the lysis solution is 2 w / w%. The concentration of the inorganic salt in the pyrolysis solution is 4 w / v%. The concentration of the tris(hydroxymethyl)aminomethane in the pyrolysis solution was 0.15 M; The concentration of PEG8000 in the lysis solution is 10 w / w%. The concentration of Triathon X-100 in the lysis solution is 7 v / v%. The concentration of Tween 60 in the lysis buffer is 0.15 v / v.

2. A nucleic acid extraction kit, characterized in that, The kit includes the lysis buffer of claim 1; it also includes one or more of proteinase K, adsorbent, first washing buffer, second washing buffer, and elution buffer; the first washing buffer includes guanidine salt, alcohol, and tris(hydroxymethyl)aminomethane; the second washing buffer includes tris(hydroxymethyl)aminomethane and alcohol.

3. The reagent kit according to claim 2, characterized in that, The eluent is tris(hydroxymethyl)aminomethane; and / or the adsorbent is magnetic beads.

4. The reagent kit according to claim 2, characterized in that, The guanidine salt includes one or both of guanidine hydrochloride and guanidine isothiocyanate. And / or, the alcohol is ethanol.

5. The reagent kit according to claim 3, characterized in that, The magnetic beads are silanol-modified magnetic beads; And / or, based on the volume of the mixture of the sample to be tested and the lysis buffer, the working concentration of the magnetic beads is 0.5-5 mg / mL; And / or, the particle size of the magnetic beads is 100–500 nm.

6. A product for extracting microbial nucleic acids, characterized in that, The product comprises the lysis buffer of claim 1 or the kit of any one of claims 2 to 5.

7. The product according to claim 6, characterized in that, The product is a reagent, kit, or nucleic acid extraction and / or detection system; and / or, the product also includes other tools that can provide an external magnetic field.

8. The product according to claim 7, characterized in that, The nucleic acid extraction system can be a fully automated nucleic acid extractor or a manual nucleic acid extraction device.

9. The use of the lysis buffer of claim 1, the kit of any one of claims 3-5, or the product of any one of claims 6-8 in the extraction of microbial nucleic acids; wherein the microorganisms include: One or more of bacteria, viruses, and fungi.

10. A method for extracting microbial nucleic acid, comprising extracting microbial nucleic acid from a sample to be tested using the lysis buffer of claim 1, the kit of any one of claims 3-5, or the product of any one of claims 6-8; wherein the microorganism comprises: One or more of bacteria, viruses, and fungi.

11. The method according to claim 10, characterized in that, The method specifically includes: mixing lysis buffer, proteinase K, adsorbent and test sample to obtain adsorbent with adsorbed nucleic acid; washing the adsorbent with adsorbed nucleic acid with a first washing solution and a second washing solution, and then eluting with an elution solution to obtain microbial nucleic acid.

12. The method according to claim 11, characterized in that, The mixing time is 15-25 minutes.

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