Lysate, kit and method for extracting microbial nucleic acid

Abstract

The invention provides a lysis solution, a kit and a method for extracting microbial nucleic acid. The lysis solution comprises guanidine salt, an anionic surfactant, inorganic salt, a nonionic surfactant, trihydroxymethyl aminomethane and phosphite. The phosphite is compounded with other components in the lysis solution, so that the lysis effect on microorganisms with complex structures such as fungi is improved, and meanwhile, nucleic acid of microorganisms with simple structures such as viruses released in advance is not influenced; the invention further provides a nucleic acid extraction kit containing the lysate, and co-extraction of nucleic acid of various microorganisms is achieved. The kit provided by the invention is matched with an extraction program for use, so that high-quality nucleic acid of different microorganisms in various infected samples can be obtained; the nucleic acid extraction time of different microorganisms is shortened, the operation process is simplified, and the extraction cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid extraction, and in particular to a lysis solution, a kit and a method for extracting nucleic acid from microorganisms. Background Art

[0002] Nucleic acid detection technology is widely used in the field of biology. It is not only an important means of scientific research, but also an important source of information support for clinical management. Before nucleic acid analysis, it is necessary to obtain the nucleic acid of the microorganism before starting subsequent downstream tests such as PCR, isothermal amplification, and sequencing. The acquisition of nucleic acid is the starting step of the entire detection system. The quality and yield of the isolated nucleic acid have a decisive influence on the subsequent test results.

[0003] There are rich varieties of microorganisms in the natural environment, and the structural components of their cells or viruses vary. The structure of viruses is relatively simple, consisting only of DNA or RNA wrapped in a protein shell. In addition to the matrix, organelles and nuclear structure wrapped by the cell membrane, bacteria also have cell walls, capsules and other structures outside the plasma membrane, which are rich in substances such as peptidoglycan and teichoic acid. The cell wall of fungi is divided into a tangible microfibril part and an amorphous matrix part, which is mainly composed of substances such as chitin, glucan and mannan.

[0004] When performing nucleic acid separation, the difficulty in co-extracting nucleic acids from various microorganisms lies in the fact that microorganisms with different structures have different requirements for lysis conditions, which makes it difficult to take into account the nucleic acid extraction effects of different microorganisms at the same time. Microorganisms with complex cell structures such as fungi have high requirements for lysis intensity, otherwise the yield of nucleic acid will be affected; while pathogenic microorganisms such as viruses have a simple structure, and general lysis conditions can release nucleic acids. Conversely, an overly strong lysis environment or lysis time will damage the released nucleic acids.

[0005] To address this problem, currently most methods use different extraction reagents or different use procedures of the same extraction reagent to obtain nucleic acids of microorganisms such as viruses, bacteria, and fungi. However, this solution has complicated operation procedures and requires a large number of samples, and the repeatability of the results in actual operations is not ideal. Therefore, how to ensure the nucleic acid yield of different microorganisms in the same operation process is the research focus of this field. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a lysis solution and a kit that can ensure 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 objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0008] In a first aspect of the present invention, a lysis solution is provided, wherein the components of the lysis solution include: guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, tris(hydroxymethyl)aminomethane and phosphite.

[0009] In certain embodiments, the concentration of the guanidine salt in the lysate is 1-5M.

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

[0011] In certain embodiments, the concentration of the anionic surfactant in the lysate is 1-10 w / w %.

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

[0013] In certain embodiments, the inorganic salt comprises either or both of sodium chloride and potassium chloride.

[0014] In certain embodiments, the concentration of the inorganic salt in the lysate is 0.1-5 w / v %.

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

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

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

[0018] In certain embodiments, the molecular weight of the polyethylene glycol is 2000-10000; further, the molecular weight of the polyethylene glycol is 6000-8000.

[0019] In certain embodiments, the concentration of polyethylene glycol in the lysate is 5-15 w / w %.

[0020] In certain embodiments, the concentration of Triton in the lysate is 0.1-10 v / v%.

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

[0022] In certain embodiments, the Tween comprises one or more of Tween-20 (Tween-20) and Tween-80 (Tween-80).

[0023] In certain embodiments, the concentration of Tween in the lysate is 0.1-4 v / v%.

[0024] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the lysate is 0.1-1M.

[0025] In certain embodiments, the concentration of the phosphite in the lysate is 0.01-2M.

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

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

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

[0029] In certain embodiments, based on the volume of the mixture of the test sample and the lysate, the working concentration of the proteinase K is 1-100 mg / mL.

[0030] In certain 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 certain embodiments, the concentration of the guanidine salt in the first washing solution is 1-5M.

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

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

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

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

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

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

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

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

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

[0041] In certain embodiments, the eluent is tris(hydroxymethyl)aminomethane. The eluent elutes the nucleic acid from the magnetic beads and serves as a matrix for nucleic acid storage to be used in downstream experiments.

[0042] In certain embodiments, the concentration of Tris in the eluent is 1-100 mM.

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

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

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

[0046] In some embodiments, the particle size of the magnetic beads is 100-500 nm.

[0047] The present invention also provides the use of the above-mentioned lysis solution or the above-mentioned kit in extracting microbial nucleic acid or preparing a product for extracting microbial nucleic acid.

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

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

[0050] In certain 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 acid, comprising using the kit as described above to extract the microbial nucleic acid in the sample to be tested.

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

[0053] In certain embodiments, the mixing time is 15-25 min.

[0054] In certain embodiments, the mixing temperature is 90-98°C.

[0055] In certain 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 comprises: one or more of whole blood, serum, plasma, saliva, urine, pleural effusion, ascites, sputum, tissue fluid, follicular fluid, and tissue samples.

[0057] Beneficial effects:

[0058] The present invention provides a lysing solution, which contains guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, trishydroxymethylaminomethane, and phosphite; by using phosphite in the lysing solution and compounding with other components, the lysis effect on complex microorganisms such as fungi is improved while not affecting the nucleic acids of simple microorganisms such as viruses released first. The present invention also provides a nucleic acid extraction kit containing the lysing solution, which realizes the co-extraction of nucleic acids of multiple microorganisms. The kit and the extraction procedure in the present invention are used in combination to obtain high-quality nucleic acids of different microorganisms in multiple infected samples; the extraction time of nucleic acids of different microorganisms is shortened, the operation process is simplified, and the extraction cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the PCR result of Candida auris of sample 1 in Example 5.

[0060] Figure 2 This is the PCR result of Candida auris of sample 2 in Example 5.

[0061] Figure 3 This is the PCR result of Candida auris of sample 3 in Example 5.

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

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

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

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

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

[0067] Fig. 9 This is the PCR result of Klebsiella pneumoniae of sample 3 in Example 5. DETAILED DESCRIPTION

[0068] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0069] Before further describing the 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 terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0070] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0071] The first aspect of the present invention provides a lysate, the components of which include: guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, tris(hydroxymethyl)aminomethane and phosphite. The lysate is used to destroy microbial cells and virus structures, release nucleic acids, maintain the stability of nucleic acid structures, and provide a favorable environment for nucleic acids to bind to magnetic beads. The present invention optimizes the formula of the lysate to ensure that the nucleic acids of microorganisms with complex structures such as fungi are released, and does not affect the nucleic acids of simple microorganisms such as viruses that are released in advance. The sodium phosphite in the lysate has good solubility and stability in aqueous solution; sodium phosphite has strong reducing properties and can provide a reducing environment. When used in combination with guanidine salt, it can improve the lysis effect of complex cell structures; anionic surfactants can destroy cell membranes and nuclear membranes, and separate histones from DNA. The synergistic effect of nonionic surfactants and anionic surfactants can promote the release of nucleic acids; tris(hydroxymethyl)aminomethane provides a stable liquid environment for the nucleic acid extraction process to avoid large fluctuations in the pH value in the extraction liquid environment that affect the nucleic acid extraction efficiency.

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

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

[0074] In certain embodiments, the concentration of the anionic surfactant in the lysate is 1 to 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 certain embodiments, the anionic surfactant includes at least one of sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium lauryl sulfonate; preferably sodium lauryl sulfate.

[0076] In certain embodiments, the inorganic salt comprises either or both of sodium chloride and potassium chloride.

[0077] In certain embodiments, the concentration of the inorganic salt in the lysate is 0.1 to 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%, or 1-3 w / v%; it can also be 0.1, 0.5, 1, 2, 3, 4 or 5 w / v%.

[0078] In certain 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; further preferably, the nonionic surfactant includes polyethylene glycol, Triton, and Tween.

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

[0080] In certain embodiments, the concentration of polyethylene glycol in the lysate is 5 to 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 certain embodiments, the concentration of Triton in the lysate is 0.1-10 v / v%; it can also be 0.1-1 v / v%, it can also be 1-5 v / v%, it can also be 5-10 v / v%; it can also be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 v / v%.

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

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

[0084] In certain embodiments, the concentration of Tween in the lysate 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 certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the lysate is 0.1-1M; it can also be 0.3-0.8M, or it can be 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M or 1M.

[0086] In certain embodiments, the concentration of the phosphite in the lysate is 0.01 to 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, 0.01 M, 0.1 M, 0.5 M, 1 M, 1.5 M or 2 M.

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

[0088] The present invention also provides a kit for extracting nucleic acid from microorganisms, the kit comprising the lysis solution as described above.

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

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

[0091] In certain embodiments, based on the volume of the mixture of the sample to be tested and the lysate, the working concentration of the proteinase K is 1-100 mg / mL; it can also be 5-80 mg / mL, 10-50 mg / mL, 15-30 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL. The proteinase K is a protein hydrolyzing agent that can hydrolyze proteins in cell or virus structures and proteins bound to DNA, fully exposing the DNA and facilitating DNA extraction.

[0092] In certain 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 certain embodiments, the concentration of the guanidine salt in the first washing solution is 1-5M; it can also be 2-4M, or it can be 1M, 2M, 3M, 4M or 5M.

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

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

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

[0097] In certain 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 certain embodiments, the first washing solution comprises components in the following concentrations: 1-5 M guanidine salt, 60-80 v / v% alcohol, and 1-50 mM tris(hydroxymethyl)aminomethane.

[0099] In certain 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 certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the second washing solution is 1-100 mM; it can also be 1-60 mM, it can also be 60-100 mM, it can also be 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 certain embodiments, the concentration of the alcohol is 60-80 v / v%; it may also be 65-75 v / v%, or it may be 60 v / v%, 65 v / v%, 70 v / v%, 75 v / v% or 80 v / v%.

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

[0103] In certain embodiments, the eluent is tris(hydroxymethyl)aminomethane. The eluent elutes the nucleic acid from the magnetic beads and serves as a matrix for nucleic acid storage to be used in downstream experiments.

[0104] In certain 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 certain embodiments, the adsorbent comprises magnetic beads.

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

[0107] In certain embodiments, based on the volume of the mixture of the test sample and the lysate, the working concentration of the magnetic beads is 0.5-5 mg / mL; it can also be 1-3 mg / mL, it can also be 2-5 mg / mL, it can also be 0.8-4.5 mg / mL, it can also be 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 100-500 nm, or 100-300 nm, or 300-500 nm, or 150-400 nm, or 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm.

[0109] The present invention also provides a product for extracting microbial nucleic acid, wherein the product comprises the lysis solution or the kit as described above.

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

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

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

[0113] In certain embodiments, the product further comprises other means for providing an external magnetic field.

[0114] The present invention also provides the use of the lysis solution, kit or product as described above in extracting microbial nucleic acid or preparing a product for extracting microbial nucleic acid.

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

[0116] In certain 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 inf b, Helicobacter pylori, Klebsiella oxytoca, K. pneumonia (MDR / CRE), Legionella pneumophila, Neisseria meningitidis, meningitides), Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella typhi, Paratyphi, Typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridium neoformans, C. difficile, C. perfringens, Corynebacterium spp., Enterococcus faecalis, Enterococcus faecium faecium), vancomycin-resistant Enterococci (VRE), Listeria monocytogenes, Mycobacterium avium, Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium leprae, Nocardia farcinica, Propionibacterium acnes, Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), and Group C Streptococcus.

[0117] Rickettsiae include, but are not limited to, Rickettsia prowazekii, Rickettsia morozekii, Rickettsia conorii, Rickettsia amurensis, Rickettsia rickettsii, Rickettsia sibirica, Ehrlichia chaffeensis, Ehrlichia canis, Orientia tsutsugamushi and Coxiella burnetii.

[0118] Viruses include, but are not limited to, Dengue virus, Ebola virus, EBV, Hepitis A virus, Hepitis B virus, Hepitis C virus, Hepitis 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, C. parapsilosis, Candida tropicalis, Cryptococcus, Fusarium spp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

[0120] Mycoplasmas include, but are 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 muris, and Chlamydia suis.

[0122] Treponema include, but are not limited to, Treponema pallidum, Treponema yaws, Treponema pinta, Treponema pallidum, Treponema burgdorferi, Treponema hermsii, or Leptospira interrogans.

[0123] Actinomycetes include but are not limited to actinomycetes of the genus Streptomyces, for example, selected from Streptomyces muridarum, Streptomyces celluloflavus, and Streptomyces griseus.

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

[0125] In certain 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 acid, comprising using the lysis solution or the kit or the product as described above to extract the microbial nucleic acid in the sample to be tested.

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

[0128] It should be noted that the above method is not limited to mixing the lysate with the sample to be extracted and then adding the magnetic beads for mixing; the lysate and magnetic beads can also be mixed and then adding the sample to be extracted for mixing and reaction; the lysate, magnetic beads and sample to be extracted can also be mixed at the same time.

[0129] In certain embodiments, when the mixing time is 15-25 min, or 18-23 min, or 20 min, the extraction effect on microbial nucleic acid is the best.

[0130] In certain embodiments, the mixing temperature is 90-98°C, and can 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 sample to be tested, the lysate, the first washing solution, the second washing solution and the eluent is 3:3-7:3-7:5-8:1-6; preferably 3:5:5:6:4; the addition amount of each component can be appropriately adjusted according to the experimental conditions.

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

[0133] In certain embodiments, based on the volume of the mixture of the test sample and the lysate, the working concentration of the proteinase K is 1-100 mg / mL.

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

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

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

[0137] In some embodiments, the volume ratio of the sample to be tested to proteinase K, lysis solution, magnetic bead working solution, first washing solution, second washing solution and elution solution 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 addition amount of each component can be appropriately adjusted according to the experimental conditions.

[0138] In certain 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 comprises: one or more of whole blood, serum, plasma, saliva, urine, pleural effusion, ascites, sputum, tissue fluid, follicular fluid, and tissue samples.

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

[0141] Example 1 A kit for extracting microbial nucleic acid

[0142] 1. The formula of the kit is as follows:

[0143] (1) Lysis solution: guanidine thiocyanate 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: 3 M guanidine hydrochloride, 70 v / v% anhydrous ethanol, 10 mM tris(hydroxymethyl)aminomethane;

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

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

[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 mixed solution of sample and lysis buffer.

[0148] (6) Magnetic beads: The magnetic beads are stored in the form of a magnetic bead suspension. When in use, the magnetic bead suspension and 75 v / v% ethanol are first mixed in a volume ratio of 1:9 as the magnetic bead working solution (75 v / v% ethanol can also be replaced with DEPC water). The magnetic beads are silanol-modified ferroferric oxide magnetic beads with a particle size of 200 nm. The working concentration of the magnetic beads is 2 mg / mL, where the working concentration of the magnetic beads is based on the volume of the mixed solution of the sample and the lysate.

[0149] The kit can be prepared in a pre-packed state, and each reagent can be filled into a deep-well plate or a reagent strip in advance, sealed and stored at room temperature, and the sealing film can be removed before use. The preparation of the kit 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 dispensed into each well of the first deep-well plate at 500 μL / well, the lysate is dispensed into each well of the second deep-well plate at 500 μL / well, the first washing solution is dispensed into each well of the third deep-well plate at 500 μL / well, the second washing solution is dispensed into each well of the fourth deep-well plate at 600 μL / well, and the elution solution is dispensed into each well of the fifth deep-well plate at 400 μL / well.

[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 deep-well plate containing magnetic beads, the first washing solution, the second washing solution, and the elution solution, and place each reagent in the corresponding plate position of the nucleic acid extraction instrument according to Table 1.

[0154] Table 1 Reagent placement

[0155] Instrument board position Reagents 1 Magnetic bead working solution and magnetic rod set 2 Lysis buffer plate with samples added 3 The third deep well plate 4 The fourth deep well plate 5 The fifth deep well plate

[0156] (3) Set up the program according to Table 2 or open the program 1 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 eluate plate (plate position 5) to a clean nuclease-free centrifuge tube for later use.

[0160] Example 2 Optimization of lysate formulation

[0161] In order to improve the lysis effect on fungal microorganisms while not affecting the nucleic acids of viral microorganisms released first, the formula of the lysis solution was adjusted to form different formulas L1-L9, as shown in the following table:

[0162] Table 3: Formula of Scheme L1-L9

[0163]

[0164] The remaining components and the use process were kept the same as those in Example 1. The cultures of Candida auris, influenza A virus and Klebsiella pneumoniae were mixed together to simulate a mixed infection sample.

[0165] Schemes L1 to L9 were used to extract three mixed infection samples (samples 1 to 3). The "Influenza A and B Virus Nucleic Acid Detection Kit (Fluorescence PCR Method)" was used to detect influenza A virus nucleic acid, and the Ct value of the FAM channel was used to reflect the concentration of influenza A virus nucleic acid; the "Candida auris nucleic acid detection reagent (fluorescence PCR method)" was used to detect Candida auris nucleic acid, and the Ct value of the FAM channel was used to reflect the concentration of Candida auris nucleic acid; the "Klebsiella pneumoniae detection kit (real-time fluorescence PCR method)" was used to detect Klebsiella pneumoniae nucleic acid, and the Ct value of the FAM channel was used to reflect the concentration of Klebsiella pneumoniae nucleic acid; a 2-fold concentration difference corresponds to a 1 Ct value difference, and a 2-fold concentration difference corresponds to a n-fold Ct value difference. The lower the concentration, the greater the Ct value. The above-mentioned PCR detection reagents are all produced by Jiangsu Merle Biotechnology Co., Ltd.

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

[0167] Table 4

[0168]

[0169] The results showed that: comparing the Candida auris results of schemes L1 to L9, the extraction result of scheme L7 was the best, followed by L5 and L6, indicating that the addition of sodium phosphite played a key role in improving the extraction efficiency of Candida auris nucleic acid, significantly improving the nucleic acid yield of Candida auris. At the same time, 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 using other reducing agents commonly used in nucleic acid extraction experiments such as dithiothreitol or β-mercaptoethanol to replace sodium phosphite, the CT value results of nucleic acid detection of Candida auris are significantly delayed, indicating that not all substances of the same type can achieve the effect of sodium phosphite.

[0171] Example 3 Screening of magnetic beads

[0172] Except for the magnetic beads, the other components are consistent with Example 1. Differently modified magnetic beads are used for extraction to form different extraction schemes, as shown in the following table:

[0173] Table 5

[0174] Solution Name Magnetic beads Magnetic beads working concentration C1 Silane Magnetic Beads 2mg / mL C2 Amino Magnetic Beads 2mg / mL C3 Thiol Magnetic Beads 2mg / mL C4 Epoxy Magnetic Beads 2mg / mL

[0175] Keep the remaining components and the use process consistent with Example 1. Use C1 to C4 schemes to extract mixed infection samples 1 to 3 as in Example 2. Use the "Influenza A and B Virus Nucleic Acid Detection Kit (Fluorescent PCR Method)" to detect influenza A virus nucleic acid, and the Ct value result of the FAM channel reflects the concentration of influenza A virus nucleic acid; use the "Candida auris nucleic acid detection reagent (fluorescent PCR method)" to detect Candida auris nucleic acid, and the Ct value result of the FAM channel reflects the concentration of Candida auris nucleic acid; use the "Klebsiella pneumoniae detection kit (real-time fluorescent PCR method)" to detect Klebsiella pneumoniae nucleic acid, and the Ct value result of the FAM channel reflects the concentration of Klebsiella pneumoniae nucleic acid; wherein a difference of 2 times the concentration corresponds to a difference of 1 Ct value, and a difference of 2 to the power of n times the concentration corresponds to a difference of n Ct values, and the lower the concentration, the greater the Ct value. The above-mentioned PCR detection reagents are all produced by Jiangsu Merle Biotechnology Co., Ltd.

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

[0177] Table 6

[0178]

[0179] The results show that: compared with the results, the nucleic acid product detection Ct value obtained by the extraction method C1 is the smallest, indicating that the nucleic acid yield is the highest. Therefore, the preferred magnetic beads are silicon hydroxy magnetic beads.

[0180] Example 4 Optimization of extraction procedure

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

[0182] Table 7

[0183]

[0184] Keep the remaining components and the use process consistent with Example 1, and extract the mixed infection samples 1 to 3 in Example 2 respectively. Use the "Influenza A and B Virus Nucleic Acid Detection Kit (Fluorescence PCR Method)" to detect influenza A virus nucleic acid, and use the Ct value result of the FAM channel to reflect the concentration of influenza A virus nucleic acid; use the "Candida auris nucleic acid detection reagent (fluorescence PCR method)" to detect Candida auris nucleic acid, and use the Ct value result of the FAM channel to reflect the concentration of Candida auris nucleic acid; use the "Klebsiella pneumoniae detection kit (real-time fluorescence PCR method)" to detect Klebsiella pneumoniae nucleic acid, and use the Ct value result of the FAM channel to reflect the concentration of Klebsiella pneumoniae nucleic acid; wherein a difference of 2 times the concentration corresponds to a difference of 1 Ct value, and a difference of 2 to the power of n times the concentration corresponds to a difference of n Ct values, and the lower the concentration, the greater the Ct value. The above-mentioned detection reagents are all produced by Jiangsu Merle Biotechnology Co., Ltd.

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

[0186] Table 8

[0187]

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

[0189] Example 5 is compared with commercial reagents

[0190] Use Tiangen's commercial fungal microbial extraction reagent "Yeast Genomic DNA Extraction Kit"

[0191] (DP307) (referred to as commercial reagent 1), Novezan's commercial extraction reagent for viral microorganisms "VAMNEVirus DNA / RNA Extraction Kit 3.0" (RM501) (referred to as commercial reagent 2), Thermo Fisher's commercial extraction reagent for bacterial microorganisms "Genomic DNA Purification Kit" (K0512) (referred to as commercial reagent 3) and the kit method of Example 1 were used to extract mixed infection samples 1 to 3 as in Example 2, respectively.

[0192] Similarly, the "Candida auris nucleic acid detection reagent (fluorescence PCR method)" was used to detect Candida auris nucleic acid, and the Ct value result of the FAM channel was used to reflect the concentration of Candida auris nucleic acid (referred to as detection 1); the "Influenza A and B Virus Nucleic Acid Detection Kit (Fluorescence PCR Method)" was used to detect influenza A virus nucleic acid, and the Ct value result of the FAM channel was used to reflect the concentration of influenza A virus nucleic acid (referred to as detection 2); the "Klebsiella pneumoniae Detection Kit (Real-time Fluorescence PCR Method)" was used to detect Klebsiella pneumoniae nucleic acid, and the Ct value result of the FAM channel was used to reflect the concentration of Klebsiella pneumoniae nucleic acid (referred to as detection 3), where a difference of 2 times the concentration corresponds to a difference of 1 Ct value, and a difference of 2 to the power of n times the concentration corresponds to a difference of n Ct values, and the lower the concentration, the greater the Ct value. The above-mentioned PCR detection reagents are all produced by Jiangsu Merle Biotechnology Co., Ltd.

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

[0194] in Figure 1 to Figure 3 The PCR results for Candida auris are: Figures 4 to 6 The results of influenza A virus PCR are as follows: Figure 7 to Figure 9 This is the PCR result of Klebsiella pneumoniae;

[0195] Table 9

[0196]

[0197] The results show that: compared with the results of Candida auris, the extraction results of commercial reagent 1 and Example 1 are relatively the best; compared with the results of influenza A virus, the extraction results of commercial reagent 2 and Example 1 are relatively the best; compared with the results of Klebsiella pneumoniae, the extraction results of commercial reagents 1, 2, 3 and Example 1 are similar. In summary, the present invention combines the extraction advantages of commercial reagents 1, 2 and 3, can take into account the nucleic acid extraction of various types of microorganisms, and has a 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 present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A lysis solution, characterized in that The components of the lysate include: guanidine salt, anionic surfactant, inorganic salt, nonionic surfactant, tris(hydroxymethyl)aminomethane and phosphite.

2. The lysate according to claim 1, characterized in that The concentration of the guanidine salt in the lysate is 1-5 M; and / or, The concentration of the anionic surfactant in the lysate is 1 to 10 w / w%; and / or, The concentration of the inorganic salt in the lysate is 0.1 to 5 w / v%; and / or, The concentration of tris(hydroxymethyl)aminomethane in the lysate is 0.1 to 1 M; and / or, The concentration of the phosphite in the lysate is 0.01-2M.

3. The lysate according to claim 1 or 2, characterized in that The guanidine salt includes one or both of guanidine hydrochloride and guanidine isothiocyanate; and / or, The inorganic salt includes one or both of sodium chloride and potassium chloride; and / or, The anionic surfactant includes one or more of sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium lauryl sulfonate; and / or, The nonionic surfactant includes one or more of polyethylene glycol, Triton, Tween, nonylphenol polyoxyethylene ether, and hexadecyl polyoxyethylene ether; and / or, The phosphite includes one or more of sodium phosphite, potassium phosphite and magnesium phosphite.

4. The lysate according to claim 3, characterized in that The nonionic surfactants include polyethylene glycol, Triton and Tween; Preferably, the concentration of polyethylene glycol in the lysate is 5 to 15 w / w%; Preferably, the concentration of Triton in the lysate is 0.1 to 10 v / v%; Preferably, the concentration of Tween in the lysate is 0.1-4 v / v%.

5. A nucleic acid extraction kit, characterized in that: The kit comprises the lysate according to any one of claims 1 to 4; It also includes one or more of proteinase K, adsorbent, first washing solution, second washing solution, and elution solution.

6. The kit according to claim 5, characterized in that The first washing liquid includes guanidine salt, alcohol, and tris(hydroxymethyl)aminomethane; Preferably, the second washing liquid comprises tris(hydroxymethyl)aminomethane and alcohols; Preferably, the eluent is tris(hydroxymethyl)aminomethane; Preferably, the guanidine salt includes one or both of guanidine hydrochloride and guanidine isothiocyanate; Preferably, the alcohol is ethanol; Preferably, the adsorbent is a magnetic bead; Preferably, the magnetic beads are silanol-modified magnetic beads; Preferably, based on the volume of the mixture of the sample to be tested and the lysate, the working concentration of the magnetic beads is 0.5-5 mg / mL; Preferably, the particle size of the magnetic beads is 100-500 nm.

7. A product for extracting microbial nucleic acid, characterized in that: The product comprises the lysate according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 6; The product is preferably a reagent, a kit or a system; Preferably, the product is a nucleic acid extraction and / or detection system; Preferably, the nucleic acid extractor is a fully automatic nucleic acid extractor or a manual nucleic acid extraction device; Preferably, the product also includes other tools that can provide an external magnetic field.

8. Use of the lysate according to any one of claims 1 to 4, the kit according to any one of claims 5 to 6, or the product according to claim 7 in extracting microbial nucleic acid; Preferably, the microorganisms include: One or more of bacteria, viruses, rickettsia, mycoplasma, chlamydia, spirochetes, fungi, and actinomycetes.

9. A method for extracting microbial nucleic acid, comprising using the lysis solution according to any one of claims 1 to 4, the kit according to any one of claims 5 to 6, or the product according to claim 7 to extract the microbial nucleic acid in a sample to be tested.

10. The method according to claim 9, characterized in that The method specifically comprises: mixing a lysate, proteinase K, an adsorbate and a sample to be tested to obtain the adsorbate adsorbed with nucleic acid; washing the adsorbate adsorbed with nucleic acid with a first washing solution and a second washing solution, and then eluting with an elution solution to obtain microbial nucleic acid; Preferably, the mixing time is 15-25 min.

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