Pathogen and pathogen nucleic acid enrichment buffer solution and method for enriching pathogen and nucleic acid of pathogen in large number of samples

Abstract

The invention relates to a method for enriching nucleic acid and / or pathogen, in particular to a method for enriching nucleic acid and / or pathogen, a method for extracting nucleic acid and / or pathogen and a corresponding kit thereof. By using the method, nucleic acids and / or pathogens with high concentration can be enriched from a complex and large number of samples. The method is high in recovery rate, suitable for large-volume samples and wide in application scene.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a pathogen and pathogen nucleic acid enrichment buffer and a method for enriching pathogens and nucleic acids in a large number of samples. Background Art

[0002] In the field of biotechnology, especially in virology, microbiology and molecular biology, the enrichment and analysis of viruses, Gram-negative bacteria and free nucleic acids is an important technology. Current molecular biology techniques can basically solve the problem of extracting genomic information required for research or diagnosis from samples of normal volume (generally 200μL). There are also specialized extraction reagents compatible with 1mL-5mL samples to improve detection sensitivity. Such reagents are usually limited by consumables and instruments. However, there is no good solution when faced with larger sample volumes.

[0003] For example, in the process of pathogen monitoring in urban sewage, the schemes given in the industry standards include polyethylene glycol precipitation, aluminum salt coagulation precipitation, centrifugal ultrafiltration (Standard for the enrichment and concentration of novel coronavirus and nucleic acid detection in sewage WS / T799-2022), etc. However, these schemes have the disadvantages of being time-consuming, inefficient, and involving many chemical reagents. At the same time, there are many kinds of impurities in sewage, and there is currently no better method to enrich pathogens and nucleic acids in a relatively complex matrix.

[0004] Urine is a body fluid sample that is very easy to obtain in the clinic, and it contains a lot of nucleic acid and pathogen information in the body. However, the current nucleic acid extraction reagents that can process more than 5mL of urine have complicated operation steps and are time-consuming. Most of them are extracted by centrifugal column method, which is not conducive to large-scale screening.

[0005] Therefore, the present application aims to develop an efficient, interference-resistant, and easy-to-operate method for enriching nucleic acid and pathogen samples. Summary of the invention

[0006] The purpose of the present invention is to provide a method for enriching nucleic acid and pathogen samples which is efficient, interference-resistant and easy to operate.

[0007] In a first aspect of the present invention, a method for enriching nucleic acids and / or pathogens from a sample is provided, comprising the steps of:

[0008] (s1) mixing the sample, enrichment magnetic beads and enrichment buffer to obtain a first mixture, and performing enrichment treatment so that the enrichment magnetic beads adsorb the nucleic acid and / or pathogen to be enriched in the sample to form enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched (enrichment magnetic beads after enrichment treatment);

[0009] (s2) separating the enrichment magnetic beads adsorbed with the nucleic acid and / or pathogen to be enriched, and washing them to obtain washed magnetic beads; and

[0010] (s3) separating the washed magnetic beads and performing an elution treatment to elute the enriched nucleic acid and / or pathogen from the magnetic beads to obtain an eluate containing the enriched nucleic acid and / or pathogen;

[0011] Wherein, the enrichment buffer contains the following components: a cationic surfactant, a cation exchange resin and a reducing agent;

[0012] The washing treatment comprises washing with a washing liquid containing the following components: a cationic surfactant and a neutral surfactant;

[0013] The elution treatment includes eluting using an elution buffer containing the following components: guanidine thiocyanate, EDTA, anionic surfactant, nonionic surfactant, chaotropic salt and buffer.

[0014] In another preferred embodiment, the sample (for pathogens) has been pre-processed for nucleic acid extraction and purification.

[0015] In another preferred embodiment, the sample (for pathogens) is pre-treated with a universal nuclease.

[0016] In another preferred example, the sample contains nucleic acid and / or pathogens.

[0017] In another preferred embodiment, the pathogen is selected from the group consisting of bacteria, viruses, mycoplasmas, chlamydia, or a combination thereof.

[0018] In another preferred embodiment, the virus includes DNA virus and / or RNA virus.

[0019] In another preferred embodiment, the pathogen is selected from the following group: norovirus, influenza A virus, influenza B virus, new coronavirus, syncytial virus, human metapneumovirus, parainfluenza virus, human rhinovirus, adenovirus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycoplasma pneumoniae, or a combination thereof.

[0020] In another preferred example, the sample is an environmental sample.

[0021] In another preferred embodiment, the sample is selected from the group consisting of domestic sewage, plasma, serum, urine, water sample, swab wash fluid, or a combination thereof.

[0022] In another preferred embodiment, in step (s1), the volume of the sample is 0.1-500 mL, preferably 1-200 mL, preferably 5-100 mL, for example, about 10 mL.

[0023] In another preferred embodiment, the enrichment buffer comprises enrichment buffer A and enrichment buffer B.

[0024] In another preferred embodiment, the enrichment buffer A includes a cationic surfactant, and the cationic surfactant is selected from the following group: hexadecyl ammonium bromide, hexadecyltrimethylammonium bromide chloride, hexadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, or a combination thereof, preferably CTAB, more preferably 1% to 20% CTAB, more preferably 1% to 15% CTAB, more preferably 1% to 10% CTAB, for example, about 5% CTAB.

[0025] In another preferred embodiment, the enrichment buffer B comprises a cation exchange resin and a reducing agent, wherein the cation exchange resin is selected from the following group: 50-100 mesh chelex-100, 100-200 mesh chelex-100, 200-400 mesh chelex-100, or a combination thereof, preferably 100-200 mesh chelex-100; the reducing agent comprises acetylcysteine.

[0026] In another preferred embodiment, the step (s1) comprises mixing the sample with enrichment buffer A, adding enrichment buffer B for mixing, and then adding magnetic beads to obtain a first mixture.

[0027] In another preferred embodiment, in step (s1), the amount of enrichment buffer A added is 10 to 3000 μL, preferably 100 to 1000 μL, more preferably 200 to 800 μL, for example, about 500 μL.

[0028] In another preferred embodiment, in step (s1), the volume ratio of the enrichment buffer B to the sample is 1:10-100, preferably 1:20-80, more preferably 1:30-60, for example, about 1:40.

[0029] In another preferred embodiment, in step (s1), in the first mixture, the concentration of acetylcysteine ​​is 1-100 mM, preferably 10-80 mM, more preferably 10-50 mM, for example, about 25 mM; the concentration of chelex is 0.1-500 mg / mL, preferably 1-300 mg / mL, more preferably 1-100 mg / mL, for example, about 25 mg / mL.

[0030] In another preferred embodiment, the enrichment magnetic beads are magnetic beads with surface modified cationic groups, preferably magnetic beads with surface modified amino groups.

[0031] In another preferred embodiment, in step (s2), the enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched are obtained by standing, magnetically adsorbing, and removing the supernatant.

[0032] In another preferred embodiment, in step (s1), the volume ratio of the enriched magnetic beads to the sample is 1:50-200, preferably 1:70-150, more preferably 1:80-120, for example, about 1:100.

[0033] In another preferred embodiment, in step (s2), the standing time is 1 to 10 min, preferably 3 to 8 min, more preferably 5 to 8 min, for example, about 6 min.

[0034] In another preferred embodiment, in step (s2), vortex mixing is performed once every 1 to 3 minutes during the standing process, for example, about 2 minutes.

[0035] In another preferred embodiment, in step (s2), the volume of the washing solution is 0.01-10 mL, preferably 0.1-5 mL, more preferably 0.5-2 mL, for example, about 1 mL.

[0036] In another preferred embodiment, in step (s3), the volume of the eluent is 10 to 1000 μL, preferably 100 to 800 μL, more preferably 100 to 500 μL, for example, about 200 μL.

[0037] In another preferred embodiment, in the washing liquid, the cationic surfactant includes CTAB.

[0038] In another preferred embodiment, the concentration of the cationic surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0039] In another preferred embodiment, in the washing solution, the neutral surfactant includes Triton.

[0040] In another preferred embodiment, the concentration of the neutral surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0041] In another preferred embodiment, the washing solution comprises the following components: 0.1-5% CTAB and 0.1-5% Triton.

[0042] In another preferred embodiment, the washing solution comprises the following components: 0.5-2.5% CTAB and 0.5-2.5% Triton.

[0043] In another preferred embodiment, the washing solution comprises the following components: 0.5-2% CTAB and 0.5-2% Triton.

[0044] In another preferred embodiment, the washing solution comprises the following components: 1% CTAB and 1% Triton.

[0045] In another preferred embodiment, in step (s3), the washed magnetic beads are separated by adsorbing the magnetic beads and discarding the supernatant.

[0046] In another preferred embodiment, in the eluent, the anionic surfactant includes sodium lauroyl sarcosinate.

[0047] In another preferred embodiment, in the eluent, the nonionic surfactant includes NP-40.

[0048] In another preferred embodiment, in the eluent, the chaotropic salt is selected from the following group: potassium iodide, sodium perchlorate, or a combination thereof.

[0049] In another preferred embodiment, the eluent comprises the following components: 20% to 40% guanidine thiocyanate, 0.1 to 50 mM EDTA, 0.01 to 1 M Tris-HCl, 0.01% to 0.1% (v / v) sodium lauroyl sarcosinate, 0.1% to 5% (v / v) NP-40, and 0.5 to 2 M potassium iodide.

[0050] In another preferred embodiment, the eluent comprises the following components: 25% to 35% guanidine thiocyanate, 0.5 to 25 mM EDTA, 0.01 to 0.1 M Tris-HCl, 0.03% to 0.08% sodium lauroyl sarcosinate, 0.1% to 3% NP-40, and 0.8 to 1.5 M potassium iodide.

[0051] In another preferred embodiment, the eluent comprises the following components: 28% to 32% guanidine thiocyanate, 0.25 to 10 mM EDTA, 0.01 to 0.07 M Tris-HCl, 0.04% to 0.06% sodium lauroyl sarcosinate, 0.1% to 1% NP-40, and 0.8 to 1.2 M potassium iodide.

[0052] In another preferred embodiment, the eluent comprises the following components: 30% guanidine thiocyanate, 5 mM EDTA, 0.05 M Tris-HCl, 0.05% sodium lauroyl sarcosinate, 0.5% NP-40, and 1 M potassium iodide.

[0053] A second aspect of the present invention provides a method for extracting nucleic acid from a pathogen sample, comprising the steps of:

[0054] (s1) mixing the sample, enrichment magnetic beads and enrichment buffer to obtain a first mixture, and performing enrichment treatment so that the enrichment magnetic beads adsorb the nucleic acid and / or pathogen to be enriched in the sample to form enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched (enrichment magnetic beads after enrichment treatment);

[0055] (s2) separating the enrichment magnetic beads adsorbed with the nucleic acid and / or pathogen to be enriched, and washing them to obtain washed magnetic beads;

[0056] (s3) separating the washed magnetic beads and performing an elution treatment to elute the enriched nucleic acid and / or pathogen from the magnetic beads to obtain an eluate containing the enriched nucleic acid and / or pathogen; and

[0057] (s4) extracting nucleic acid from the enriched nucleic acid and / or pathogen to obtain the nucleic acid;

[0058] Wherein, the enrichment buffer contains the following components: a cationic surfactant, a cation exchange resin and a reducing agent;

[0059] The washing treatment comprises washing with a washing liquid containing the following components: a cationic surfactant and a neutral surfactant;

[0060] The elution treatment includes eluting using an elution buffer containing the following components: guanidine thiocyanate, EDTA, anionic surfactant, nonionic surfactant, chaotropic salt and buffer.

[0061] In another preferred embodiment, the enrichment magnetic beads, enrichment buffer, washing solution and elution solution are as described in the first aspect of the present invention.

[0062] In another preferred embodiment, the sample has been pre-processed for nucleic acid extraction and purification.

[0063] In another preferred embodiment, the sample is pre-treated with a universal nuclease.

[0064] In another preferred embodiment, the sample contains pathogens.

[0065] In another preferred embodiment, the pathogen is selected from the group consisting of bacteria, viruses, mycoplasmas, chlamydia, or a combination thereof.

[0066] In another preferred embodiment, the virus includes DNA virus and / or RNA virus.

[0067] In another preferred embodiment, the pathogen is selected from the following group: norovirus, influenza A virus, influenza B virus, new coronavirus, syncytial virus, human metapneumovirus, parainfluenza virus, human rhinovirus, adenovirus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycoplasma pneumoniae, or a combination thereof.

[0068] In another preferred embodiment, the sample is selected from the group consisting of domestic sewage, plasma, serum, urine, water sample, swab wash fluid, or a combination thereof.

[0069] In another preferred embodiment, in step (s1), the volume of the sample is 0.1-500 mL, preferably 1-200 mL, preferably 5-100 mL, for example, about 10 mL.

[0070] In another preferred embodiment, the enrichment buffer comprises enrichment buffer A and enrichment buffer B.

[0071] In another preferred embodiment, the enrichment buffer A includes a cationic surfactant, and the cationic surfactant is selected from the following group: hexadecyl ammonium bromide, hexadecyltrimethylammonium bromide chloride, hexadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, or a combination thereof, preferably CTAB, more preferably 1% to 20% CTAB, more preferably 1% to 15% CTAB, more preferably 1% to 10% CTAB, for example, about 5% CTAB.

[0072] In another preferred embodiment, the enrichment buffer B comprises a cation exchange resin and a reducing agent, wherein the cation exchange resin is selected from the following group: 50-100 mesh chelex-100, 100-200 mesh chelex-100, 200-400 mesh chelex-100, or a combination thereof, preferably 100-200 mesh chelex-100; the reducing agent comprises acetylcysteine.

[0073] In another preferred embodiment, the step (s1) comprises mixing the sample with enrichment buffer A, adding enrichment buffer B for mixing, and then adding magnetic beads to obtain a first mixture.

[0074] In another preferred embodiment, in step (s1), the amount of enrichment buffer A added is 10 to 3000 μL, preferably 100 to 1000 μL, more preferably 200 to 800 μL, for example, about 500 μL.

[0075] In another preferred embodiment, in step (s1), the volume ratio of the enrichment buffer B to the sample is 1:10-100, preferably 1:20-80, more preferably 1:30-60, for example, about 1:40.

[0076] In another preferred embodiment, in step (s1), in the first mixture, the concentration of acetylcysteine ​​is 1-100 mM, preferably 10-80 mM, more preferably 10-50 mM, for example, about 25 mM; the concentration of chelex is 0.1-500 mg / mL, preferably 1-300 mg / mL, more preferably 1-100 mg / mL, for example, about 25 mg / mL.

[0077] In another preferred embodiment, in step (s1), the volume ratio of the enriched magnetic beads to the sample is 1:50-200, preferably 1:70-150, more preferably 1:80-120, for example, about 1:100.

[0078] In another preferred embodiment, the enrichment magnetic beads are magnetic beads with surface modified cationic groups, preferably magnetic beads with surface modified amino groups.

[0079] In another preferred embodiment, in step (s2), the enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched are obtained by standing, magnetically adsorbing, and removing the supernatant.

[0080] In another preferred embodiment, in step (s2), the standing time is 1 to 10 min, preferably 3 to 8 min, more preferably 5 to 8 min, for example, about 6 min.

[0081] In another preferred embodiment, in step (s2), vortex mixing is performed once every 1 to 3 minutes during standing, for example, about 2 minutes.

[0082] In another preferred embodiment, in step (s2), the volume of the washing solution is 0.01-10 mL, preferably 0.1-5 mL, more preferably 0.5-2 mL, for example, about 1 mL.

[0083] In another preferred embodiment, in step (s3), the volume of the eluent is 10 to 1000 μL, preferably 100 to 800 μL, more preferably 100 to 500 μL, for example, about 200 μL.

[0084] In another preferred embodiment, the recovery rate of the method is ≥80%, preferably ≥90%, and more preferably ≥95%.

[0085] In another preferred embodiment, the recovery rate of the method is 75% to 98%.

[0086] In another preferred embodiment, in the washing liquid, the cationic surfactant includes CTAB.

[0087] In another preferred embodiment, the concentration of the cationic surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0088] In another preferred embodiment, in the washing solution, the neutral surfactant includes Triton.

[0089] In another preferred embodiment, the concentration of the neutral surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0090] In another preferred embodiment, the washing solution comprises the following components: 0.1-5% CTAB and 0.1-5% Triton.

[0091] In another preferred embodiment, the washing solution comprises the following components: 0.5-2.5% CTAB and 0.5-2.5% Triton.

[0092] In another preferred embodiment, the washing solution comprises the following components: 0.5-2% CTAB and 0.5-2% Triton.

[0093] In another preferred embodiment, the washing solution comprises the following components: 1% CTAB and 1% Triton.

[0094] In another preferred embodiment, in step (s3), the washed magnetic beads are separated by adsorbing the magnetic beads and discarding the supernatant.

[0095] In another preferred embodiment, in the eluent, the anionic surfactant includes sodium lauroyl sarcosinate.

[0096] In another preferred embodiment, in the eluent, the nonionic surfactant includes NP-40.

[0097] In another preferred embodiment, in the eluent, the chaotropic salt is selected from the following group: potassium iodide, sodium perchlorate, or a combination thereof.

[0098] In another preferred embodiment, the eluent comprises the following components: 20% to 40% guanidine thiocyanate, 0.1 to 50 mM EDTA, 0.01 to 1 M Tris-HCl, 0.01% to 0.1% (v / v) sodium lauroyl sarcosinate, 0.1% to 5% (v / v) NP-40, and 0.5 to 2 M potassium iodide.

[0099] In another preferred embodiment, the eluent comprises the following components: 25% to 35% guanidine thiocyanate, 0.5 to 25 mM EDTA, 0.01 to 0.1 M Tris-HCl, 0.03% to 0.08% sodium lauroyl sarcosinate, 0.1% to 3% NP-40, and 0.8 to 1.5 M potassium iodide.

[0100] In another preferred embodiment, the eluent comprises the following components: 28% to 32% guanidine thiocyanate, 0.25 to 10 mM EDTA, 0.01 to 0.07 M Tris-HCl, 0.04% to 0.06% sodium lauroyl sarcosinate, 0.1% to 1% NP-40, and 0.8 to 1.2 M potassium iodide.

[0101] In another preferred embodiment, the eluent comprises the following components: 30% guanidine thiocyanate, 5 mM EDTA, 0.05 M Tris-HCl, 0.05% sodium lauroyl sarcosinate, 0.5% NP-40, and 1 M potassium iodide.

[0102] A third aspect of the present invention provides a kit for enriching nucleic acids and / or pathogens from a sample, comprising:

[0103] (i) Enrichment of magnetic beads;

[0104] (ii) an enrichment buffer comprising a cationic surfactant, a cation exchange resin and a reducing agent;

[0105] (iii) a washing liquid comprising a cationic surfactant and a neutral surfactant; and

[0106] (iv) an eluting solution, which comprises guanidine thiocyanate, EDTA, an anionic surfactant, a nonionic surfactant, a chaotropic salt and a buffer.

[0107] In another preferred embodiment, the enrichment buffer comprises enrichment buffer A and enrichment buffer B.

[0108] In another preferred embodiment, the enrichment buffer A includes a cationic surfactant, and the cationic surfactant is selected from the following group: hexadecyl ammonium bromide, hexadecyltrimethylammonium bromide chloride, hexadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, or a combination thereof, preferably CTAB, more preferably 1% to 20% CTAB, more preferably 1% to 15% CTAB, more preferably 1% to 10% CTAB, for example, about 5% CTAB.

[0109] In another preferred embodiment, the enrichment buffer B comprises a cation exchange resin and a reducing agent, wherein the cation exchange resin is selected from the following group: 50-100 mesh chelex-100, 100-200 mesh chelex-100, 200-400 mesh chelex-100, or a combination thereof, preferably 100-200 mesh chelex-100; the reducing agent comprises acetylcysteine.

[0110] In another preferred embodiment, the enrichment buffer A and the enrichment buffer B are located in different containers.

[0111] In another preferred embodiment, in the washing liquid, the cationic surfactant includes CTAB.

[0112] In another preferred embodiment, the concentration of the cationic surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0113] In another preferred embodiment, in the washing solution, the neutral surfactant includes Triton.

[0114] In another preferred embodiment, the concentration of the neutral surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0115] In another preferred embodiment, the washing solution comprises the following components: 0.1-5% CTAB and 0.1-5% Triton.

[0116] In another preferred embodiment, the washing solution comprises the following components: 0.5-2.5% CTAB and 0.5-2.5% Triton.

[0117] In another preferred embodiment, the washing solution comprises the following components: 0.5-2% CTAB and 0.5-2% Triton.

[0118] In another preferred embodiment, the washing solution comprises the following components: 1% CTAB and 1% Triton.

[0119] In another preferred embodiment, in the eluent, the anionic surfactant includes sodium lauroyl sarcosinate.

[0120] In another preferred embodiment, in the eluent, the nonionic surfactant includes NP-40.

[0121] In another preferred embodiment, in the eluent, the chaotropic salt is selected from the following group: potassium iodide, sodium perchlorate, or a combination thereof.

[0122] In another preferred embodiment, the eluent comprises the following components: 20% to 40% guanidine thiocyanate, 0.1 to 50 mM EDTA, 0.01 to 1 M Tris-HCl, 0.01% to 0.1% (v / v) sodium lauroyl sarcosinate, 0.1% to 5% (v / v) NP-40, and 0.5 to 2 M potassium iodide.

[0123] In another preferred embodiment, the eluent comprises the following components: 25% to 35% guanidine thiocyanate, 0.5 to 25 mM EDTA, 0.01 to 0.1 M Tris-HCl, 0.03% to 0.08% sodium lauroyl sarcosinate, 0.1% to 3% NP-40, and 0.8 to 1.5 M potassium iodide.

[0124] In another preferred embodiment, the eluent comprises the following components: 28% to 32% guanidine thiocyanate, 0.25 to 10 mM EDTA, 0.01 to 0.07 M Tris-HCl, 0.04% to 0.06% sodium lauroyl sarcosinate, 0.1% to 1% NP-40, and 0.8 to 1.2 M potassium iodide.

[0125] In another preferred embodiment, the eluent comprises the following components: 30% guanidine thiocyanate, 5 mM EDTA, 0.05 M Tris-HCl, 0.05% sodium lauroyl sarcosinate, 0.5% NP-40, and 1 M potassium iodide.

[0126] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0127] After extensive and in-depth research, a large number of experiments and screening, the inventors unexpectedly discovered for the first time a kit and method for enriching nucleic acids and / or pathogens, the kit comprising an enrichment buffer, a washing solution and an eluent. The inventors optimized the components and contents of the enrichment buffer, the washing solution and the eluent, respectively, and finally obtained a nucleic acid / pathogen enrichment kit and method with high recovery rate, suitable for large volume samples, and wide application scenarios. The present invention was completed on this basis.

[0128] the term

[0129] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0130] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., are understood to include the stated elements or components but do not exclude other elements or components.

[0131] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0132] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0133] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0134] As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid.

[0135] As used herein, the term "NP-40" refers to ethylphenyl polyethylene glycol.

[0136] As used herein, the term "DTT" refers to dithiothreitol.

[0137] As used herein, the term "chelex-100" refers to chelex-100 resin. The terms "mesh" and "mesh" are used interchangeably and refer to the size of chelex-100 resin particles, the larger the mesh number, the smaller the particles.

[0138] As used herein, the term "CTAB" refers to cetyltrimethylammonium bromide.

[0139] Method for enriching nucleic acid and / or pathogens of the present invention

[0140] As used herein, the terms "method for enriching nucleic acids and / or pathogens according to the present invention" and "method for enriching nucleic acids and / or pathogens from a sample according to the present invention" are used interchangeably and include the steps of:

[0141] (s1) mixing the sample, enrichment magnetic beads and enrichment buffer to obtain a first mixture, and performing enrichment treatment so that the enrichment magnetic beads adsorb the nucleic acid and / or pathogen to be enriched in the sample to form enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched (enrichment magnetic beads after enrichment treatment);

[0142] (s2) separating the enrichment magnetic beads adsorbed with the nucleic acid and / or pathogen to be enriched, and washing them to obtain washed magnetic beads; and

[0143] (s3) separating the washed magnetic beads and performing an elution treatment to elute the enriched nucleic acid and / or pathogen from the magnetic beads to obtain an eluate containing the enriched nucleic acid and / or pathogen;

[0144] Wherein, the enrichment buffer contains the following components: a cationic surfactant, a cation exchange resin and a reducing agent;

[0145] The washing treatment comprises washing with a washing liquid containing the following components: a cationic surfactant and a neutral surfactant;

[0146] The elution treatment includes eluting using an elution buffer containing the following components: guanidine thiocyanate, EDTA, anionic surfactant, nonionic surfactant, chaotropic salt and buffer.

[0147] In a preferred embodiment, the volume of the sample is 0.1-500 mL, preferably 1-200 mL, preferably 5-100 mL, for example, about 10 mL.

[0148] In a preferred embodiment, the enrichment buffer comprises enrichment buffer A and enrichment buffer B. In a preferred embodiment, the enrichment buffer A comprises a cationic surfactant, the cationic surfactant is selected from the group consisting of hexadecyl ammonium bromide, hexadecyl trimethyl ammonium bromide chloride, hexadecyl trimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, or a combination thereof, preferably CTAB, more preferably 1% to 20% CTAB, more preferably 1% to 15% CTAB, more preferably 1% to 10% CTAB, for example, about 5% CTAB. In a preferred embodiment, the enrichment buffer B comprises a cation exchange resin and a reducing agent, the cation exchange resin is selected from the group consisting of 50-100 mesh chelex-100, 100-200 mesh chelex-100, 200-400 mesh chelex-100, or a combination thereof, preferably 100-200 mesh chelex-100; the reducing agent comprises acetylcysteine.

[0149] In a preferred embodiment, the step (s1) comprises mixing the sample with enrichment buffer A, adding enrichment buffer B for mixing, and then adding magnetic beads to obtain a first mixture.

[0150] In a preferred embodiment, the amount of enrichment buffer A added is 10-3000 μL, preferably 100-1000 μL, more preferably 200-800 μL, for example, about 500 μL. In a preferred embodiment, the volume ratio of enrichment buffer B to sample is 1:10-100, preferably 1:20-80, more preferably 1:30-60, for example, about 1:40.

[0151] In a preferred embodiment, in step (s1), in the first mixture, the concentration of acetylcysteine ​​is 1-100 mM, preferably 10-80 mM, more preferably 10-50 mM, for example, about 25 mM; the concentration of chelex is 0.1-500 mg / mL, preferably 1-300 mg / mL, more preferably 1-100 mg / mL, for example, about 25 mg / mL.

[0152] In a preferred embodiment, in step (s2), magnetic attraction and removal of supernatant are performed to obtain enrichment magnetic beads that adsorb nucleic acids and / or pathogens to be enriched.

[0153] In a preferred embodiment, in step (s2), the volume of the washing solution is 0.01-10 mL, preferably 0.1-5 mL, more preferably 0.5-2 mL, for example, about 1 mL.

[0154] In a preferred embodiment, in step (s3), the volume of the eluent is 10 to 1000 μL, preferably 100 to 800 μL, more preferably 100 to 500 μL, for example, about 200 μL.

[0155] In a preferred embodiment, in the washing liquid, the cationic surfactant includes CTAB. In a preferred embodiment, the concentration of the cationic surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0156] In a preferred embodiment, in the washing liquid, the neutral surfactant includes Triton. In a preferred embodiment, the concentration of the neutral surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0157] In a preferred embodiment, the washing solution comprises the following components: 0.1-5% CTAB and 0.1-5% Triton, preferably, 0.5-2.5% CTAB and 0.5-2.5% Triton; more preferably, 0.5-2% CTAB and 0.5-2% Triton, for example, about 1% CTAB and 1% Triton.

[0158] In a preferred embodiment, in the eluent, the anionic surfactant includes sodium lauroyl sarcosinate. In a preferred embodiment, in the eluent, the nonionic surfactant includes NP-40. In a preferred embodiment, in the eluent, the chaotropic salt is selected from the group consisting of potassium iodide, sodium perchlorate, or a combination thereof.

[0159] Kit of the present invention

[0160] The kit of the present invention comprises:

[0161] (i) Enrichment of magnetic beads;

[0162] (ii) an enrichment buffer comprising a cationic surfactant, a cation exchange resin and a reducing agent;

[0163] (iii) a washing solution comprising a cationic surfactant and a neutral surfactant; and

[0164] (iv) an eluting solution, which comprises guanidine thiocyanate, EDTA, an anionic surfactant, a nonionic surfactant, a chaotropic salt and a buffer.

[0165] In a preferred embodiment, the enrichment buffer comprises enrichment buffer A and enrichment buffer B. In a preferred embodiment, the enrichment buffer A comprises a cationic surfactant, the cationic surfactant is selected from the group consisting of hexadecyl ammonium bromide, hexadecyl trimethyl ammonium bromide chloride, hexadecyl trimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, or a combination thereof, preferably CTAB, more preferably 1% to 20% CTAB, more preferably 1% to 15% CTAB, more preferably 1% to 10% CTAB, for example, about 5% CTAB. In a preferred embodiment, the enrichment buffer B comprises a cation exchange resin and a reducing agent, the cation exchange resin is selected from the group consisting of 50-100 mesh chelex-100, 100-200 mesh chelex-100, 200-400 mesh chelex-100, or a combination thereof, preferably 100-200 mesh chelex-100; the reducing agent comprises acetylcysteine.

[0166] In another preferred embodiment, the enrichment buffer A and the enrichment buffer B are located in different containers.

[0167] In a preferred embodiment, in the washing liquid, the cationic surfactant includes CTAB. In a preferred embodiment, the concentration of the cationic surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0168] In a preferred embodiment, in the washing liquid, the neutral surfactant includes Triton. In a preferred embodiment, the concentration of the neutral surfactant in the washing liquid is 0.1-5%, preferably 0.5-2.5%, more preferably 0.5-2%, for example, about 1%.

[0169] In a preferred embodiment, the washing solution comprises the following components: 0.1-5% CTAB and 0.1-5% Triton, preferably, 0.5-2.5% CTAB and 0.5-2.5% Triton; more preferably, 0.5-2% CTAB and 0.5-2% Triton, for example, about 1% CTAB and 1% Triton.

[0170] In a preferred embodiment, in the eluent, the anionic surfactant includes sodium lauroyl sarcosinate. In a preferred embodiment, in the eluent, the nonionic surfactant includes NP-40. In a preferred embodiment, in the eluent, the chaotropic salt is selected from the group consisting of potassium iodide, sodium perchlorate, or a combination thereof.

[0171] In a preferred embodiment, the eluent comprises the following components: 20% to 40% guanidine thiocyanate, 0.1 to 50 mM EDTA, 0.01 to 1 M Tris-HCl, 0.01% to 0.1% (v / v) sodium lauroyl sarcosinate, 0.1% to 5% (v / v) NP-40, and 0.5 to 2 M potassium iodide.

[0172] In a preferred embodiment, the eluent comprises the following components: 25% to 35% guanidine thiocyanate, 0.5 to 25 mM EDTA, 0.01 to 0.1 M Tris-HCl, 0.03% to 0.08% sodium lauroyl sarcosinate, 0.1% to 3% NP-40, and 0.8 to 1.5 M potassium iodide.

[0173] In a preferred embodiment, the eluent comprises the following components: 28% to 32% guanidine thiocyanate, 0.25 to 10 mM EDTA, 0.01 to 0.07 M Tris-HCl, 0.04% to 0.06% sodium lauroyl sarcosinate, 0.1% to 1% NP-40, and 0.8 to 1.2 M potassium iodide.

[0174] In a preferred embodiment, the eluent comprises the following components: 30% guanidine thiocyanate, 5 mM EDTA, 0.05 M Tris-HCl, 0.05% sodium lauroyl sarcosinate, 0.5% NP-40, and 1 M potassium iodide.

[0175] The main advantages of the present invention include:

[0176] 1. The method of the present invention can simultaneously enrich viruses and nucleic acids.

[0177] 2. The method of the present invention improves the efficiency of enriching nucleic acids, viruses, and bacteria from samples and shortens the time for enriching nucleic acids in samples.

[0178] 3. The method of the present invention has many application scenarios and a wide range, and still has a high enrichment efficiency for samples containing more impurities.

[0179] 4. The method of the present invention is suitable for nucleic acid and / or pathogen enrichment in large volume samples.

[0180] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0181] Example 1

[0182] 1.1 Methods

[0183] After the pseudovirion of Norovirus is treated with universal nuclease, it is divided into two parts, one part is used as virus particles, and the other part is extracted with nucleic acid extraction and purification reagent (Shanghai Fengxiebei No. 20180202) and used as nucleic acid. The present invention is based on positively charged magnetic microspheres. The positive microspheres can bind to negatively charged substances through charge action. At the same time, in a high salt concentration environment, due to the change of charge, this binding is reversible.

[0184] The present invention evaluates the efficiency of adsorbing viruses and nucleic acids through the above samples, and uses the eluents of the formulas shown in Table 1 for testing respectively:

[0185] Table 1

[0186] serial number formula Recipe 1 1M NaCl, 1% 0.5M EDTA, 5% 1M Tris-HCl Recipe 2 1M guanidine thiocyanate, 1% 0.5M EDTA, 5% 1M Tris-HCl Recipe 3 2M guanidine thiocyanate, 1% 0.5M EDTA, 5% 1M Tris-HCl Recipe 4 3M guanidine thiocyanate, 1% 0.5M EDTA, 5% 1M Tris-HCl

[0187] Note: 1% 0.5M EDTA means adding 1% 0.5M EDTA, and the converted concentration is 5mM EDTA; 5% 1M Tris-HCl means adding 5% 1M Tris-HCl, and the converted concentration is 0.05M Tris-HCl.

[0188] The specific operation process is as follows:

[0189] (1) Sample processing

[0190] All samples in this experiment were subjected to two biological replicates.

[0191] For the virus particle samples, the virus particle samples were diluted 1000 times, and then the pseudovirus particle samples treated with universal nuclease were used for the experiment. The samples were divided into 5 portions, one portion of 200 μL was directly used with nucleic acid extraction and purification reagents (Shanghai Fengxiebei No. 20180202) (S1), and the other four portions of 200 μL were added to 9800 μL of DEPC-treated water and eluted with elution buffers of different formulas after enrichment.

[0192] The nucleic acid sample (the nucleic acid obtained after nucleic acid extraction of the same pseudovirus as above) was divided into 5 portions, one portion of 200 μL (as an undiluted and unenriched control), and the other four portions of 200 μL were added to 9800 μL of DEPC-treated water and eluted using elution solutions of different formulas after enrichment.

[0193] (2) Enrichment operation

[0194] Add 100 μL of positively charged magnetic microspheres to each of the two 10,000 μL samples, vortex and mix, then let stand for 6 minutes, mixing every 2 minutes. After mixing, magnetize and discard the supernatant. Add 200 μL of 1M NaCl solution, mix the magnetic beads thoroughly and let stand for 2 minutes, magnetize and take the supernatant as a sample for nucleic acid extraction. Different formulas are used to elute the enriched samples of pseudovirus and nucleic acid.

[0195] (3) Nucleic acid extraction

[0196] The above-mentioned enriched samples and undiluted, unenriched samples (control) were extracted simultaneously using nucleic acid extraction and purification reagents (Shanghai Fengxiebei No. 20180202), and detected using the Norovirus Nucleic Acid Detection Kit (Fluorescence PCR Method) (National Medical Device Registration No. 20213400566).

[0197] 1.2 Results

[0198] The results are shown in Table 2.

[0199] Table 2 Eluent screening results

[0200]

[0201]

[0202] According to the results in Table 2, guanidine isothiocyanate of the same concentration has a stronger chaotropic effect and a stronger ability to shield charges, and its elution effect is better than that of 1M NaCl. It can also be seen that with the increase in the concentration of guanidine isothiocyanate, the elution effect on nucleic acids gradually increases, but the increase in the elution effect on pseudoviruses is not obvious.

[0203] Example 2

[0204] 2.1 Methods

[0205] The sample processing was the same as that in Example 1, and the elution test was performed using the formulations shown in Table 3.

[0206] Table 3

[0207]

[0208] Note: 1% 0.5M EDTA means adding 1% 0.5M EDTA, and the concentration after conversion is 5mM EDTA; 5% 1MTris-HCl means adding 5% 1M Tris-HCl, and the concentration after conversion is 0.05M Tris-HCl.

[0209] 2.2 Results

[0210] The results are shown in Table 4.

[0211] Table 4 Eluent screening results

[0212]

[0213]

[0214] According to the results shown in Table 4, the addition of surfactant can effectively improve the recovery efficiency of the virus. According to the binding principle of the surface groups of the magnetic beads with nucleic acids and viruses, it is speculated that the recovery efficiency is low due to incomplete charge shielding. It is preferred to add a large molecular weight chaotropic salt to further improve the elution efficiency.

[0215] The basic formula (Formula 1 in the above table) was added with 0.5% NP-40 alone: ​​the recovery rate of pseudovirus was 50.52%; the recovery rate of nucleic acid was 75.00%.

[0216] The basic formula (Formula 1 in the above table) was added with 0.10% sodium lauroyl sarcosinate alone: ​​the recovery rate of pseudovirus was 69.50%; the recovery rate of nucleic acid was 73.46%.

[0217] Example 3

[0218] The sample processing was the same as that in Example 1, and the elution test was performed using the formulation shown in Table 5.

[0219] Table 5

[0220]

[0221]

[0222] Note: 1% 0.5M EDTA means adding 1% 0.5M EDTA, and the concentration after conversion is 5mM EDTA; 5% 1MTris-HCl means adding 5% 1M Tris-HCl, and the concentration after conversion is 0.05M Tris-HCl.

[0223] Among them, Formula 4 and Formula 7 were not subjected to subsequent experiments due to poor solubility due to high salt concentration.

[0224] 3.2 Results

[0225] The results are shown in Table 6.

[0226] Table 6

[0227]

[0228]

[0229] By comparing the elution effects after enrichment, the elution solution in this example has a good enrichment effect on both pseudovirus and nucleic acid.

[0230] The commonly used buffer system is 1-5% 0.5M EDTA, 1-10% Tris-HCl, and the preferred eluent formula is 10-30% guanidine thiocyanate.

[0231] In order to further optimize the elution efficiency of pseudovirus, the optimized elution solution formula is 10-30% guanidine thiocyanate, 0.1%-0.2% NP-40, and 0.05%-0.1% sodium lauroyl sarcosinate. In order to further improve the efficiency and stability of elution, the optimized elution solution formula is 30% guanidine thiocyanate, 1% 0.5M EDTA, 5% 1M Tris-HCl, 0.15% sodium lauroyl sarcosinate, 0.5% NP-40, and 0.5-1M chaotropic salt (such as potassium iodide or sodium perchlorate).

[0232] Example 4

[0233] 4.1 Methods

[0234] Tests are conducted on different pathogens and matrices, including urine, serum, and domestic sewage; and pathogens include standard strains of COVID-19, Escherichia coli, and Mycoplasma pneumoniae.

[0235] All samples in this experiment were subjected to two biological replicates.

[0236] After the Norovirus, influenza A samples, Escherichia coli liquid, and Mycoplasma pneumoniae standard strain were tested with the detection reagent, they were diluted to a theoretical Ct value of 25 according to the Ct value, and mixed together at a ratio of Norovirus: influenza A virus: Escherichia coli liquid: Mycoplasma pneumoniae standard strain: water = 1:1:1:1:1. DEPC-treated water, urine, serum, and domestic sewage were used to dilute 10 times, and different types of samples were divided into two parts, one 200 μL was directly used for nucleic acid extraction and detection, and the other was diluted to 10 mL with the corresponding sample matrix.

[0237] The treatment was carried out according to the method described in Example 1. The enriched concentrate and the unenriched sample were extracted by nucleic acid extraction and purification reagents (Shanghai Fengxiebei No. 20180202), and then detected using detection reagents for norovirus, influenza A virus, Escherichia coli, and Mycoplasma pneumoniae.

[0238] 4.2 Results

[0239] The results are shown in Table 7.

[0240] Table 7

[0241]

[0242]

[0243] Using the optimized formula in Example 3, excellent recovery rates were achieved in urine and serum matrices, but poor recovery efficiency was achieved in the matrix of domestic sewage. For more complex matrices, it is speculated that the ions or proteins present in the matrix may have affected the enrichment. At the same time, since it contains more inhibitors, it may also have affected the subsequent extraction.

[0244] Example 5

[0245] 5.1 Methods

[0246] According to the binding principle of amino magnetic beads, common cationic surfactants and reducing agents are added during enrichment to reduce other negatively charged substances in the sample. The specific test formula is shown in Table 8.

[0247] Table 8

[0248] serial number formula Recipe 1 Add 500 μL 1% CTAB Recipe 2 Add 500 μL 2.5% CTAB Recipe 3 Add 500 μL 5% CTAB Recipe 4 Add β-mercaptoethanol at a ratio of 1:125 volume:sample volume Recipe 5 Add 1M DTT at a ratio of volume: sample volume = 1:40 Recipe 6 Add 1M acetylcysteine ​​at a ratio of volume: sample volume = 1:40

[0249] Sample processing was the same as in Example 4, and norovirus particles were used for evaluation. The enriched concentrate and the unenriched sample were extracted using nucleic acid extraction and purification reagents (Shanghai Fengxiebei No. 20180202), and then tested using norovirus, influenza A virus, Escherichia coli, and Mycoplasma pneumoniae detection reagents.

[0250] 5.2 Results

[0251] The results are shown in Table 9.

[0252] Table 9

[0253]

[0254] According to the results, the addition of cationic surfactants and reducing agents can effectively improve the enrichment efficiency in different matrices, and the improvement effect is obvious in simpler biological samples such as urine and serum. However, there is still a problem of low efficiency in sewage.

[0255] Example 6

[0256] 6.1 Methods

[0257] According to the results of Example 5, adding positively charged substances to the solution as much as possible can effectively improve the binding efficiency. Cationic exchange resins are added during enrichment, mainly chelex of different particle sizes, including chelex-100 (50-100 mesh), chelex-100 (100-200 mesh), and chelex-100 (200-400 mesh). Tests were carried out in domestic sewage. Considering that sewage samples are more complex, the tests were carried out in combination with the above optimal conditions. The specific test formula is shown in Table 10.

[0258] Table 10

[0259]

[0260] Acetylcysteine ​​and chelex can be mixed and added to ensure that the final concentration of acetylcysteine ​​is 24.4 mM and the final concentration of chelex is 0.025 g / mL.

[0261] The sample processing was the same as in Example 4. The enriched concentrate and the unenriched sample were extracted using a nucleic acid extraction and purification reagent (Shanghai Fengxiebei No. 20180202), and then tested using a Norovirus detection reagent.

[0262] 6.2 Results

[0263] The results are shown in Table 11.

[0264] Table 11

[0265]

[0266]

[0267] According to the above results, in order to improve the enrichment efficiency and the suitable sample types, an enrichment buffer composed of a cationic surfactant, a reducing agent, and a cation exchange resin can be added during enrichment.

[0268] Example 7

[0269] 7.1 Methods

[0270] Considering the complexity of sewage samples, after the sewage samples are enriched in the above-mentioned embodiment 6, the eluent is sometimes turbid or colored. Considering the complexity of sewage, a washing liquid can be designed according to the principle of enrichment. The matrix of the washing liquid is ultrapure water, and other ions in the water are removed as much as possible. At the same time, cationic surfactants are continued to be added to keep nucleic acids or virus particles closely connected to magnetic beads. A small amount of nonionic surfactants, Triton-X-100, NP-40, are added to remove impurities such as proteins and pigments in order to obtain better enrichment effects. The formula tested is shown in Table 12.

[0271] Table 12

[0272] serial number formula Recipe 1 No washing Recipe 2 Ultrapure water, 1% CTAB Recipe 3 Ultrapure water, 2.5% CTAB Recipe 4 Ultrapure water, 1% CTAB, 1% Triton Recipe 5 Ultrapure water, 1% CTAB, 2.5% Triton Recipe 6 Ultrapure water, 1% CTAB, 5% Triton

[0273] The washing step is performed after the enrichment is completed and before elution. Different formulas are used for washing. 1 mL of washing solution is used for washing. The magnetic beads are mixed and then left to stand for 2 minutes. After the magnetic beads are adsorbed on a magnetic stand, the washing solution is discarded and elution is performed according to the elution step.

[0274] 7.2 Results

[0275] The results are shown in Table 13.

[0276] Table 13

[0277]

[0278]

[0279] Adding a washing step can effectively improve the enrichment effect. Using 1% CTAB is more effective. High concentrations of CTAB are prone to produce more bubbles during the mixing process, which may cause the loss of magnetic beads.

[0280] Example 8

[0281] 8.1 Methods

[0282] The enrichment volume of this method was confirmed using sewage samples. Norovirus was used as the sample and domestic sewage was used to dilute the norovirus to a Ct value of around 30. 200 μL of the sample was left as the non-extracted treatment group, and the other samples were diluted according to Table 14.

[0283] Table 14

[0284] Samples of different volumes Sample volume (μL) Sewage volume (μL) Total volume (μL) Sample 1 200 4800 5000 Sample 2 200 9800 10000 Sample 3 200 14800 15000 Sample 4 200 24800 25000 Sample 5 200 49800 50000 Sample 6 200 99800 100000

[0285] Take the above sample, add enrichment buffer (Formula 4) according to the method in Example 6, and vortex mix, then add 100 μL of surface-modified amino-containing cationic magnetic microspheres with cores, mix thoroughly and let stand for 6 minutes, vortex mix every 2 minutes, use a magnetic stand to adsorb the magnetic beads, and discard the supernatant.

[0286] For the magnetic beads obtained in the previous step, add 500 μL of the washing solution (Formula 4) in Example 7, mix the magnetic beads thoroughly, use a magnetic stand to adsorb the magnetic beads, and discard the supernatant.

[0287] For the magnetic beads obtained in the previous step, add 200 μL of the eluent (Formula 3) in Example 3, repeatedly disperse the magnetic beads, and let stand for 2 minutes. The obtained liquid is extracted using a nucleic acid extraction reagent, and the nucleic acid obtained after extraction is the nucleic acid concentrate in the sample. Use a Norovirus nucleic acid detection kit for detection.

[0288] 8.2 Results

[0289] The results are shown in Table 15.

[0290] Table 15

[0291]

[0292]

[0293] According to the above results, good enrichment effects can be achieved within a sample volume of 50 mL.

[0294] Example 9

[0295] 9.1 Methods

[0296] The compatibility of the eluate obtained after enrichment with different extraction reagents was tested. The sample used norovirus, and domestic sewage was used to dilute the norovirus to a Ct value of about 30. Three 200 μL samples were prepared, and nucleic acid extraction and purification reagents (Shanghai Feng Xiebei No. 20180202), QIAamp Viral RNA Mini Kit, Tianlong nucleic acid extraction and purification reagents (Shaanxi Xiebei No. 20140007) were used for extraction, and the three kits were represented by Kit 1, Kit 2, and Kit 3, respectively. After diluting the other three 200 μL samples to 50 mL with domestic sewage, they were enriched according to the steps in Example 7, and extracted with nucleic acid extraction and purification reagents (Shanghai Feng Xiebei No. 20180202), QIAamp Viral RNA Mini Kit, Tianlong nucleic acid extraction and purification reagents (Shaanxi Xiebei No. 20140007). The nucleic acid obtained after extraction was detected using a norovirus nucleic acid detection reagent.

[0297] 9.2 Results

[0298] The results are shown in Table 16.

[0299] Table 16

[0300]

[0301] According to the above results, the eluate enriched according to this method is compatible with the above three types of nucleic acid extraction and purification reagents.

[0302] Comparative Example 1

[0303] Compared with the method in the industry standard, the norovirus in sewage was enriched, and the norovirus was first diluted with domestic sewage to a Ct value of about 30. The method in the industry standard follows the operation recorded in WS / T 799-2022.

[0304] (1) Polyethylene glycol precipitation method

[0305] Take 200 μL of diluted Norovirus sample and dilute it to 35 mL with domestic sewage. Add 3.5 g ± 0.1 g of polyethylene glycol and 0.79 g ± 0.01 g of sodium chloride to the 50 mL centrifuge tube containing the sample and mix thoroughly until the reagent is completely dissolved. The dissolution process takes about 15 minutes. Then centrifuge at 4 ° C and 12 000 g for 120 minutes. The braking force should not be used when the centrifuge slows down. After the centrifuge stops, pour and discard the supernatant in the centrifuge tube until no supernatant flows out.

[0306] Centrifuge the remaining sample after the second centrifugation again at 4°C and 12 000g for 5 min. The centrifuge should not be braked when slowing down. After the centrifuge stops, remove the remaining supernatant from the centrifuge tube with a pipette and discard.

[0307] Pipette 0.2mL of nuclease-free water into one of the centrifuge tubes, repeatedly blow and suck the precipitate, centrifuge instantly, and make all the liquid gather at the bottom of the tube. The final suspension is the concentrated solution of the water sample, with a volume of about 0.2mL. Transfer the concentrated solution to a 1.5mL centrifuge tube, store it at 0℃~4℃, and complete nucleic acid extraction and real-time fluorescence RT-PCR detection within 24h.

[0308] (2) Aluminum salt coagulation precipitation method

[0309] Take 200 μL of norovirus sample and dilute it to 50 mL. Add 0.5 mL of 0.3 mol / L aluminum chloride solution to the 50 mL centrifuge tube containing the sample, and adjust the water sample pH to 6.0 ± 0.1 with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution. Place the sample centrifuge tube in a constant temperature shaking incubator and mix at room temperature for 15 minutes at a speed of 150 r / min.

[0310] The sample was centrifuged at 4°C and 1900g for 5 minutes. The supernatant in the centrifuge tube was discarded, and 0.20g±0.01g of disodium ethylenediaminetetraacetic acid dihydrate was added to the remaining colloid, and the colloid was shaken dozens of times until it became liquid, and then transferred to a 10mL centrifuge tube. The 10mL centrifuge tube was placed in a water bath and water bathed at 60°C for 10 minutes. The liquid in the centrifuge tube after the water bath is the concentrated solution of the water sample, with a volume of about 0.2mL. It was stored at 0°C to 4°C, and nucleic acid extraction and real-time fluorescence RT-PCR detection were completed within 24 hours.

[0311] The enrichment method of the present invention is carried out according to the method in Example 8, and enrichment is carried out from 35mL and 50mL of domestic sewage, respectively. The enriched concentrate and the unenriched sample are extracted by nucleic acid extraction and purification reagents (Shanghai Fengxiebei No. 20180202), and then detected using a Norovirus detection reagent.

[0312] The results are shown in Table 17.

[0313] Table 17

[0314]

[0315]

[0316] As shown in Table 17, the enrichment efficiency of the method of the present invention is much higher than the method recommended by the industry standard.

[0317] Comparative Example 2

[0318] Conventional enrichment methods usually use the positive charge on the surface of magnetic beads for enrichment, such as the magnetic beads product with catalog number 10700D from Thermo Fisher Scientific. The magnetic beads can be used to enrich various viruses in sewage samples, and then the virus particles are released through the buffer. However, the product has poor adsorption capacity for nucleic acids in samples, which may cause missing results.

[0319] The enrichment performance of the two kits was evaluated using simulated samples of domestic sewage. Norovirus and norovirus nucleic acid were used as simulated samples. A total of two samples were prepared, one of which had a final concentration of 28 for the pseudovirus Ct value and 30 for the nucleic acid, and was recorded as sample 1; the other had a final concentration of 30 for the pseudovirus Ct value and 28 for the nucleic acid, and was recorded as sample 2.

[0320] Thermo Fisher's enrichment products are used in combination with Thermo Fisher's extraction reagents (A52610) for enrichment and extraction.

[0321] The specific operation method is as follows:

[0322] Take 40mL of domestic sewage, add 2mL of formula 4 (enrichment buffer) in Example 6 and vortex mix, then add 100μL of surface-modified amino-containing cationic core-containing magnetic microspheres, mix thoroughly and let stand for 6min, vortex mix once every 2min, use a magnetic stand to adsorb the magnetic beads, and discard the supernatant. Add 500μL of formula 4 (washing solution) in Example 7, mix the magnetic beads thoroughly, use a magnetic stand to adsorb the magnetic beads, and discard the supernatant. Add 200μL of formula 3 (elution solution) in Example 3, repeatedly break up the magnetic beads, and let stand for 2min. The obtained liquid is extracted using a nucleic acid extraction reagent, and the nucleic acid obtained after extraction is the nucleic acid concentrate in the sample.

[0323] The results are shown in Table 18.

[0324] Table 18

[0325]

[0326] As shown in Table 18, the method of the present invention has relatively stable results in different sewage and can simultaneously adsorb viral particles and nucleic acids in sewage.

[0327] Comparative Example 3

[0328] For the enrichment of nucleic acids in urine, the existing kits mainly include the urine nucleic acid extraction kit with the Zymo catalog number D3061. The enrichment effects of the two kits were evaluated using urine from adult males and adult females. The Zymo kit was operated according to its instructions, and the method of the present invention was operated according to the following steps:

[0329] Take a sample, add Formula 4 in Example 6 (add according to the proportion of 10 ml sample), then add 100 μL of amino magnetic beads, mix thoroughly and let stand for 6 minutes, mixing every 2 minutes. Use a magnetic stand to absorb the magnet, discard the supernatant, and use 200 μL of Formula 3 in Example 3 for elution. The eluted nucleic acid is extracted using a nucleic acid extraction and purification reagent (Shanghai Fengxiebei No. 20180202).

[0330] The nucleic acid concentrations obtained by both kits were detected using Qubit Flex.

[0331] The results are shown in Table 19.

[0332] Table 19

[0333]

[0334] The results show that the method of the present invention is better than the zymo reagent in enriching nucleic acids in the same urine sample. At the same time, the method provided by the present invention is combined with the extraction reagent, and the operation time is within 30 minutes, while the operation time of the zymo reagent is more than 1 hour.

[0335] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for enriching nucleic acids and / or pathogens from a sample, characterized in that: Includes steps: (s1) mixing the sample, enrichment magnetic beads and enrichment buffer to obtain a first mixture, and performing enrichment treatment so that the enrichment magnetic beads adsorb the nucleic acid and / or pathogen to be enriched in the sample to form enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched (enrichment magnetic beads after enrichment treatment); (s2) separating the enrichment magnetic beads adsorbed with the nucleic acid and / or pathogen to be enriched, and washing them to obtain washed magnetic beads; and (s3) separating the washed magnetic beads and performing an elution treatment to elute the enriched nucleic acid and / or pathogen from the magnetic beads to obtain an eluate containing the enriched nucleic acid and / or pathogen; Wherein, the enrichment buffer contains the following components: a cationic surfactant, a cation exchange resin and a reducing agent; The washing treatment comprises washing with a washing liquid containing the following components: a cationic surfactant and a neutral surfactant; The elution treatment includes eluting using an elution buffer containing the following components: guanidine thiocyanate, EDTA, an anionic surfactant, a nonionic surfactant, a chaotropic salt and a buffer.

2. The method according to claim 1, characterized in that The volume of the sample is 0.1-500 mL.

3. The method according to claim 1, characterized in that The enrichment buffer comprises an enrichment buffer A and an enrichment buffer B, wherein the enrichment buffer A comprises a cationic surfactant; and the enrichment buffer B comprises a cation exchange resin and a reducing agent.

4. The method according to claim 3, characterized in that The cationic surfactant is selected from the group consisting of hexadecyl ammonium bromide, hexadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, or a combination thereof.

5. The method according to claim 3, characterized in that The cation exchange resin is selected from the group consisting of 50-100 mesh chelex-100, 100-200 mesh chelex-100, 200-400 mesh chelex-100, or a combination thereof; and the reducing agent comprises acetylcysteine.

6. The method according to claim 1, characterized in that The step (s1) comprises mixing the sample with enrichment buffer A, adding enrichment buffer B to mix, and then adding magnetic beads to obtain a first mixture.

7. The method according to claim 1, characterized in that In the washing solution, the cationic surfactant includes CTAB; and / or the neutral surfactant includes Triton.

8. The method according to claim 1, characterized in that The washing solution comprises the following components: 0.1-5% CTAB and 0.1-5% Triton.

9. The method according to claim 1, characterized in that In the eluent, the anionic surfactant includes sodium lauroyl sarcosinate; the nonionic surfactant includes NP-40; and / or the chaotropic salt is selected from the group consisting of potassium iodide, sodium perchlorate, or a combination thereof.

10. The method according to claim 1, characterized in that The eluent comprises the following components: 20% to 40% guanidine thiocyanate, 0.1 to 50 mM EDTA, 0.01 to 1 M Tris-HCl, 0.01% to 0.1% sodium lauroyl sarcosinate, 0.1% to 5% NP-40, and 0.5 to 2 M potassium iodide.

11. The method according to claim 1, characterized in that In the first mixture, the concentration of acetylcysteine ​​is 1-100 mM and the concentration of chelex is 0.1-500 mg / mL.

12. The method according to claim 1, characterized in that In the first mixture, the concentration of acetylcysteine ​​is 10-80 mM and the concentration of chelex is 1-300 mg / mL.

13. The method according to claim 1, characterized in that In step (s3), the volume of the washing solution is 0.01 to 10 mL.

14. The method according to claim 1, wherein: In step (s4), the volume of the eluent is 10 to 1000 μL.

15. The method according to claim 1, wherein: The pathogen is selected from the group consisting of bacteria, viruses, mycoplasmas, chlamydia, or a combination thereof.

16. The method according to claim 1, wherein: The sample is selected from the group consisting of domestic sewage, plasma, serum, urine, water sample, swab wash fluid, or a combination thereof.

17. A method for extracting nucleic acid from a pathogen sample, characterized in that: Includes steps: (s1) mixing the sample, enrichment magnetic beads and enrichment buffer to obtain a first mixture, and performing enrichment treatment so that the enrichment magnetic beads adsorb the nucleic acid and / or pathogen to be enriched in the sample to form enrichment magnetic beads adsorbing the nucleic acid and / or pathogen to be enriched (enrichment magnetic beads after enrichment treatment); (s2) separating the enrichment magnetic beads adsorbed with the nucleic acid and / or pathogen to be enriched, and washing them to obtain washed magnetic beads; (s3) separating the washed magnetic beads and performing an elution treatment to elute the enriched nucleic acid and / or pathogen from the magnetic beads to obtain an eluate containing the enriched nucleic acid and / or pathogen; and (s4) extracting nucleic acid from the enriched nucleic acid and / or pathogen to obtain the nucleic acid; Wherein, the enrichment buffer contains the following components: a cationic surfactant, a cation exchange resin and a reducing agent; The washing treatment comprises washing with a washing liquid containing the following components: a cationic surfactant and a neutral surfactant; The elution treatment includes eluting using an elution buffer containing the following components: guanidine thiocyanate, EDTA, an anionic surfactant, a nonionic surfactant, a chaotropic salt and a buffer.

18. A kit for enriching nucleic acids and / or pathogens from a sample, characterized in that: include: (i) Enrichment of magnetic beads; (ii) an enrichment buffer; the enrichment buffer comprises a cationic surfactant, a cation exchange resin and a reducing agent; (iii) a washing liquid; the washing liquid comprising a cationic surfactant and a neutral surfactant; and (iv) an eluting solution, which comprises guanidine thiocyanate, EDTA, an anionic surfactant, a nonionic surfactant, a chaotropic salt and a buffer.

19. The kit according to claim 18, wherein The enrichment buffer comprises an enrichment buffer A and an enrichment buffer B, wherein the enrichment buffer A comprises a cationic surfactant; and the enrichment buffer B comprises a cation exchange resin and a reducing agent.

20. The kit according to claim 19, characterized in that The enrichment buffer A and the enrichment buffer B are located in different containers.

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